Proefschrift van den Brom

Charissa Esmé van den Brom

Dietary modulation of the effects of sevoflurane on myocardial perfusion, function and ischemic injuryin rats

Dietary m

odulation of the effects of sevoflurane on myocardial perfusion, function and ischem

ic injury in rats Charissa Esm

é van den Brom

UITNODIGING

voor het bijwonen van de openbare verdediging van

het proefschrift

Dietary modulation of the effects of sevoflurane on

myocardial perfusion, function and ischemic

injury in rats

door Charissa van den Brom

Donderdag 30 januari 2014 om 15.45 uur

In de aula van het Hoofdgebouw

Vrije UniversiteitDe Boelelaan 1105

te Amsterdam

Receptie na afloop van de promotie

Paranimfen

Ester [email protected]

Marianne de Gruil-van [email protected]

Dietary modulation of the

effects of sevoflurane on

myocardial perfusion,

function and ischemic

injury in rats


Het effect van dieetsamenstelling en sevofluraan op de perfusie, functie en

ischemische schade van het hart in ratten

Printed by: Gildeprint Drukkerijen www.gildeprint.nl

ISBN: 978-90-9027989-3

Copyright © 2013 by C.E. van den Brom ([email protected])

All rights reserved. No part of this book may be reproduced or transmitted in any

form or by any means, without prior written permission of the author.

The work presented in this thesis was performed at the Department of

Anesthesiology and Laboratory for Physiology, VU University Medical Center /

Institute of Cardiovascular Research (ICaR-VU), Amsterdam, the Netherlands.

Financial support by the Dutch Heart Foundation for the publication of this thesis is

gratefully acknowledged.

Additional financial support for this thesis was kindly provided by the following

sponsors: UNO roestvaststaal B.V. and AbbVie B.V.

VRIJE UNIVERSITEIT

Dietary modulation of the effects of sevoflurane on myocardial perfusion, function and ischemic

injury in rats

ACADEMISCH PROEFSCHRIFT

ter verkrijging van de graad Doctor aan de Vrije Universiteit Amsterdam,

op gezag van de rector magnificus prof.dr. F.A. van der Duyn Schouten,

in het openbaar te verdedigen ten overstaan van de promotiecommissie

van de Faculteit der Geneeskunde op donderdag 30 januari 2014 om 15.45 uur

in de aula van de universiteit, De Boelelaan 1105

door


geboren te Nijkerk

promotor: prof.dr. S.A. Loer copromotoren: dr. C. Boer dr. R.A. Bouwman

Voor papa

Contents

Chapter 1: 9

General introduction & Outline of this thesis

Chapter 2: 19

Metabolic disease and perioperative ischemia in the experimental setting:

Consequences of derangements in myocardial substrate metabolism

Chapter 3: 49

High fat diet-induced glucose intolerance impairs myocardial function, but

not myocardial perfusion during hyperemia: a pilot study

Chapter 4: 67

Sevoflurane impairs myocardial systolic function, but not myocardial

perfusion in diet-induced prediabetic rats

Chapter 5: 85

Diet composition modulates sevoflurane-induced myocardial depression in

rats

Chapter 6: 109

Western diet modulates the susceptibility of the heart to ischemic injury

and sevoflurane-induced cardioprotection in rats

Chapter 7: 129

Conclusions & General discussion

Chapter 8: 143

Summary

Chapter 9: 149

Samenvatting

List of abbreviations 155

Dankwoord 161

List of publications 167

Curriculum Vitae 177

1General introduction & Outline of this

thesis

Introduction 11

Perioperative cardiovascular risks

In the Netherlands, about 1.3 million surgical procedures are performed every year

under general or locoregional anesthesia. Surgery and anesthesia are associated

with alterations in systemic and regional perfusion and oxygenation. These

alterations are due to the cardiovascular effects of anesthetics, loss and replacement

of intravascular volume, mechanical positive pressure ventilation and application of

vasoactive drugs. Although the mortality risk of surgery and anesthesia is nowadays

considerably low, patients are still at risk for perioperative cardiovascular

complications. Bainbridge et al. showed a total perioperative mortality of 0.12% and

anesthetic-related mortality of 0.0034% in the 1990s-2000s in developed and

developing countries.1 The risk for cardiovascular complications increases with age

and in the presence of comorbidities like heart disease, pulmonary disease or

diabetes mellitus.2-4 Other well-known predictors of perioperative cardiac

complications are coronary disease, angina pectoris, prior myocardial infarction,

heart failure, stroke/transient ischemic attack, renal dysfunction and diabetes

mellitus requiring insulin therapy.4;5 It is estimated that cardiovascular complications

like myocardial infarction and cardiac arrest occur in 2-5% of all noncardiac surgical

procedures, and globally affect 5-12 million patients each year.4;5

Myocardial ischemia and reperfusion

The most common cardiovascular complication during or after noncardiac surgery is

the development of myocardial ischemia,4;5 which is associated with a significant risk

for morbidity and mortality. Myocardial ischemia occurs when coronary perfusion is

inadequate to maintain a sufficient oxygen supply/demand ratio in the heart. During

surgery, maintenance of the myocardial oxygen balance is challenged by anesthetics

and surgical stress, which directly affect myocardial oxygen supply and consumption.

This altered balance may increase the risk of myocardial ischemia.6;7

While the silent nature of perioperative myocardial ischemia hampers its diagnosis,

Landesberg et al. showed that patients with a large increase in troponin following

major surgery are at risk for cardiac complications and unfavorable outcome.8 More

recently, McFalls et al. described that perioperative elevated cardiac troponin levels

strongly predict long-term mortality.9 From these findings it might be concluded that

myocardial ischemia is often present during and after surgery, and prevention of an

oxygen supply/demand mismatch may contribute to improved postoperative

outcome.

Volatile anesthesia

There is an ongoing debate whether the type of anesthesia could modulate the risk

of perioperative cardiovascular complications. Surgical procedures under general

1

12 Chapter 1

anesthesia are either performed using intravenous anesthetics, like propofol, or

inhalational anesthesics using volatile agents like isoflurane or sevoflurane. Volatile

anesthetics are acknowledged for their cardioprotective effects, which consist of

preservation of cardiovascular function in case of reduced tissue oxygenation.

Volatile anesthetics exert several effects on the heart, peripheral vasculature and

the autonomic nervous system. Sevoflurane alters calcium (Ca2+) handling in the

heart. To be more specific, sevoflurane reduces myocardial Ca2+ availability and

increases sarcoplasmic reticulum Ca2+ content,10 depresses Ca2+ currents,11 reduces

Ca2+ influx via the L-type Ca2+ channels12 and increases Na+/Ca2+ exchanger-

mediated Ca2+ influx.13 In addition, sevoflurane prolongs the QT interval, thereby

prolonging ventricular repolarization.14;15 Moreover, inhalation of volatile anesthetics

causes a dose-dependent decrease in blood pressure, mainly due to a reduction in

systemic vascular resistance.16 In healthy humans, sevoflurane decreases myocardial

blood volume and hyperemic blood flow, while myocardial blood flow during baseline

conditions is not affected.17 Further effects of volatile anesthetics on the heart are

negative inotropic effects, such as depressed myocardial contractility and lusitropic

effects reflected by early diastolic dysfunction.18-20 One might expect that, due to the

general effects of volatile anesthetics on the heart, anesthesia may be associated

with a changed balance between myocardial pefusion and function, although this has

been rarely investigated.

Cardiometabolic disease

Patients with lifestyle risk factors, such as excessive caloric intake and a sedentary

lifestyle, which both contribute to the development of obesity and type 2 diabetes

mellitus, even show a higher risk for postoperative morbidity and mortality in case of

perioperative myocardial ischemia.3;4;9;21 Worldwide, more that 1.4 billion adults

were overweight in 2008, and of these over 500 million people were obese.

Moreover, 344 million people suffer from impaired glucose tolerance, whereas 366

million patients are diagnosed with type 2 diabetes mellitus.22 It is expected that by

the year 2030 almost 400 million people suffer from impaired glucose tolerance and

552 million people from diabetes.22

The higher risk for the development of perioperative myocardial ischemia in

patients with obesity and type 2 diabetes mellitus may partly be due to reduced

coronary vasodilation in response to pharmacological stimuli, atherosclerosis,

oxidative stress and insulin resistance.23-25 With the expanding epidemic of obesity

and type 2 diabetes mellitus it is therefore expected that the number of patients that

are prone to develop perioperative myocardial ischemia will increase within the next

decades. The number of studies focusing on the impact of cardiometabolic disease

on myocardial perfusion and function during anesthesia is however limited, and the

underlying pathophysiology is not well understood.

Introduction 13

Dietary intake

While obesity and type 2 diabetes mellitus are acknowledged as factors that

accelerate the risk of perioperative myocardial ischemia, there is only limited

information available with respect to the association between the development of

cardiometabolic disease with myocardial perfusion, function and ischemia and

reperfusion injury. It is commonly acknowledged that excessive caloric intake, in

combination with a sedentary lifestyle, is the main contributor to the development of

obesity and type 2 diabetes mellitus. Most lifestyle programs therefore focus on

changes in dietary behavior in combination with physical activity. In addition to the

number of calories that contribute to the development of cardiometabolic disease,

there is increasing interest in the role of dietary composition on the development of

cardiometabolic disease. A western or cafetaria diet, which is characterized by a high

percentage of saturated fatty acids and simple carbohydrates such as fructose or

sucrose, is one of the most common causes for overweight, obesity and diabetes

mellitus. Animal studies provide evidence that excessive availability of dietary lipids

or simple carbohydrates leads to accumulation of metabolic intermediates, thereby

inducing myocardial insulin resistance and impairment of myocardial function.26-29 In

humans, myocardial triglyceride accumulation is associated with alterations in

myocardial function and has been demonstrated to occur in the diabetic heart.30

During normal physiologic conditions the heart derives its energy requirements from

fatty acid oxidation (60-70%), glucose oxidation (30-40%) and amongst others,

lactate (10%). However, this process is restricted as myocardial substrate

metabolism is largely determined by the availability of the substrate.31-35 The above

described observations contribute to the emerging concept that myocardial substrate

metabolism is closely related to myocardial function. Moreover, in case of surgical

stress and anesthesia, diet-induced alterations in myocardial substrate metabolism

may become more abundant with respect to the balance between the supply and

demand of oxygen and cellular nutrients. It might therefore be interesting to

investigate how modulation of dietary intake influences the susceptibiltiy of the heart

to injury, and whether the protective effects of anesthetic strategies may be altered

by dietary composition.31-35

Interestingly, while the negative consequences of obesity and type 2 diabetes

mellitus for the development of cardiometabolic diseases are well acknowledged,

there is increasing evidence that overweight and obesity may also be protective in

case of postoperative risks,36 the so-called obesity paradox. The counterintuitive

observations suggest that the unfavorable cardiometabolic effects of high intake of

saturated fatty acids may shift towards a beneficial condition in case of stress.

Moreover, recent studies suggest that a change in diet before surgery and

anesthesia may be of influence on the risk of postoperative complications.37 Animal

studies further showed that short-term dietary restrictions before surgical stress are

1

14 Chapter 1

associated with improved insulin sensitivity, oxidative protection and organ

preservation during ischemia and reperfusion injury.38;39 It is however unknown

whether dietary changes in the period before exposure to an anesthetic procedure

may alter the effects of volatile anesthetics on myocardial perfusion, function and

injury.

Aim of this thesis

In the light of these considerations, the present thesis particularly focuses on the

interaction of the volatile anesthetic sevoflurane with myocardial perfusion, systolic

function as well as ischemia and reperfusion injury in rats exposed to a normal

healthy or a western diet. Myocardial perfusion and function were studied using

echocardiography as a noninvase technique for the monitoring of myocardial systolic

and diastolic function. The use of a contrast agent expands the use of the technique

for determination of myocardial perfusion.

We hypothesized that sevoflurane-induced changes in myocardial perfusion and

function are distinctly influenced by preoperative dietary composition. Moreover, we

tested the hypothesis that a high intake of saturated fatty acids in combination with

simple carbohydrates alters the cardioprotective effects of sevoflurane in case of

myocardial ischemia and reperfusion. The specific aims per chapter are summarized

below.

Outline of the thesis

This thesis focuses on the effect of dietary changes on myocardial function, perfusion

and injury during sevoflurane anesthesia. In chapter 2 the effects of volatile

anesthetics, diabetes and perioperative ischemia on myocardial substrate

metabolism are reviewed.

In chapter 3, we studied the effect of high fat diet feeding on myocardial perfusion

and function with (contrast) echocardiography. We used dipyridamole infusion to

induce conditions of hyperemia. Chapter 4 describes the effects of a more severe,

western diet on myocardial perfusion and function. Additionally, the effects of

sevoflurane were studied.

Furthermore, we studied the effect of changing dietary intake on myocardial

function and myocardial ischemia and reperfusion injury. Chapter 5 was designed to

study the effect of dietary alterations in combination with sevoflurane exposure on

myocardial function. In Chapter 6 the hypothesis that the cardioprotective effects of

sevoflurane on myocardial ischemia and reperfusion injury are reduced by western

diet-feeding has been tested.

Finally, Chapter 7 provides a general discussion of the findings presented in this

thesis and places these findings in perspective.

Introduction 15

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1

16 Chapter 1

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IDF diabetes atlas 2011. 5th ed. Brussels: International Diabetes Federation; 2011.

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anaesthetic implications. Br J Anaesth 2009, 103 Suppl 1:i23-i30.

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considerations. Anesthesiology 2008, 108:506-523.

26.Ouwens DM, Diamant M, Fodor M, Habets DD, Pelsers MM, El Hasnaoui M, Dang ZC, van den

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mediated fatty acid uptake and esterification. Diabetologia 2007, 50:1938-1948.

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Introduction 17

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Roos A, Lamb HJ: The ageing male heart: myocardial triglyceride content as independent

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CF, Lammertsma AA, Ouwens DM, Diamant M, Boer C: Diabetic cardiomyopathy in Zucker

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1

CE van den Brom, CSE Bulte, SA Loer, RA Bouwman,

C Boer

Cardiovascular Diabetology, 2013 12:47

2Metabolic disease and perioperative

ischemia in the experimental setting:

Consequences of derangements in

myocardial substrate metabolism

Metabolic disease and perioperative ischemia 21

Abstract

Volatile anesthetics exert protective effects on the heart against perioperative

ischemic injury. However, there is growing evidence that these cardioprotective

properties are reduced in case of type 2 diabetes mellitus. A strong predictor of

postoperative cardiac function is myocardial substrate metabolism. In the type 2

diabetic heart, substrate metabolism is shifted from glucose utilization to fatty acid

oxidation, resulting in metabolic inflexibility and cardiac dysfunction. The ischemic

heart also loses its metabolic flexibility and can switch to glucose or fatty acid

oxidation as its preferential state, which may deteriorate cardiac function even

further in case of type 2 diabetes mellitus.

Recent experimental studies suggest that the cardioprotective properties of volatile

anesthetics partly rely on changing myocardial substrate metabolism. Interventions

that target at restoration of metabolic derangements, like lifestyle and

pharmacological interventions, may therefore be an interesting candidate to reduce

perioperative complications. This review will focus on the current knowledge

regarding myocardial substrate metabolism during volatile anesthesia in the obese

and type 2 diabetic heart during perioperative ischemia.

2

22 Chapter 2

Introduction

Perioperative cardiac complications occur in 2-5% of all non-cardiac surgical

procedures, which globally affect 5-12 million patients each year.1 More specifically,

0.65% of these patients develop perioperative myocardial infarction or cardiac

arrest.2 Perioperative cardiac complications are an economical, medical and social

burden that warrants optimization of perioperative health and cardiovascular care to

improve patient outcome and reduce health care costs. There are several well-known

predictors for perioperative cardiac complications identified, such as type of surgery,

ASA classification and increasing age.1,2 Additionally, lifestyle risk factors associated

with metabolic alterations, such as excessive dietary intake and physical inactivity,

are strongly associated with clinical risk factors that predict perioperative

cardiovascular complications.1

Lifestyle risk factors related to obesity and type 2 diabetes mellitus (T2DM) have

become an epidemic over the last decade. Worldwide, 366 million people have

T2DM.3 It is predicted that in the year 2030 about 552 million people will have overt

diabetes, mainly T2DM.3 Patients with T2DM are more likely to develop coronary

artery disease and myocardial ischemia4 and have an increased cardiovascular

complication rate after major non-cardiac surgery.5

In addition to prevention programs to reduce the burden of metabolic disease on

the perioperative process, there are intraoperative cardioprotective strategies

available that may reduce the impact of ischemic injury during and after surgery, like

the application of the volatile anesthetics sevoflurane and isoflurane. These volatile

anesthetics exert multiple protective effects that enhance perioperative preservation

of the heart in patients6 and rats.7 Although exposure to volatile anesthetics reduced

infarct size and improved post-ischemic recovery in healthy rats,7 the

cardioprotective effects of these agents are reduced in obese8 and hyperglycemic9

rats. Derangements in myocardial substrate metabolism are one of the hypothetical

mechanisms that may explain the suppressed cardioprotective capacity in T2DM.10-12

It is however not yet understood how these myocardial metabolic alterations affect

intraoperative cardioprotective mechanisms.

In order to elucidate the impact of altered myocardial substrate metabolism on

intraoperative myocardial protection, this review will focus on available preclinical

knowledge regarding myocardial substrate metabolism during volatile anesthesia in

the obese/T2DM heart under normal conditions and in the context of ischemia. We

first describe myocardial substrate metabolism under healthy, obese/T2DM and

ischemic conditions, followed by an overview of the interaction between substrate

metabolism and volatile anesthetics in the context of perioperative ischemia and

reperfusion injury. Finally, we propose strategies to modulate myocardial substrate


metabolism that may contribute to an improvement of myocardial protective capacity

and perioperative and postoperative outcome in obesity and T2DM.

Myocardial substrate metabolism

Fatty acids and carbohydrates are essential for the pump function of the heart.13

Under physiological conditions, myocardial contractile function relies on oxidation of

fatty acids (60-70%), glucose (30-40%) and to a lesser extent lactate, ketones,

amino acids and pyruvate (10%) to generate adenosine triphosphate (ATP).14-16 The

heart exerts a metabolic flexibility, and myocardial substrate utilization depends on

substrate availability, nutritional status, and exercise level. With glucose as the more

energetically efficient substrate, the healthy heart is able to switch to glucose under

conditions of stress, such as ischemia, pressure overload or in heart failure.

Glucose metabolism is regulated through multiple steps, including uptake,

glycolysis and pyruvate decarboxylation. Myocardial glucose supply is regulated 1)

via circulating glucose levels or 2) by release of glucose from intracellular glycogen

stores.17 Myocardial glucose uptake depends on the sarcolemmal glucose transporter

GLUT1 (insulin-independent) and the dominant glucose transporter GLUT4 (insulin-

dependent) (Figure 2.1).18 After uptake, glucose is broken down into pyruvate by

glycolysis, consumed by the mitochondria and decarboxylated into acetyl-CoA by

pyruvate dehydrogenase. Acetyl-CoA enters the tricarboxylic acid cycle with entry of

reducing equivalents to the electron transport chain and oxidative phosphorylation,

which finally leads to ATP formation (Figure 2.1).

Fatty acid metabolism consists of uptake, oxidation and esterification. There are

two sources of fatty acids for myocardial metabolism: 1) circulating albumin bound

fatty acids derived from adipose tissue via lipolysis or 2) released from triglyceride-

rich lipoproteins from the liver.19 Fatty acids enter cardiomyocytes by simple

diffusion and via transport through three different membrane fatty acid transporters

fatty acid translocase (FAT)/CD36, fatty acid transport protein (FATP1/6) and

plasma membrane fatty acid binding protein (FABPpm) (Figure 2.1).19 After

sarcolemmal uptake, intracellular fatty acids are activated to form fatty acyl-CoA,

which can undergo beta-oxidation or esterification to form intracellular

triglycerides.20 Fatty acid oxidation requires fatty acyl-CoA entry into the

mitochondria, which is dependent on the activity of carnitine palmitoyl transferase

(CPT-1).21 After translocation into the mitochondria, fatty acyl-CoA can enter the

beta-oxidation pathway to form acetyl-CoA and subsequently ATP (Figure 2.1).

Under physiological conditions, 70-90% of the fatty acids that enter cardiomyocytes

are oxidized for ATP generation, whereas 10-30% is converted to triglycerides by

lipoprotein lipase.22 In case of energy expenditure, intracellular triglyceride stores

can be hydrolyzed as an endogenous fatty acid source, which is explanatory for 10%

of the total fatty acid utilization in the heart.23

2

24 Chapter 2

Figure 2.1: Glucose and fatty acid metabolism in the cardiomyocyte

Glucose uptake into the cell occurs through the glucose transporters GLUT1 and GLUT4. Once

inside, glucose is broken down into pyruvate by glycolysis. Pyruvate is subsequently transported

into the mitochondria and decarboxylated to acetyl-CoA. Non-esterified fatty acids are taken up

through fatty acid transporter (FAT)/CD36, fatty acid transport protein (FATP) and plasma

membrane fatty acid binding protein (FABPpm). Intracellular fatty acids form fatty acyl-CoA and

can either be esterified into triglycerides (TG) or enter the mitochondria via carnitine palmitoyl

transferase (CPT-1). Fatty acyl- -oxidation pathway, forming acetyl-CoA. Glucose

or fatty acid-derived acetyl-CoA enters the tricarboxylic acid (TCA) cycle with entry of reducing

equivalents to the electron transport chain and oxidative phosphorylation, and finally ATP is

formed.


Type 2 diabetes mellitus

Alterations in myocardial substrate metabolism in T2DM hearts are extensively

reviewed by others.15,22,24 In short, myocardial fatty acid metabolism is initially

enhanced in T2DM hearts, with increased rates of fatty acid oxidation and

esterification.25,26 There are two proposed mechanisms that may underlie this

derangement: 1) increased fatty acid uptake due to increased substrate supply and

augmented expression and localization of sarcolemmal fatty acid transporters26 and

2) increased oxidation and esterification due to changes in regulation at both the

enzymatic and transcriptional level.26

In addition, a decreased myocardial glucose metabolism is a concomitant feature of

the T2DM heart.25,26 The slow rate of glucose transport across the sarcolemmal

membrane due to decreased glucose transporters leads to a restriction of glucose

oxidation. Accordingly, fatty acid oxidation has an inhibitory effect on the pyruvate

dehydrogenase complex due to increased fatty acid supply. Taken together, the

T2DM heart has a distinct metabolic phenotype, characterized by enhanced

myocardial fatty acid metabolism and a concomitant reduction in myocardial glucose

metabolism.

Ischemia

Myocardial ischemia occurs when coronary perfusion is inadequate to maintain a

sufficient oxygen supply/demand ratio. Ischemia influences both myocardial

substrate metabolism and myocardial function. The pathophysiological mechanisms

underlying this phenomenon have been reviewed previously.24,27

In the event of ischemia, high-energy phosphates are depleted, ionic homeostasis

is disturbed and contractile dysfunction is caused. The energetic demand of the heart

changes in case of myocardial ischemia. The heart usually responds to injury by

increasing myocardial glucose metabolism to improve its energetic efficiency.22,24

However, increased adipose tissue lipolysis results in increased plasma free fatty acid

concentrations, which may increase myocardial fatty acid utilization and

esterification.27 In this context, glycolysis becomes an important source of energy

due to its ATP-generating ability in the absence of oxygen. It is also suggested that

in the early phase of ischemia, fatty acid oxidation shifts to the more efficient

glucose oxidation, followed by a decrease in total substrate oxidation.24 Increased

glycolysis can parallel depression of myocardial glucose and fatty acid oxidation

depending on the severity of ischemia. Overall, the ischemic heart favors the

energetically more efficient glucose (3.17 ATP/oxygen molecule) over fatty acid

oxidation (2.83 ATP/oxygen molecule).28 This flexibility additionally depends on

substrate availability, oxygen supply, tissue vascularization and myocardial

workload. In conclusion, the metabolic state of the ischemic heart is characterized by

2

26 Chapter 2

imbalances in substrate availability and utilization and is also influenced the severity

of ischemia.

The combination of type 2 diabetes mellitus and ischemia

The cardiometabolic profile of patients with T2DM makes them more prone to

develop plaque formation and intravascular stenosis, leading to the development of

stroke or myocardial infarction. In addition, these patients are more susceptible to

subsequent episodes of ischemia.29,30 Whereas the metabolic undisturbed heart

usually responds to injury by increasing myocardial glucose metabolism,22,24 this

adaptive response is inhibited by insulin resistance, which is a characteristic of

obesity and T2DM. This inhibition results in increased myocardial fatty acid

metabolism,31;32 increased oxygen consumption, decreased cardiac efficiency31 and

altered myocardial perfusion.33 In obese or T2DM animals subjected to myocardial

ischemia the findings are inconclusive. It has been shown that obesity reduced

ischemia and reperfusion injury34 and myocardial function during ischemia (and

reperfusion),35-40 but also similar ischemia and reperfusion injury was found.41

Additionally, increased glucose oxidation and decreased fatty acid oxidation after

myocardial infarction was found, which was ameliorated in obese rats.40 Obese rats

with insulin resistance resulted in preserved myocardial function36 or aggravated36,42-

44 ischemia and reperfusion injury. Moreover, the combination of insulin resistance,

dyslipidaemia and hypertension in obese animals seems to increase the susceptibility

of the heart to ischemia (and reperfusion) injury.45-48 Others however reported that

myocardial injury during ischemia was unaffected in T2DM rats, independent of the

severity of T2DM.49 In case of genetically induced T2DM rats in combination with a

high cholesterol diet, ischemic injury was however exacerbated.50 As stated earlier,

these inconclusive results in animal experiments suggest that the type and severity

of T2DM may influence the sensitivity of the heart to ischemic insults.

With regard to myocardial substrate metabolism, endogenous glycogen stores may

support increased glucose availability as substrate for the heart, and may thus be

beneficial in case of ischemic injury. However, whether pre-ischemic glycogen levels

are beneficial or detrimental depends on the duration of T2DM51 and to the extent of

glycogen depletion during ischemia.52

Overall, the effects of imbalanced myocardial substrate metabolism during ischemia

in T2DM are inconclusive. These observed contrasts may be due to differences in the

severity of ischemia, the measured outcome parameter, exogenous circumstances

and the severity of the experimental model for T2DM.32,53


Effects of volatile anesthetics in animals

Cardioprotective effects during ischemia

Sevoflurane and isoflurane are commonly used volatile anesthetics. Sevoflurane and

isoflurane make the rat heart more resistant to ischemia and reperfusion injury.54-58

It has been shown that proteins related to myocardial substrate metabolism are,

amongst others, affected by sevoflurane-induced cardioprotection. PI3K and Akt,

which regulate translocation of glucose transporter 4 (GLUT4) to the sarcolemma for

glucose uptake, are increased during sevoflurane in the isolated ischemic rat heart.59

Moreover, sevoflurane enhances GLUT4 expression in lipid rafts, increases glucose

oxidation and decreases fatty acid oxidation after ischemia and reperfusion injury in

isolated working rat hearts compared to untreated ischemic hearts.10 In the same

study, no alterations in AMP activated protein kinase (AMPK) phosphorylation,

pyruvate dehydrogenase activity and glycogen content were found, whereas

sevoflurane decreased triglycerides and ceramide levels after ischemia and

reperfusion injury.10

Moreover, volatile anesthetics are also known to alter mitochondrial function, which

is nicely reviewed by Stadnicka et al.60 In short, it has been shown that sevoflurane

and isoflurane open mitochondrial ATP-activated potassium (mito K+ATP)

channels,61,62 activates reactive oxygen species62 and thereby alters mitochondrial

metabolism.63

Together, these results suggest a role for myocardial substrate metabolism in the

cardioprotective effects of volatile anesthesia during ischemia and reperfusion injury

in animals, although evidence is limited.

Myocardial substrate metabolism during volatile anesthesia

In rats, it has been shown that in vivo myocardial glucose uptake was increased in

the heart during isoflurane (2 vol%) when compared to sevoflurane (3.5 vol%).64 An

explanation could be the differences by more stable blood glucose levels during

sevoflurane. However, a limitation of this study was that the effects were not

compared with findings in awake rats or using non-volatile anesthetics. Others found

that isoflurane (2 vol%) increased myocardial glucose uptake compared to awake

mice.65

The effects of sevoflurane on myocardial substrate metabolism have only been

studied ex vivo. Sevoflurane (2 vol%) decreased FAT/CD36 in lipid rafts and fatty

acid oxidation in isolated rat hearts.12 And, although studied in skeletal muscle cells,

sevoflurane (2.6-5.2%) increased glucose uptake.66 Altogether, these results

suggest that isoflurane and sevoflurane might switch myocardial metabolism to

glucose as energetically more efficient substrate.

2

28 Chapter 2

Volatile anesthesia is also known to affect pancreatic insulin release. In isolated rat

pancreatic islets, enflurane67 and isoflurane68 have an inhibitory effect on glucose-

stimulated insulin release. In rats, isoflurane impaired glucose-induced insulin

release,69 whereas sevoflurane impaired glucose tolerance,70 which both resulted in

hyperglycemia. Therefore it seems that impaired insulin release during volatile

anesthesia might have a negative effect on substrate metabolism. However, the

beneficial cardioprotective effects may outweigh the adverse effects of impaired

insulin secretion, as the American Heart Association 2007 guidelines on

that it can be beneficial to use volatile anesthetics during non cardiac surgery for

maintenance of general anesthesia in hemodynamically stable patients at risk for

myocardial ischemia.1

Alterations in cardioprotective mechanisms in the metabolic altered heart

The healthy heart is capable of protecting itself against stressors like ischemia by the

flexibility to switch between circulating substrates. These cardioprotective properties

might be enlarged during volatile anesthesia. On the other hand, the obese/T2DM

heart is less capable of switching between circulating substrates, which may

contribute to a reduced intrinsic protective capacity. It is generally acknowledged

that the incidence of perioperative cardiovascular complications is increased in

patients with T2DM after non-cardiac surgery.5 Accordingly, blood glucose

concentrations at admission correlated with long-term mortality in diabetic patients

with acute myocardial infarction,71 suggesting that T2DM may affect perioperative

cardiovascular risk. The next paragraphs focus on available experimental knowledge

whether obesity, insulin resistance, hyperlipidemia and hyperglycemia, important

hallmarks of T2DM, exert a cumulative effect on endogenous and exogenous

cardioprotective mechanisms.

Obesity and insulin resistance

It has been shown that obesity and insulin resistance inhibit the cardioprotective

effects of ischemic pre-72 and postconditioning.73 In high fat diet-induced obese rats,

sevoflurane preconditioning failed to induce cardioprotection during myocardial

ischemia and reperfusion injury.41 Moreover, sevoflurane postconditioning did not

protect the heart against myocardial and reperfusion injury in obese and insulin

resistant Zucker rats,8 however, more research is necessary to draw a conclusion.

Hyperlipidemia

The hyperlipidemic heart has difficulties to adapt to stressors like ischemia,

suggesting that cardioprotective mechanisms are impaired. In rats it has been shown

that pacing-induced cardioprotection74 and ischemic-induced preconditioning75 was

inhibited by hypercholesterolemia. Sevoflurane preconditioning reduced myocardial


infarct size in normocholesterolemic rats, which was blocked in hypercholesterolemic

rats.76 Further research is warranted to study the impact of hyperlipidemia on

anesthesia-induced cardioprotection.

Acute hyperglycemia

Hyperglycemia is an independent predictor of cardiovascular risk.71 The

glycometabolic state upon hospital admission is associated with the mortality risk in

T2DM patients with acute myocardial infarction.77 It has further been shown that

hyperglycemia inhibits the cardioprotective capacity during desflurane-induced

preconditioning,78 isoflurane-induced preconditioning,9,79 and sevoflurane-induced

postconditioning in the experimental setting.80 Accordingly, infarct size was directly

related to the severity of hyperglycemia,81;82 whereas the inhibited cardioprotective

effects of isoflurane-induced preconditioning are concentration dependent and

related to the severity of acute hyperglycemia.9 Moreover, it has been shown that

hyperglycemia attenuated cardioprotection via inhibition of Akt and endothelial nitric

oxide synthase (eNOS) phosphorylation.83 However, interpretation of

abovementioned findings in relation to T2DM is difficult, because experiments were

performed during acute hyperglycemia in otherwise healthy animals without the

typical characteristics of T2DM, such as obesity and insulin resistance.

Type 2 diabetes mellitus

T2DM hinders the cardioprotective effects of ischemic preconditioning,84 which has

been reviewed by Miki et al.85 However, the diabetic rat heart may still benefit when

the preconditioning stimulus is enlarged.86 The effects of anesthesia-induced

cardioprotection in T2DM have however never been studied. In type 1 diabetes, the

protective effects of isoflurane-induced preconditioning were inhibited in case of low

isoflurane concentrations, but not at high concentrations.82 Further, sevoflurane-

induced postconditioning in the type 1 diabetic heart was disturbed, whereas insulin

treatment to reach normoglycemia did not restore the cardioprotective capacity.87

Mechanisms that are suggested to be involved include the inhibition of PI3K/Akt86,87

and inactivity of mito K+ATP.

87 Furthermore, AMPK activation during ischemia protects

the non-obese T2DM Goto-Kakizaki rat heart against reperfusion injury,88 suggesting

a role for AMPK in the cardioprotective properties of the diabetic heart. A limitation

of the above-described studies is that anesthesia-induced cardioprotection is only

studied in type 1 diabetes with insulinopenia and hyperglycemia, but without

characteristics such as obesity, insulin resistance and hyperinsulinemia. Although

current findings suggest that the degree of T2DM, dependent on the presence and

severity of hyperglycemia and hyperlipidemia, is of influence for the cardioprotective

capacity of anesthetics, there are no direct studies available that investigated

cardioprotective strategies in animals with this diabetic entity.

2

30 Chapter 2

Figure 2.2: (Hypothetical) Glucose and fatty acid metabolism under different conditions

Glucose and fatty acid metabolism in the healthy heart (A), during volatile anesthesia (B), in the

metabolic altered heart (C), in the ischemic heart (D), during volatile anesthesia in the ischemic

heart (E) and during volatile anesthesia in the ischemic metabolic altered heart (F). The healthy

heart utilizes 70% of fatty acids and 30% of glucose for ATP generation (A). We hypothesize that

one of the mechanisms of volatile anesthesia is the effect on increased glucose metabolism (B). In

the metabolic altered heart it is suggested that myocardial substrate metabolism is shifted to

increased fatty acid metabolism (C), whereas it is suggested that the ischemic heart is shifted to

increased glucose metabolism, however, also contrasting results exist (D). We hypothesize that

exposure of volatile anesthetics in the ischemic heart might increase myocardial glucose

metabolism even more (E), which is disturbed in the ischemic and metabolic altered heart (F).


Experimental options to improve perioperative myocardial

metabolism

The reduced adaptability of the metabolic altered heart to ischemic injury and

cardioprotective interventions warrants further investigation of treatment strategies

that optimize myocardial substrate metabolism before surgery. It is suggested that

volatile anesthesia induces a switch from myocardial fatty acid to glucose

metabolism. In the metabolically altered heart, however, myocardial substrate

metabolism is shifted to increased fatty acid and decreased glucose metabolism.

Accordingly, the effect of volatile anesthetics seems blunted in the metabolic altered

heart. As a consequence, an improvement of the metabolic flexibility of the heart

may be an important target. Figure 2.2 shows a hypothetical overview of the effects

of different conditions on myocardial substrate metabolism.

Pharmacological interventions

Improvement of myocardial metabolic flexibility may be achieved by shifting

myocardial substrate metabolism to glucose metabolism. This can be induced by 1)

altering substrate supply, 2) inhibition of fatty acid oxidation and/or 3) improving

insulin sensitivity. The next paragraphs provide an overview of pharmacological

interventions in the experimental setting in the treatment of T2DM and/or

myocardial ischemic injury, which might reduce perioperative risk due to

normalization of metabolic derangements (Table 2.1).

Inhibition of fatty acid metabolism

Carnitine palmitoyl transferase 1 (CPT-1) is a rate-limiting step of fatty acid

oxidation. Several inhibitors of CPT-1 have shown beneficial effects during ischemia

and reperfusion in rats, such as etomoxir,89-91 perhexiline92 and oxfenicine.92;93

However, not all of these variants of CPT-1 inhibitors are yet registered for clinical

use. Other possibilities to reduce fatty acid oxidation are trimetazidine (3-ketoacyl

CoA thiolase inhibitor),94,95 ranolazine (partial fatty acid oxidation inhibitor)96,97 and

dichloroacetate (DCA; pyruvate dehydrogenase kinase inhibitor),98 which have

protective characteristics during myocardial ischemia in rats. One of the suggested

mechanisms underlying the beneficial effects of these substances is the stimulation

of myocardial glucose oxidation.96,98,99 However, as insulin resistance is a hallmark of

the metabolic altered heart, stimulation of glucose metabolism via inhibition of fatty

acid metabolism may be blunted during insulin resistance. Unfortunately, the effect

of volatile anesthesia in combination with inhibition of fatty acid metabolism on

ischemic injury in T2DM hearts has not been studied yet, however, based on the use

of these fatty acid inhibitors in models of T2DM it may be deduced that insulin

2

32 Chapter 2

resistance might be improved, thereby improving the impact of anesthesia-induced

cardioprotection.

Insulin

Glucose-insulin-potassium (GIK) infusion has been shown to reduce mortality in non-

diabetic100,101 and diabetic patients,102 and to reduce infarct size in rats.103 However,

also other results exist.104,105 In the perioperative context, GIK infusion lowered

glucose levels and other metabolic parameters106 and improved perioperative

outcomes, enhanced survival, decreased the incidence of ischemic events107 in T2DM

patients during coronary artery bypass grafting (CABG).

The beneficial effects of GIK include increasing myocardial glucose uptake and

glycogen content. It is suggested that insulin itself might be the major

cardioprotective component. In isolated rat hearts, administration of insulin

protected against ischemia and reperfusion injury.108,109 However, insulin treatment

was not able to restore the lost cardioprotective capacity of sevoflurane in the type 1

diabetic heart.87

Disadvantages of insulin infusion might be hypoglycemia, which could be

circumvented by additional glucose infusion (hyperinsulinemic euglycemic clamping).

Insulin and dextrose infusion normalized postoperative whole body insulin sensitivity

and substrate utilization in healthy patients during elective surgery.110 During cardiac

surgery, insulin and dextrose infusion maintained normoglycemia in healthy111 and

T2DM112 patients, however, hypolipidemia was observed.113 Further, it was shown in

diabetic patients that isoflurane reduced postoperative markers of ischemic injury

after CABG, indicating a cardioprotective effect of isoflurane.114 Preoperative

treatment with glibenclamide prevented this protective effect, which was restored by

changing glibenclamide preoperatively to insulin.114 Taken together, these data

suggest that perioperative glucose control by insulin may decrease the risk of

postoperative mortality and morbidity.

Peroxisome proliferator-activated receptor agonists

Fibrates are selective peroxisome proliferator-activated receptor

which have lipid lowering effects, thereby improving insulin sensitivity. PPAR

activation has been shown to reduce myocardial ischemia and reperfusion injury in

rat hearts.115,116 -Kakizaki rat hearts reduced

ischemic injury,117 whereas in T2DM db/db

sensitivity to ischemia and reperfusion even while myocardial glucose oxidation was

increased and myocardial fatty acid oxidation reduced.47 Moreover, sevoflurane

,118 whereas during CABG

right atrial tissue compared to propofol.11 Based on

agonists combined with volatile anesthesia.


Insulin-sensitizing drugs, such as thiazolidinediones have beneficial effects by

myocardial ischemia and reperfusion injury in rats.48,115,119,120 Rosiglitazone has been

shown to increase myocardial GLUT4 translocation121 and glucose metabolism122 in

healthy and T2DM rat hearts. During myocardial ischemia and reperfusion, it was

shown that rosiglitazone treatment normalized ischemic injury by improvement of

the reduced glucose uptake in obese Zucker rats,44 and reduced ischemic injury by

improved myocardial insulin sensitivity and glucose oxidation in T2DM Zucker

diabetic fatty rats,48 suggesting a role f

metabolism to optimize metabolic flexibility during myocardial ischemia and

reperfusion. Accordingly, it was shown that desflurane-induced cardioprotection

during ischemia hibition in rabbits,123

Metformin

Metformin, a biguanide with antihyperglycemic properties, has been widely used in

the treatment of obesity and T2DM and exerts its actions by enhancing insulin

sensitivity. It is suggested that the glucose-lowering effects of metformin are

mediated through the activation of AMPK, which has also been indicated to play an

important protective role in the ischemic mouse heart.124,125 In non-diabetic rat

hearts, metformin protects against ischemic injury.126,127 Accordingly, metformin

provides cardioprotection against ischemic injury in T2DM hearts from animals in

vivo,125 but not in vitro.128 The effects of volatile anesthesia and metformin in

ischemic and T2DM hearts has not been studied yet. However, it has been shown

that AMPK is involved in anesthetic cardioprotection.41;129

Glucagon-like peptide 1

Glucagon-like peptide 1 (GLP1) is a gut incretin hormone that is released in response

to nutrient intake, stimulates insulin secretion and exerts insulinotropic and

insulinomimetic properties. GLP1 has been shown to be protective in ischemic rat

hearts.130

GLP1 has a short half-life of several minutes, due to rapid breakdown by dipeptidyl

peptidase IV (DPP4). Exendin-4 is a peptide derived from the saliva of the gila

monster which mimics GLP1, but is resistant to degradation by DPP4. Exenatide and

liraglutide are synthetic GLP1 analogues, which mimic human GLP1 and are currently

used for blood glucose-lowering therapy in T2DM. Exendin-4,131 exenatide132 and

liraglutide133 have been shown to reduce infarct size in animals, but also a neutral

effect of liraglutide on myocardial infarct size was found.134 Another possibility to

circumvent the rapid breakdown of GLP1 is the use of a DPP4 inhibitor. However,

inhibition of DPP4 by valine pyrrolidide in rats130 or in DPP4 knockout mice135 was not

2

34 Chapter 2

protective during myocardial infarction. It is suggested that the cardioprotective

effect is a consequence of insulin, however, GLP1 has cardioprotective effects both in

vivo and in vitro, whereby the latter is in absence of circulating insulin levels,130

suggesting a role for GLP1 in cardioprotection.

The mechanism behind the cardioprotective properties of GLP1 may, amongst

others,136 rely on improving myocardial glucose metabolism. GLP1 increased glucose

uptake in isolated mouse137 and isolated healthy,138 hypertensive139 and

ischemic/reperfused138 rat hearts. Moreover, exenatide increased myocardial glucose

uptake in healthy140 and insulin resistant dilated cardiomyopathy141 mice, whereas it

did not alter myocardial glucose uptake in type 2 diabetic patients.142

Exposure of healthy rats to isoflurane anesthesia decreased GLP1 levels, without

affecting DPP4 activity, insulin and glucose levels,143 suggesting impaired GLP1

secretion during isoflurane anesthesia. However, the effect of volatile anesthetics on

GLP1 is scarcely studied and therefore no conclusion van be drawn.

Taken together, the above-discussed pharmacological interventions suggest that

improving insulin sensitivity, and thereby improving myocardial flexibility, may be

the most beneficial option in metabolically altered hearts in order to restore

cardioprotective mechanisms. However, according to current clinical practice, oral

hypoglycemic agents are usually withheld before surgery in order to avoid associated

adverse effects, such as perioperative hypoglycemia or lactic acidosis. Therefore the

(clinical) feasibility and safety of the proposed interventions should be carefully

studied and weighted against the potential risk of these adverse effects.


Tab

le 2

.1:

Overv

iew

of

ph

arm

aco

log

ical

inte

rven

tion

s in

th

e e

xp

eri

men

tal

sett

ing

Dru

g

Ap

pli

cab

ilit

y

Ad

van

tag

es

Sid

e-e

ffect

s

Fatt

y a

cid

meta

boli

sm i

nh

ibit

ors

Eto

moxi

r T2D

M,

infa

rction

89-9

1

Stim

ula

tion g

luco

se o

xidat

ion

99

- Per

hex

iline

T2D

M,

infa

rction

92

Stim

ula

tion g

luco

se o

xidat

ion

- O

xfen

icin

e

T2D

M,

infa

rction

92,9

3

Stim

ula

tion g

luco

se o

xidat

ion

- Trim

etazi

din

e

T2D

M,

infa

rction

94,9

5

Stim

ula

tion g

luco

se o

xidat

ion

- Ran

ola

zine

T2D

M,

infa

rction

96,9

7

Stim

ula

tion g

luco

se o

xidat

ion

96

- D

ichlo

roac

etat

e T2D

M,

infa

rction

98

Stim

ula

tion g

luco

se o

xidat

ion

98

- In

suli

n

Glu

cose

-insu

lin-p

ota

ssiu

m

Infa

rction

103

Stim

ula

tion g

luco

se o

xidat

ion

Hyp

ogly

cem

ia

Insu

lin

T2D

M

Infa

rction

108,1

09

Red

uct

ion g

luco

se

Stim

ula

tion g

luco

se o

xidat

ion lev

els

Hyp

ogly

cem

ia

Hyp

ogly

cem

ia

PP

AR

ag

on

ists

T2D

M47

Infa

rction

115-1

17

Red

uct

ion lip

ids

Red

uct

ion lip

ids

Myo

pat

hy

M

yopat

hy

T2D

M121,1

22

Infa

rction

44,4

8,1

15,1

19,1

20

Insu

lin s

ensi

tize

r

Insu

lin s

ensi

tize

r In

crea

sed r

isk

hea

rt a

ttac

ks

Incr

ease

d r

isk

hea

rt a

ttac

ks

Big

uan

ide

Met

form

in

T2D

M,

infa

rction

125-1

28

Stim

ula

tion g

luco

se o

xidat

ion

Lact

ic a

cidosi

s G

LP

1

GLP

1

T2D

M,

infa

rction

130

Red

uct

ion g

luco

se

Short

hal

f-lif

e Exe

ndin

-4

T2D

M,

infa

rction

131

Red

uct

ion g

luco

se

Hyp

ogly

cem

ia

Exe

nat

ide

T2D

M,

infa

rction

132

Red

uct

ion g

luco

se

Hyp

ogly

cem

ia

Lira

glu

tide

T2D

M,

infa

rction

133,1

34

Red

uct

ion g

luco

se

Hyp

ogly

cem

ia

T2D

M,

type

2 d

iabet

es m

ellit

us;

PPA

R,

per

oxi

som

e pro

lifer

ators

-act

ivat

ed r

ecep

tor;

GLP

-1,

glu

cagon-l

ike

pep

tide

1.

2

36 Chapter 2

Preoperative health risk improvement

Based on 7 risk factors (physical inactivity, dietary pattern, obesity, smoking, high

cholesterol, hypertension and elevated blood glucose levels), the 2020 impact goal of

the American Heart Association is: "to improve the cardiovascular health by 20%

while reducing deaths from cardiovascular diseases and stroke by 20%".144 Another

possibility besides pharmacological intervention is preoperative lifestyle intervention,

such as changing the dietary intake and stimulation of physical activity thereby

losing weight and improving insulin sensitivity.

It has been shown by reducing dietary fat in rodents that diet-induced obesity is

reversible.145-147 In contrast, diet-induced obesity was not reversed by withdrawal of

an energy dense diet.148 Reversibility of diet-induced obesity is independent of the

duration of the obese state,146 whereas long-term diet feeding did not reversed

obesity.145 Overall, these data suggest that changing dietary intake may have

beneficial effects on health. However, there is only limited literature available that

describes the effects of changing dietary balance on the heart.

In western diet-fed rats, lowering caloric intake improved systolic and diastolic

function and prevented sevoflurane-induced cardiodepression (van den Brom et al.,

unpublished observations). Accordingly, pacing-induced cardioprotection was lost by

diet-induced hypercholesterolemia, but restored after reversion to control diet,149

whereas caloric restriction by itself in healthy rats also has cardioprotective

properties.150 In conclusion, restriction of dietary fat seems an effective treatment to

improve metabolic flexibility of the heart and thereby may be a possibility to reduce

perioperative risk.

Obesity and T2DM are closely related to physical inactivity, and exercise could be a

possible lifestyle intervention to reduce perioperative risk. The benefits of exercise

with respect to obesity and T2DM are already recognized clinically.151 However, the

effects of exercise on myocardial infarction are contradictory. Exercise did not reduce

myocardial ischemic injury in rats,152 whereas others showed that exercise had

protective effects in rat hearts.153-155 The question remains if exercise has beneficial

effects in obese and T2DM on myocardial function and ischemia and reperfusion

injury. Exercise was shown to reverse diet-induced obesity, insulin resistance and

cardiomyocyte dysfunction,147 however, the effects of exercise on myocardial

infarction in obese and T2DM with and without the effects of volatile anesthesia is

not known. Based on the above described results exercise might be a possible

lifestyle intervention to reduce perioperative risk.


Conclusions

Over the years, several mechanisms that are involved in anesthesia-induced

cardioprotection have been evaluated in the experimental setting. The existing

evidence suggests that the obese and/or T2DM heart is less adaptable to

cardioprotective interventions and that anesthesia-induced cardioprotection is just a

Differences between experimental models, the type of metabolic disease and the

severity of myocardial substrate derangements challenge the identification of

unifying mechanisms related to anesthesia-induced cardioprotection in cases of

obesity and T2DM. It might be deduced that interventional options should focus on

recovery of the metabolic flexibility of the heart, especially by improving insulin

sensitivity. Although changing lifestyle seems promising to reduce the susceptibility

of the heart to intraoperative ischemia and reperfusion injury, experimental data has

not been translated into clinical data. Therefore more studies are required to

elucidate whether these interventions have beneficial effects on perioperative

outcome.

2

38 Chapter 2

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metabolic phenotyping of myocardial substrate metabolism in rodents: differential efficacy of

metformin and rosiglitazone monotherapy. Circ Cardiovasc Imaging 2009, 2:373-381.

123.Lotz C, Lange M, Redel A, Stumpner J, Schmidt J, Tischer-Zeitz T, Roewer N, Kehl F:

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protect the heart against ischemia/reperfusion injury. Diabetes 2005, 54:146-151.

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Kerver M, Piek JJ, Doevendans PA, Pasterkamp G, Hoefer IE: Exenatide reduces infarct size

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2

CE van den Brom, CSE Bulte, BM Kloeze, SA Loer, C Boer,

RA Bouwman

Cardiovascular Diabetology 2012, 11:74

High fat diet-induced glucose intolerance

impairs myocardial function, but not

myocardial perfusion during hyperemia:

a pilot study

3

Myocardial function and perfusion during glucose intolerance 51

Abstract

Introduction

Glucose intolerance is a major health problem and is associated with increased risk

of progression to type 2 diabetes mellitus and cardiovascular disease. However,

whether glucose intolerance is related to impaired myocardial perfusion is not

known. The purpose of the present study was to study the effect of diet-induced

glucose intolerance on myocardial function and perfusion during baseline and

pharmacological induced hyperemia.

Methods

Male Wistar rats were randomly exposed to a high fat diet (HFD) or control diet (CD)

(n=8 per group). After 4 weeks, rats underwent an oral glucose tolerance test.

Subsequently, rats underwent (contrast) echocardiography to determine myocardial

function and perfusion during baseline and dipyridamole-induced hyperemia (20

mg/kg for 10 min).

Results

Four weeks of HFD feeding resulted in glucose intolerance compared to CD-feeding.

Contractile function as represented by fractional shortening was not altered in HFD-

fed rats compared to CD-fed rats under baseline conditions. However, dipyridamole

increased fractional shortening in CD-fed rats, but not in HFD-fed rats. Basal

myocardial perfusion, as measured by estimate of perfusion, was similar in CD- and

HFD-fed rats, whereas dipyridamole increased estimate of perfusion in CD-fed rats,

but not in HFD-fed rats. However, flow reserve was not different between CD- and

HFD-fed rats.

Conclusions

Diet-induced glucose intolerance is associated with impaired myocardial function

during conditions of hyperemia, but myocardial perfusion is maintained. These

findings may result in new insights into the effect of glucose intolerance on

myocardial function and perfusion during hyperemia.

3

52 Chapter 3

Introduction

Glucose intolerance defines the intermittent stage between transition from normal

glucose levels to type 2 diabetes mellitus.1 Glucose intolerance is a predictor of

cardiovascular disease2,3 and known to associate with vascular dysfunction and

consequent impairment of organ perfusion as one of the appearing consequences.4

Myocardial perfusion in combination with myocardial performance plays a central role

in the balance between myocardial energy supply and demand. Under physiological

conditions, myocardial blood flow and function are in balance,5 while

pathophysiological conditions leading to vascular dysfunction, such as glucose

intolerance, could alter balance between energy supply and demand. Although

glucose intolerance-induced vascular dysfunction may impair organ perfusion, data

elucidating the effects of glucose intolerance on the perfusion of vital organs like the

heart are limited. Up to now, only one group showed that insulin resistance in

prediabetic patients was associated with myocardial perfusion defects.6,7

Contrast echocardiography is a non-invasive method for assessment and

quantification of myocardial perfusion that is clinically applied for specific indications

in the cardiology setting. Using this technique it has been shown that myocardial

blood flow reserve, as a measure for vascular function, is reduced in type 1 diabetic

rats8 and type 1 diabetic patients9 when compared to normoglycemic controls. There

are currently limited data available on myocardial perfusion in glucose intolerance. In

the present study we therefore studied myocardial function and perfusion under

baseline and pharmacological-induced hyperemic conditions in a rat model for diet-

induced glucose intolerance using contrast echocardiography. Our hypothesis is that

glucose intolerance reduces the myocardial vasodilating capacity, which blunts the

increase in myocardial perfusion during hyperemia. This study demonstrates that

diet-induced glucose intolerance is associated with impaired myocardial function

during conditions of hyperemia, but not myocardial perfusion.


Methods

Animals and experimental set-up

All experiments were approved by the Institutional Animal Care and Use Committee

of the VU University, and were conducted following the European Convention for the

Protection of Vertebrate Animals used for Experimental and Other Scientific

Purposes.10 The performed research is in compliance with the modern ARRIVE

guidelines on animal research.11

Adult male Wistar rats (n=16; body weight 262±1 g; Harlan CBP, Horst, the

Netherlands) were fed a high fat diet (HFD) for a period of 4 weeks (n=8). Animals

that received a diet low in fat and sugars (control diet; CD) for 4 weeks served as

controls (n=8). Animals were housed in a temperature-controlled room (20-23°C;

40-60% humidity) under a 12/12 h light/dark cycle starting at 6.00 am. After 4

weeks, rats underwent an oral glucose tolerance test and (contrast)

echocardiography during baseline and after dipyridamole infusion.

Diets

High fat diet was obtained from Research Diets (D12451, New Brunswick, NJ). The

HFD consisted of 24 wt% protein, 24 wt% fat and 41 wt% carbohydrates (8.5 wt%

starch, 20.1 wt% sucrose). Control diet (Teklad 2016) was obtained from Harlan

(Horst, The Netherlands) and consisted of 17 wt% protein, 4 wt% fat and 61 wt%

carbohydrates (45.1 wt% starch, 5.0 wt% sucrose).

Oral glucose tolerance test

Rats fasted overnight received an oral glucose load (2 g/kg of body weight). Blood

glucose levels were measured from tail bleeds with a Precision Xceed Blood Glucose

monitoring system (MediSense, UK) before (0) and 15, 30, 60, 90 and 120 min after

glucose ingestion.12 At similar time points, plasma insulin (LINCO research, St.

Charles, Missouri) levels were measured as described previously.12

Plasma measurements

Plasma hematocrit levels were determined using microcentrifugation. Plasma free

fatty acids (WAKO NEFA-C, Wako Pure Chemical Industries, Osaka, Japan), plasma

high-density lipoprotein (HDL) cholesterol and plasma low-density lipoprotein

(LDL)/very-low-density lipoprotein (VLDL) cholesterol (Abcam, Cambridge, MA) were

measured after overnight fasting as previously described.13,14

3

54 Chapter 3

Cannulation of the jugular vein

For infusion of the contrast agent for echocardiography, a catheter was placed in the

jugular vein under S-Ketamine (Ketanest®, 150 mg/kg, Pfizer, the Netherlands) and

diazepam (3 mg/kg, Centrafarm, the Netherlands) anesthesia intraperitoneally. After

surgery, echocardiography and contrast echocardiography to determine myocardial

function and perfusion, respectively, were performed during baseline and

dipyridamole-induced hyperemia (20 mg/kg for 10 minutes) (Figure 3.1).

Figure 3.1: Protocol (contrast) echocardiography

After 4 weeks of control diet (CD) or high fat diet (HFD) feeding, rats received anesthesia for

cannulation of the jugular vein followed by echocardiography (echo) and contrast echocardiography

(contrast echo) during baseline and dipyridamole-induced hyperemia.

Echocardiography

Echocardiography (Siemens, ACUSON, Sequoia 512) was performed as previously

described.14 Briefly, wall thickness (WT) and left ventricular (LV) dimensions during

end-systole (ES) and end-diastole (ED) were determined in the M- (motion) mode of

the parasternal short-axis view at the level of the papillary muscles. Left ventricular

contractile function was calculated by the fractional shortening = (EDD-

ESD)/E -line (Image-Arena 2.9.1, TomTec

Imaging Systems, Unterschleissheim / Munich, Germany). All parameters were

averaged over at least three cardiac contractile cycles.

Myocardial contrast echocardiography

Contrast echocardiography was performed using a Siemens (ACUSON, Sequoia 512)

equipped with a 14 MHz linear array transducer (Philips Healthcare, Best, The

Netherlands). The contrast agent Sonovue® (Bracco Imaging, Italy), which contains

2x108-5x108/ml sulphur hexafluoride-filled, phospholipid-coated microbubbles with a

diameter of 1

diluted twice by adding 5 ml NaCl (0.9%). Microbubbles were continuously infused

using a dedicated syringe pump

(Vueject, Bracco SA, Switzerland). After two minutes of microbubble infusion,

perfusion images were taken of the short axis view of the left ventricle at the level of

the papillary muscles.


Low acoustic power (mechanical index [MI] 0.20 max) was used for microbubble

detection. A perfusion sequence consisted of about 10 cardiac cycles of low MI

imaging, followed by a burst of high acoustic power (MI 1.8) for complete contrast

destruction. Subsequently, on average 20 cardiac cycles of low MI images were

acquired at a frame rate of 14 Hz to allow contrast replenishment in the

myocardium. All data were stored for offline analysis.

Myocardial contrast echocardiography analysis

Custom-designed software was used for analysis of the estimate of perfusion

(Matlab, 7.10, R2010A, MathWorks Inc. Massachusetts, USA) with special thanks to

R. Meijer. For each cardiac cycle the end-systolic frame was selected manually and

regions of interest were drawn in the posterior wall in the short axis view of the left

ventricle. Myocardial signal intensity extracted from the frames before microbubble

destruction were used to calculate microvascular blood volume A. Myocardial signal

intensities from the frames after microbubble destruction were corrected for

background noise by subtracting the signal intensity of the first frame after

microbubble destruction (Y0). These intensities were then fitted (Y=Y0+(A-Y0)*(1-

exp(- ) for calculation of the microvascular filling velocity

into min-1 for further analysis (Figure 3.2). The estimate of perfusion was calculated

as A * ß. The flow reserve was calculated as the ratio of hyperemic and baseline

estimate of perfusion.

Statistical analysis

All data are presented as mean±SD. Between group comparisons (CD vs. HFD) were

performed using a student t-test, whereas baseline vs. dipyridamole intervention

was tested with a paired student t-test. Oral glucose tolerance test was tested with

one-way ANOVA with repeated measurements and Bonferroni post-hoc test. p<0.05

was considered as statistically significant.

3

56 Chapter 3

Figure 3.2: Typical example of contrast echocardiography with replenishment curve in

rats


Results

Short-term high fat diet feeding results in mild glucose intolerance

Characteristics of rats after the diet intervention are summarized in Table 3.1. HFD

feeding did not affect body weight, but increased perirenal and epidydimal fat

weight. No changes were found in heart weight and tibia length between groups.

Haematocrit, blood glucose, plasma insulin, free fatty acid and LDL/VLDL cholesterol

levels were unaltered, whereas plasma HDL cholesterol levels were significantly

decreased compared to controls. Post-load blood glucose levels were increased in

HFD-fed rats compared to CD-fed rats, whereas post-load plasma insulin levels

remained unchanged after HFD and CD feeding (Figure 3.3), indicating mild glucose

intolerance.

Table 3.1: Characteristics after short-term diet intervention

Control diet High fat diet

Body composition

Body weight (g) 400±18 408±9

Perirenal fat (g) 6.3±1.5 7.5±1.2

Epidydimal fat (g) 7.4±1.2 9.4±1.4 *

Heart weight (g) 1.22±0.13 1.31±0.18

Tibia length (cm) 3.97±0.15 3.89±0.15

Blood/plasma characteristics

Hematocrit (%) 45.2±1.9 44.8±1.9

Blood glucose (mmol/L) 4.4±0.5 4.5±0.6

Plasma insulin (pmol/L) 95.2±37.4 130.8±82.4

Plasma free fatty acid (mmol/L) 1.22±0.21 1.09±0.19

Plasma HDL cholesterol (mg/dL) 318.7±23.9 242.6±40.8 *

Plasma LDL/VLDL cholesterol (mg/dL) 7.73±2.13 7.45±1.40

Data are mean±SD, n=8, student t-test, * p<0.05 vs. control diet.

3

58 Chapter 3

Figure 3.3: High fat diet-feeding induced mild glucose intolerance

Blood glucose (A,B) and plasma insulin levels (C,D) following an oral glucose load in rats fed a

control diet (CD) or high fat diet (HFD). Data are expressed as mean±SD, n=7-8. One-way ANOVA

with repeated measurements and Bonferroni post-hoc test (A,C) or student t-test (B,D), * p<0.05

vs. CD.


Tab

le 3

.2:

Ech

oca

rdio

gra

ph

ic p

ara

mete

rs a

fter

sho

rt-t

erm

die

t in

terv

en

tion

C

D

HFD

B

ase

lin

e

Hyp

ere

mia

Base

lin

e

Hyp

ere

mia

ED

lum

en d

iam

eter

(m

m)

7.4

1±

0.5

3

7.4

6±

0.3

5

6.5

0±

0.7

0 *

7.0

7±

0.7

9 #

ES lum

en d

iam

eter

(m

m)

3.1

8±

0.5

4

2.5

8±

0.4

9

2.9

9±

0.7

8

2.8

9±

0.4

9

Dia

stol

ic W

T (

mm

)

1.3

0±

0.1

2

1.4

1±

0.1

4 #

1.3

4±

0.2

5

1.4

2±

0.1

1

Sys

tolic

WT (

mm

) 2.9

6±

0.3

9

3.4

0±

0.1

6 #

2.8

5±

0.3

7

3.1

0±

0.2

4 *

Data

are

mea

n±

SD

, n=

8,

studen

t t-

test

, * p

<0.0

5 d

iet

effe

ct,

# p

<0.0

5 h

yper

emia

effec

t. C

D,

contr

ol d

iet;

HFD

, hig

h f

at d

iet;

ED

, en

d-d

iast

olic

; ES,

end-s

ysto

lic;

WT,

wal

l th

ickn

ess.

3

60 Chapter 3

High fat diet feeding impaired contractile function during dipyridamole-

induced hyperemia

Table 3.2 shows a summary of the echocardiographic parameters measured after 4

weeks of HFD feeding during baseline and dipyridamole-induced hyperemia. HFD

feeding resulted in decreased lumen diameter during end diastole at baseline and

reduced systolic wall thickness after dipyridamole infusion. Dipyridamole infusion

increased systolic and diastolic wall thickness in CD-fed rats, whereas in HFD-fed rats

the end diastolic lumen diameter was increased.

Contractile function, as represented by fractional shortening, was unaffected by

HFD feeding under baseline conditions (Figure 3.4). During dipyridamole infusion,

fractional shortening was increased in CD-fed rats, but not in HFD-fed rats, whereas

fractional shortening was even decreased in HFD vs. CD-fed rats, suggesting

impaired contractile function during dipyridamole-induced hyperemia.

Figure 3.4: Contractile function during baseline and dipyridamole-induced hyperemia

Representative examples of the m-mode of the left ventricle at the level of the papillary muscle

(A). Contractile function, as represented by the fractional shortening, measured in rats fed a

control diet (CD) and high fat diet (HFD) for 4 weeks during baseline and dipyridamole-induced

hyperemia (B). Data are expressed as mean±SD, n=6-8, (paired) student t-test, * p<0.05 diet

effect, # p<0.05 hyperemia effect.

A


Unchanged myocardial perfusion after high fat diet feeding

HFD-feeding did not alter microvascular blood volume A, microvascular filling

velocity the estimate of perfusion compared to CD-fed rats (Figure 3.5A-C).

Dipyridamole infusion increased microvascular blood volume A, microvascular filling

velocity the estimate of perfusion in CD-fed rats (Figure 3.5A-C), however,

failed to reach significance. In HFD-

dipyridamole infusion (Figure 3.5A-C). No differences were found in flow reserve

between CD- and HFD-fed rats (Figure 3.5D), suggesting unaltered myocardial

perfusion after 4 weeks of HFD feeding.

Figure 3.5: Myocardial perfusion measured with contrast echocardiography

Microvascular blood volume A (A), microvascular filling velocity B), estimate of perfusion (C)

and flow reserve (D) in rats fed a control (CD) or high fat diet (HFD) measured during baseline and

dipyridamole-induced hyperemia. Data are expressed as mean±SD, n=4-8, (paired) student t-test, # p<0.05 vs. CD.

3

62 Chapter 3

Discussion

In the present study, we examined the effect of glucose intolerance on myocardial

function and perfusion. We found that short-term high fat diet feeding resulted in

glucose intolerance without affecting myocardial function and perfusion under

baseline conditions. Diet-induced glucose intolerance impaired myocardial contractile

function during conditions of hyperemia, in the absence of alterations in myocardial

perfusion.

The present study showed that short-term high fat diet feeding resulted in glucose

intolerance without obesity affecting myocardial contractile function under resting

conditions. Our results confirm previous findings, showing that 4 weeks of high fat

diet feeding in rats resulted in glucose intolerance without affecting function,15

whereas 8 weeks of high fat diet feeding resulted in glucose intolerance with mildly

impaired contractile function.15 One of the suggested mechanisms that may

contribute to alterations in myocardial blood flow during prediabetes or diabetes are

changes in nitric oxide availability. High fat diet feeding in male rats decreased nitric

oxide availability,16 whereas increased nitric oxide bioavailability in eNOS-/- mice has

been shown to attenuate high fat diet-induced metabolic alterations associated with

insulin resistance.17 Taken together, these results suggest that 4 weeks of high fat

diet feeding in a rat results in a mild model of glucose intolerance without impaired

myocardial function.

Under physiological conditions, myocardial perfusion and function are in balance.5

However, a perfusion-contraction mismatch might exist in glucose intolerance. In the

present study, diet-induced glucose intolerance did not affect myocardial perfusion

measured with contrast echocardiography during baseline conditions. These results

are in agreement with Menard et al.,18 who found no differences in myocardial

perfusion index measured by [13N] ammonia PET in rats fed a high fructose and high

fat diet with additional streptozotocin. Accordingly, in patients with insulin resistance

or glucose tolerance it was found that insulin resistance was associated with a defect

in myocardial perfusion measured by single photon emission computed tomography,

which was independent of glucose tolerance and obesity.6 Together, these results

suggest that under baseline conditions diet-induced glucose intolerance does not

affect myocardial function and perfusion.

Dipyridamole is used to pharmacologically induce maximal vasodilation.

Dipyridamole blocks the uptake of adenosine, thereby increasing the circulating

levels of adenosine, which results in maximal coronary blood flow due to decreased

coronary vascular resistance.19 During hyperemia, myocardial contractile function

was increased in healthy rats, but not in rats with diet-induced glucose intolerance.

On the contrary, this hyperemic-induced increase in contractile function was not seen

in healthy and type 1 diabetic rats.8 Besides glucose intolerance, several variables


could be associated with alterations in myocardial function. Our experimental rat

model with high fat diet feeding resulted in development of glucose intolerance, but

rats did not become obese. Consequently, obesity cannot be explanatory for the

findings of the effect of high fat diet feeding on myocardial function.

Myocardial blood flow reserve is the increase in blood flow that can be achieved

from basal perfusion to maximal vasodilatation, and is therefore a measurement of

the ability of the microvasculature to respond to an increase in oxygen demand. In

type 1 diabetic rats8 and patients9 it was shown that myocardial blood flow reserve

measured with contrast echocardiography was decreased compared to healthy

controls. In the present study, myocardial blood flow reserve was unchanged

between healthy rats and rats with glucose intolerance, suggesting that glucose

intolerance does not reduce the vasodilating capacity of the myocardium. Increases

in myocardial contractile function will lead to a metabolically-mediated increase in

myocardial blood flow, however, an increase in coronary perfusion does not have to

increase myocardial contractile function,5 which might explain the hyperemic-induced

differences in myocardial function and perfusion in rats with glucose intolerance.

A limitation of this study is the small amount of rats used. Although this is a pilot

study, ideally, this data set should have been analyzed with a two-way ANOVA with

Bonferronni post-hoc testing for multiple group comparisons.

In conclusion, the present data demonstrate that diet-induced glucose intolerance

is associated with impaired myocardial function during conditions of hyperemia, but

not myocardial perfusion. These findings may result in new insights into the effect of

glucose intolerance on myocardial function and perfusion during hyperemia.

3

64 Chapter 3

References

1. Zimmet PZ, Gareeboo H, Hemraj F, Tuomilehto J, Alberti KG: Impaired fasting glucose or

impaired glucose tolerance. What best predicts future diabetes in Mauritius? Diabetes Care

1999, 22:399 402.

2. de Veire van, de Winter O, Gillebert TC, de Sutter J: Diabetes and impaired fasting glucose as

predictors of morbidity and mortality in male coronary artery disease patients with reduced

left ventricular function. Acta Cardiol 2006, 61:137 143.

3. Tominaga M, Eguchi H, Manaka H, Igarashi K, Kato T, Sekikawa A: Impaired glucose tolerance

is a risk factor for cardiovascular disease, but not impaired fasting glucose. The Funagata

Diabetes Study. Diabetes Care 1999, 22:920 924.

4. DeFronzo RA, bdul-Ghani M: Assessment and treatment of cardiovascular risk in prediabetes:

impaired glucose tolerance and impaired fasting glucose. Am J Cardiol 2011, 108:3B 24B.

5. Heusch G, Schulz R: The relation of contractile function to myocardial perfusion. Perfusion-

contraction match and mismatch. Herz 1999, 24:509 514.

6. Nasr G, Sliem H: Silent myocardial ischemia in prediabetics in relation to insulin resistance. J

Cardiovasc Dis Res 2010, 1:116 121.

7. Nasr G, Sliem H: Silent ischemia in relation to insulin resistance in normotensive prediabetic

adults: early detection by single photon emission computed tomography (SPECT). Int J

Cardiovasc Imaging 2011, 27:335 341.

8. Cosyns B, Droogmans S, Hernot S, Degaillier C, Garbar C, Weytjens C, Roosens B, Schoors D,

Lahoutte T, Franken PR, Van Camp G: Effect of streptozotocin-induced diabetes on myocardial

blood flow reserve assessed by myocardial contrast echocardiography in rats. Cardiovasc

Diabetol 2008, 7:26.

9. Rana O, Byrne CD, Kerr D, Coppini DV, Zouwail S, Senior R, Begley J, Walker JJ, Greaves K:

Acute hypoglycemia decreases myocardial blood flow reserve in patients with type 1 diabetes

mellitus and in healthy humans. Circulation 2011, 124:1548 1556.

10. http://eurlex.europa.eu/LexUriServ/LexUriServ.do?uri=CELEX:31986L0609:EN:NOT.

11. Kilkenny C, Browne WJ, Cuthill IC, Emerson M, Altman DG: Improving bioscience research

reporting: the ARRIVE guidelines for reporting animal research. PLoS Biol 2010, 8:e1000412.

12. Ouwens DM, Boer C, Fodor M, de Galan P, Heine RJ, Maassen JA, Diamant M: Cardiac

dysfunction induced by high-fat diet is associated with altered myocardial insulin signalling in

rats. Diabetologia 2005, 48:1229 1237.

13. van den Brom CE, Bosmans JW, Vlasblom R, Handoko ML, Huisman MC, Lubberink M, Molthoff



14. van den Brom CE, Huisman MC, Vlasblom R, Boontje NM, Duijst S, Lubberink M, Molthoff CF,



cardiomyopathy in rats: studies using positron emission tomography. Cardiovasc Diabetol

2009, 8:39.

15. Ouwens DM, Diamant M, Fodor M, Habets DD, Pelsers MM, El Hasnaoui M, Dang ZC, van den



mediated fatty acid uptake and esterification. Diabetologia 2007, 50:1938 1948.


16. Monteiro PF, Morganti RP, Delbin MA, Calixto MC, Lopes-Pires ME, Marcondes S, Zanesco A,

Antunes E: Platelet hyperaggregability in high-fat fed rats: a role for intraplatelet reactive-

oxygen species production. Cardiovasc Diabetol 2012, 11:5.

17. Razny U, Kiec-Wilk B, Wator L, Polus A, Dyduch G, Solnica B, Malecki M, Tomaszewska R,

Cooke JP, mbinska-Kiec A: Increased nitric oxide availability attenuates high fat diet metabolic

alterations and gene expression associated with insulin resistance. Cardiovasc Diabetol 2011,

10:68.

18. Menard SL, Croteau E, Sarrhini O, Gelinas R, Brassard P, Ouellet R, Bentourkia M, van Lier JE,

Des RC, Lecomte R, Carpentier AC: Abnormal in vivo myocardial energy substrate uptake in

diet-induced type 2 diabetic cardiomyopathy in rats. Am J Physiol Endocrinol Metab 2010,

298:E1049 E1057.

19. Leppo JA: Dipyridamole myocardial perfusion imaging. J Nucl Med 1994, 35:730 733.

3

CE van den Brom, CA Boly, CSE Bulte, RPF van den Akker,

RFJ Kwekkeboom, SA Loer, C Boer, RA Bouwman

Submitted

4Sevoflurane impairs myocardial systolic

function, but not myocardial perfusion in

diet-induced prediabetic rats

Sevoflurane and myocardial perfusion in prediabetes 69

Abstract

Introduction

Preservation of myocardial perfusion is particularly important in patients with

increased risk for perioperative cardiac complications, such as patients with diabetes.

Knowledge regarding regulation of myocardial perfusion and function during

anesthesia in subjects with cardiometabolic disease is however limited. In this study

we therefore investigated whether the effect of sevoflurane on myocardial perfusion

and function is altered in healthy and diet-induced prediabetic rats.

Methods

Male Wistar rats were randomly exposed to a western diet (WD; n=18) or control

diet (CD; n=18). After 8 weeks, rats underwent (contrast) echocardiography to

determine myocardial perfusion and function during baseline conditions and

sevoflurane (2%) exposure. Myocardial perfusion was estimated based on the

product of microvascular filling velocity ( and microvascular blood volume (A),

whereas myocardial function was determined by fractional shortening and fractional

area change.

Results

Eight weeks of WD feeding resulted in obesity and hyperglycemia compared to CD-

feeding. At baseline, WD decreased estimate of perfusion compared to control rats.

Systolic function was significantly impaired compared to CD-fed rats. Exposure to

sevoflurane increased the microvascular filling velocity in healthy controls, whereas

the overall estimate of myocardial perfusion remained unchanged in both diet

groups. Moreover, sevoflurane impaired systolic function in both diet groups.

Conclusions

Diet-induced prediabetes is associated with impaired myocardial perfusion and

function in rats. While sevoflurane further impaired systolic function, it did not affect

myocardial perfusion in prediabetic rats. Our findings suggest that sevoflurane leads

to uncoupling of myocardial perfusion and function, irrespective of the metabolic

state.

4

70 Chapter 4

Introduction

Myocardial perfusion in relation with myocardial function determines the balance

between myocardial energy supply and demand. During surgery, maintenance of the

myocardial oxygen balance is challenged, because extrinsic factors, like anesthetics

and surgical stress, and intrinsic factors, like metabolic alterations, affect myocardial

oxygen supply and consumption. This altered balance may increase the vulnerability

of the heart for an oxygen supply and demand mismatch and consequent

ischemia.1;2

Sevoflurane has vasodilating properties and is known to reduce coronary vascular

resistance3 and perfusion pressure.4;5 We recently showed that sevoflurane did not

affect myocardial blood flow in healthy patients, while myocardial flow reserve was

decreased.6 Animal studies however showed that sevoflurane, when perfusion

pressure remained constant, increased coronary blood flow in hearts of dogs7 and

decreased coronary flow reserve in isolated rat hearts.5 In contrast, sevoflurane

lowered blood pressure and decreased myocardial blood flow in healthy rats,8 dogs9

and pigs.10

While sevoflurane exerts contrasting effects on myocardial perfusion in healthy

subjects, its vasodilatory impact may be more abundant in patients with

cardiometabolic disease, like type 2 diabetes mellitus (T2DM). T2DM patients are

more likely to develop coronary artery disease11 and have an increased

cardiovascular complication rate after major non-cardiac surgery.12 Because

myocardial substrate metabolism and myocardial oxygen balance is altered in

T2DM,13 the regulation of myocardial perfusion in these patients is particularly

important during normal and intraoperative circumstances. The number of studies

focusing on myocardial perfusion during anesthesia in subjects with cardiometabolic

disease is however limited. Previously, we found that myocardial perfusion, but not

myocardial function, is preserved during hyperemia in glucose intolerant rats,14 while

others showed myocardial perfusion defects in prediabetic insulin resistant

patients15;16 or T2DM patients during the postprandial state.17;18 The aforementioned

data suggest that a cardiometabolic-compromised state may be associated with

more severe alterations in myocardial perfusion during anesthesia.

Therefore, the purpose of the present study was to investigate the effect of

sevoflurane on myocardial function and perfusion in diet-induced prediabetic rats.

While the effects in healthy subjects seem apparently conflicting, we hypothesized

cardiometabolic disease and thereby challenging myocardial perfusion regulation.


Methods


All experiments were approved by the Institutional Animal Care and Use Committee

of the VU University, and were conducted following the European Convention for the

Protection of Vertebrate Animals used for Experimental and Other Scientific

Purposes.

Adult male Wistar rats (n=36; body weight 265±7 g; Charles River Laboratories,

France) were fed a western diet (WD) for a period of 8 weeks (n=18). Rats that

received a diet low in fat and sugars (control diet; CD) for 8 weeks served as

controls (n=18). Animals were housed in a temperature-controlled room (20-23°C;

40-60% humidity) under a 12/12h light/dark cycle starting at 6.00 am. Body weight

was determined on a weekly basis. After 8 weeks, rats underwent (contrast)

echocardiography during baseline conditions and during sevoflurane (AbbVie, the

Netherlands) exposure, starting after 5 minutes of sevoflurane.

Diets

CD (Teklad 2016, Harlan, Horst, the Netherlands) consisted of 20 %kcal protein,

9 %kcal fat and 74 %kcal carbohydrates (1804 kcal/kg starch, 200 kcal/kg sugars),

whereas WD (D12451, Research Diets, New Brunswick, NJ) consisted of 20 %kcal

protein, 45 %kcal fat and 35 %kcal carbohydrates (291 kcal/kg starch, 691 kcal/kg

sugars) with 20% sucrose water (800 kcal/kg), totally containing 3300 kcal/kg and

4857 kcal/kg for CD and WD with sucrose water, respectively.

Surgery

After 8 weeks of diet exposure, rats were anesthetized with 125 mg/kg S-Ketamine

(Ketanest®, Pfizer, the Netherlands) and 4 mg/kg diazepam (Centrafarm, the

Netherlands) intraperitoneally and were intubated and mechanically ventilated (UNO,

the Netherlands; positive end-expiratory pressure, 1-2 cm H2O; respiratory rate,

~65 breaths/min; tidal volume, ~10 ml/kg) with oxygen-enriched air (40% O2/60%

N2). Anesthesia was maintained by continuous infusion of 50 mg/kg/h S-Ketamine

and 1.3 mg/kg/h diazepam intravenously via the tail vein. Respiratory rate was

adjusted to maintain pH and partial pressure of carbon dioxide within physiological

limits. Body temperature was maintained stable (36.7±1.2°C) using a warm water

underbody heating pad.

A catheter was placed in the right jugular vein for infusion of the contrast agent.

The left carotid artery was cannulated for blood sampling, blood gas analyses

(ABL50, radiometer, Copenhagen, Denmark) and for measurements of arterial blood

pressure (Safedraw Transducer Blood Sampling Set, Argon Medical Devices, Texas,

USA). Arterial blood pressure, ECG and heart rate were continuously recorded using

4

72 Chapter 4

PowerLab software (PowerLab 8/35, Chart 7.0; ADInstruments Pty, Ltd., Castle Hill,

Australia). Mean arterial blood pressure was calculated according the following

product (RPP) was calculated by the product of heart rate and systolic blood pressure

and was used as an estimate of myocardial oxygen demand.

Echocardiography

After surgery, (contrast) echocardiography was performed to determine myocardial

function and perfusion during baseline conditions and after 5 minutes of sevoflurane

(2%) exposure. Echocardiography (Siemens, ACUSON, Sequoia 512) was performed

as previously described.13 Briefly, left ventricular (LV) dimensions during end-systole

(ES) and end-diastole (ED) were determined in the M-(motion) mode of the

parasternal short-axis view at the level of the papillary muscles. LV systolic function

is represented by fractional shortening (FS) and fractional area change (FAC), which

were calculated by the equations: FS = (EDD- 2-

ESD2)/EDD2 100). All parameters were averaged over at least three cardiac

contractile cycles.

Preparation of microbubbles

Microbubbles were prepared from perfluorobutane gas and stabilized with a

monolayer of distearoyl phosphatidylcholine and PEG stearate. 1,2-distearoyl-sn-

glycero-3-phosphocholine (DSPC; Avanti Polar Lipids, Alabama, USA) and

polyoxyethylene stearate (PEG40; Sigma, St Louis, MO, USA) were dissolved in

glycerol (10 mg/ml) and sonicated (Decon FS200, Decon Ultrasonics Ltd, Sussex,

UK) at 40 kHz in an atmosphere of perfluorobutane (F2 Chemicals Ltd, Lancashire,

UK) and vials were shaken a Vialmix at 4500 rpm (Bristol-Myers Squibb Medical

Imaging, Massachusetts, USA). As the gas was dispersed in the aqueous phase,

microbubbles were formed, which were stabilized with a self-assembled

lipid/surfactant monolayer. Freshly made bubbles were then washed twice to remove

excessive DSPC and PEG40 and stored refrigerated in sealed vials in perfluorobutane

atmosphere. A Multisizer 3 coulter counter (Beckman Coulter Inc., Miami, FL, USA)

was used to measure the particle size distribution as well as the number of particles.

The average bubble concentration was 1.578 109 ± 0.364 109 and the particle

range was between 1 and 10 Microbubbles were diluted to a concentration of

200 106 with ungassed water.


Myocardial contrast echocardiography

Contrast echocardiography was performed using a Siemens (ACUSON, Sequoia 512)

equipped with a 14 MHz linear array transducer (Philips Healthcare, Best, The

Netherlands).14 Microbubbles were continuously infused into the jugular vein with a

rate of 300 μl/min using a dedicated syringe pump (Vueject, Bracco SA,

Switzerland). After two minutes of microbubble infusion, perfusion images were

taken from the long axis view of the left ventricle.

Low acoustic power (mechanical index [MI] 0.20) was used for microbubble

detection with a dynamic range of 50 dB. A perfusion sequence consisted of about 10

cardiac cycles of low MI imaging, followed by a burst of high acoustic power (MI 1.8)

for complete contrast destruction. Subsequently, on average 20 cardiac cycles of low

MI images were acquired to allow contrast replenishment in the myocardium. All

data were stored for offline analysis.

Myocardial contrast echocardiography analysis

Custom-designed software was used for analysis of the estimate of perfusion

(Matlab, 7.10, R2010A, MathWorks Inc. Massachusetts, USA).6;14 For each cardiac

cycle, regions of interest were drawn in the end-systolic frame in the posterior wall in

the long axis view of the left ventricle. Myocardial signal intensities from the frames

after microbubble destruction were corrected for background noise by subtracting

the signal intensity of the first frame after microbubble destruction (Y0). These

intensities were then fitted (Y=Y0 + (A-Y0) (1-exp(-ß x))) for calculation of

microvascular blood volume A and the microvascular filling velocity ß, which

corresponds to the capillary blood exchange rate. The estimate of perfusion was

calculated as the product of A and ß.19


Data were analyzed using Graphpad Prism 5.0 (La Jolla, USA) and presented as

mean±SD. Between group comparisons (CD vs. WD) were performed using a

student t-test, whereas the effect of sevoflurane exposure was tested with a two-

way ANOVA with Bonferonni as post-hoc test. p<0.05 was considered as statistically

significant.

4

74 Chapter 4

Results

Western diet feeding resulted in obesity and hyperglycemia

After 8 weeks of western diet feeding, bodyweight was significantly increased

compared to control rats (Table 4.1), whereas heart rate, systolic blood pressure,

diastolic blood pressure and mean arterial pressure remained unchanged (Figure

4.1). After sacrifice, blood glucose levels and left ventricular weights were higher in

western diet- compared to control diet-fed rats (Table 4.1).

Table 4.1: Characteristics after 8 weeks of diet intervention

Control diet Western diet

Body weight (g) 420±25 450±20 *

Blood glucose after sacrifice (mmol/L) 14.6±5.1 19.3±3.6 *

Tibia length (mm) 42.7±1.0 43.1±0.6

Left ventricular weight (g) 0.53±0.06 0.57±0.08 *

Data are mean±SD, n=18, student t-test, * p<0.05 vs. control diet.


Figure 4.1: Hemodynamics during sevoflurane exposure

Systolic blood pressure (A), diastolic blood pressure (B), mean arterial pressure (C), heart rate (D)

and rate pressure product (E) during baseline conditions, before sevoflurane exposure, after 5

minutes of sevoflurane and after 5 minutes of washout period in rats fed a control diet (CD) or

western diet (WD) for 8 weeks. Data are mean±SD, n=16-18, two-way ANOVA with repeated

measurements and Bonferonni post-hoc analyses, # p<0.05 sevoflurane effect, $ p<0.05 washout

effect.

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76 Chapter 4

Impaired myocardial perfusion and systolic function in prediabetic rats

Compared to healthy controls, western diet feeding tended to decrease

volume (A), which resulted in a significant reduction in the estimate of perfusion

(Figure 4.2).

Western diet feeding significantly increased end-systolic lumen diameter and

diastolic wall thickness, but did not affect end-diastolic lumen diameter and wall

thickness during systole compared to control rats (Table 4.2). Fractional shortening

and fractional area change were significantly decreased in western diet-fed rats

compared to control animals, suggesting impaired systolic function (Figure 4.3).

Table 4.2: Myocardial function after 8 weeks during baseline and after sevoflurane

exposure

Data are mean±SD, n=9-18, two-way ANOVA with Bonferroni post-hoc analyses, * p<0.05 diet

effect, # p<0.05 sevoflurane effect. CD, control diet; WD, western diet.

Baseline Sevoflurane

CD WD CD WD

Diastolic lumen diameter (mm) 5.7±0.6 5.5±0.8 5.4±0.8 5.5±0.8

Systolic lumen diameter (mm) 2.0±0.4 2.7±0.7* 2.3±0.5 3.4±0.6*#

Diastolic wall thickness (mm) 1.8±0.1 1.9±0.2* 1.6±0.2 1.9±0.2*

Systolic wall thickness (mm) 3.3±0.3 3.1±0.4 3.0±0.4 2.9±0.2


Figure 4.2: Effect of sevoflurane on myocardial perfusion in prediabetic rats

Microvascular blood volume A (A), microvascular filling velocity B) and estimate of perfusion (C)

measured with contrast echocardiography in rats fed a control diet (CD) or western diet (WD) for 8

weeks during baseline conditions and after 5 minutes of sevoflurane exposure. Data are expressed

as mean±SD, n=9-13, two-way ANOVA with Bonferonni post-hoc analyses, * p<0.05 diet effect, #

p<0.05 sevoflurane effect.

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78 Chapter 4

Sevoflurane further impaired systolic function, but not myocardial perfusion

Blood pressure, heart rate and rate pressure product were significantly decreased

after 5 minutes of sevoflurane exposure and significantly restored after a 5-minute

washout period, without differences among diet groups (Figure 4.1).

Compared to baseline conditions, sevoflurane decreased the microvascular filling

tended to increase microvascular blood volume (A) in controls, while

this observation was absent in western diet-fed animals. Overall, this resulted in an

unchanged estimate of perfusion in both diet groups (Figure 4.2).

Sevoflurane additionally increased end-systolic lumen diameter in western diet-fed

rats compared to baseline conditions (Table 4.2), which resulted in further impaired

systolic function in western diet-fed rats compared to control rats (Figure 4.3).

Figure 4.3: Effect of sevoflurane on systolic function in prediabetic rats

Systolic function, as represented by the fractional shortening (A) and fractional area change (B),

measured with echocardiography in rats fed a control diet (CD) or western diet (WD) for 8 weeks

during baseline conditions and after 5 minutes of sevoflurane exposure. Data are expressed as

mean±SD, n=9-18, two-way ANOVA with Bonferonni post-hoc analyses, * p<0.05 diet effect, #

p<0.05 sevoflurane effect.


Discussion

In the present study, we examined the effect of sevoflurane on myocardial perfusion

and function in western diet-fed rats. We found that short-term western diet feeding

resulted in a prediabetic phenotype characterized by obesity and hyperglycemia.

Further, diet-induced prediabetes was associated with impaired myocardial perfusion

and systolic dysfunction. Sevoflurane had no overall effect on myocardial perfusion in

healthy and prediabetic rats, while systolic function was even further impaired. These

results suggest that sevoflurane leads to uncoupling of myocardial perfusion and

function, irrespective of the metabolic state.

Sevoflurane did not affect myocardial perfusion, despite of decreased arterial blood

pressure, heart frequency and rate pressure product in healthy rats. Previously it has

indeed been shown that sevoflurane did not alter myocardial blood flow in healthy

rats20 and healthy subjects6 compared to the awake condition. In contrast, others

however described decreased myocardial blood flow in healthy rats,8 dogs9 and

pigs.10 In addition to species variation and the use of different experimental

techniques,19 administration of general anesthetics may explain the contrasting

observations, as this may distinctly alter hemodynamics compared to the awake

state. While myocardial perfusion remained unchanged, sevoflurane decreased the

A). The

microvascular filling velocity is a parameter of the capillary exchange rate providing

an estimate of the speed of erythrocytes through the capillaries, while microvascular

blood volume suggests the surface area for exchange of nutrients and correlates with

oxygen consumption. Our observations are in contrast with a previously performed

study in healthy subjects by our group, where we showed that sevoflurane decreased

myocardial blood volume and increased the microvascular filling velocity.6 A possible

mechanism to explain these differences may be derived by the differences in heart

rate among species. We found a decrease in heart rate, while in healthy subjects an

increase in heart rate was shown.6 However, also decreased8 or unchanged20 heart

rate in rats are found. Taken together, although sevoflurane impaired the

microvascular filling velocity, myocardial perfusion was not affected in healthy rats.

Moreover, the present study showed that myocardial perfusion and function were

both decreased in prediabetic rats compared to healthy controls. In detail,

microvascular blood volume was decreased, which resulted in impairment of

myocardial perfusion. Previously, we found that myocardial perfusion was maintained

during baseline conditions in high fat diet-induced glucose intolerant rats.14 The

present study used a more severe diet, which resulted in more pronounced

disturbances in the cardiometabolic condition of the rats. Independent of glucose

tolerance and obesity, myocardial perfusion defects were found in prediabetic insulin

resistant patients.15;16 In T2DM patients, no differences in myocardial blood flow

were found during fasting conditions.17;18 However, during the postprandial state

4

80 Chapter 4

decreased myocardial blood flow was found in T2DM patients,17;18 which is in

agreement with the present study showing decreased myocardial perfusion in non-

fasted prediabetic rats. An underlying mechanism of this impaired myocardial

perfusion might be alterations in insulin-mediated recruitment of the capillaries. In

healthy subjects, insulin increased basal21 and adenosine-stimulated22 myocardial

perfusion. Moreover, insulin directly affects myocardial perfusion by enhancing

hyperemic myocardial blood flow in a dose-dependent manner in healthy subjects.23

However, insulin resistance may block insulin-mediated capillary recruitment.24 In

T2DM patients, the use of an insulin analog partially reversed myocardial perfusion

abnormalities.18

As far as we know, we are the first to study the effect of sevoflurane anesthesia on

myocardial perfusion in prediabetic rats. Our results show that sevoflurane has a

stronger cardiodepressive effect in prediabetic rats, whereas myocardial perfusion

remained unaffected. Under physiological conditions, myocardial blood flow and

function are in balance.25 In our prediabetic rats, myocardial perfusion as well as

myocardial function are decreased. However, during sevoflurane myocardial

perfusion is maintained, while myocardial function is decreased in healthy and

prediabetic rats. This uncoupling of perfusion and function suggests that despite of

increased microvascular blood volume and decreased microvascular filling velocity,

myocardial function cannot be maintained.

In conclusion, the present study showed impaired myocardial function and

perfusion in diet-induced prediabetic rats. Moreover, sevoflurane further impaired

systolic function, but myocardial perfusion was maintained. These results suggest

that sevoflurane leads to uncoupling of myocardial perfusion and function,

irrespective of the metabolic state of the heart.


References

1. Dole WP: Autoregulation of the coronary circulation. Prog Cardiovasc Dis 1987, 29:293-323.

2. Hoffman JI, Spaan JA: Pressure-flow relations in coronary circulation. Physiol Rev 1990,

70:331-390.

3. Malan TP, Jr., DiNardo JA, Isner RJ, Frink EJ, Jr., Goldberg M, Fenster PE, Brown EA, Depa R,



4. Park KW: Cardiovascular effects of inhalational anesthetics. Int Anesthesiol Clin 2002, 40:1-14.

5. Larach DR, Schuler HG: Direct vasodilation by sevoflurane, isoflurane, and halothane alters

coronary flow reserve in the isolated rat heart. Anesthesiology 1991, 75:268-278.

6. Bulte CS, Slikkerveer J, Kamp O, Heymans MW, Loer SA, de Marchi SF, Vogel R, Boer C,

Bouwman RA: General anesthesia with sevoflurane decreases myocardial blood volume and

hyperemic blood flow in healthy humans. Anesth Analg 2013, 116:767-774.

7. Crystal GJ, Zhou X, Gurevicius J, Czinn EA, Salem MR, Alam S, Piotrowski A, Hu G: Direct

coronary vasomotor effects of sevoflurane and desflurane in in situ canine hearts.


8. Conzen PF, Vollmar B, Habazettl H, Frink EJ, Peter K, Messmer K: Systemic and regional

hemodynamics of isoflurane and sevoflurane in rats. Anesth Analg 1992, 74:79-88.

9. Hirano M, Fujigaki T, Shibata O, Sumikawa K: A comparison of coronary hemodynamics during

isoflurane and sevoflurane anesthesia in dogs. Anesth Analg 1995, 80:651-656.

10.Manohar M, Parks CM: Porcine systemic and regional organ blood flow during 1.0 and 1.5

minimum alveolar concentrations of sevoflurane anesthesia without and with 50% nitrous oxide.

J Pharmacol Exp Ther 1984, 231:640-648.

11.Preis SR, Pencina MJ, Hwang SJ, D'Agostino RB, Sr., Savage PJ, Levy D, Fox CS: Trends in



12.Lee TH, Marcantonio ER, Mangione CM, Thomas EJ, Polanczyk CA, Cook EF, Sugarbaker DJ,



Circulation 1999, 100:1043-1049.





8:39.




15.Nasr G, Sliem H: Silent myocardial ischemia in prediabetics in relation to insulin resistance. J

Cardiovasc Dis Res 2010, 1:116-121.

16.Nasr G, Sliem H: Silent ischemia in relation to insulin resistance in normotensive prediabetic

adults: early detection by single photon emission computed tomography (SPECT). Int J

Cardiovasc Imaging 2011, 27:335-341.

17.Scognamiglio R, Negut C, De Kreutzenberg SV, Tiengo A, Avogaro A: Postprandial myocardial

perfusion in healthy subjects and in type 2 diabetic patients. Circulation 2005, 112:179-184.

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18.Scognamiglio R, Negut C, De Kreutzenberg SV, Tiengo A, Avogaro A: Effects of different insulin

regimes on postprandial myocardial perfusion defects in type 2 diabetic patients. Diabetes Care

2006, 29:95-100.

19.Bulte CS, Slikkerveer J, Meijer RI, Gort D, Kamp O, Loer SA, de Marchi SF, Vogel R, Boer C,

Bouwman RA: Contrast-enhanced ultrasound for myocardial perfusion imaging. Anesth Analg

2012, 114:938-945.

20.Crawford MW, Lerman J, Saldivia V, Carmichael FJ: Hemodynamic and organ blood flow

responses to halothane and sevoflurane anesthesia during spontaneous ventilation. Anesth

Analg 1992, 75:1000-1006.

21.Liu Z: Insulin at physiological concentrations increases microvascular perfusion in human

myocardium. Am J Physiol Endocrinol Metab 2007, 293:E1250-E1255.

22.Laine H, Nuutila P, Luotolahti M, Meyer C, Elomaa T, Koskinen P, Ronnemaa T, Knuuti J: Insulin-

induced increment of coronary flow reserve is not abolished by dexamethasone in healthy young

men. J Clin Endocrinol Metab 2000, 85:1868-1873.

23.Sundell J, Nuutila P, Laine H, Luotolahti M, Kalliokoski K, Raitakari O, Knuuti J: Dose-dependent

vasodilating effects of insulin on adenosine-stimulated myocardial blood flow. Diabetes 2002,

51:1125-1130.

24.Zhang L, Vincent MA, Richards SM, Clerk LH, Rattigan S, Clark MG, Barrett EJ: Insulin

sensitivity of muscle capillary recruitment in vivo. Diabetes 2004, 53:447-453.

25.Heusch G, Schulz R: The relation of contractile function to myocardial perfusion. Perfusion-

contraction match and mismatch. Herz 1999, 24:509-514.

CE van den Brom, C Boer, RPF van den Akker, J van der Velden,

SA Loer, RA Bouwman

Submitted

5Diet composition modulates sevoflurane-

induced myocardial depression in rats

Dietary effects on the heart during sevoflurane 87

Abstract

Introduction

Cardiometabolic diseases like obesity and/or diabetes mellitus may alter the effects

of sevoflurane on the heart, but current evidence is limited to in vitro studies. This

study evaluated the influence of western diet-induced obesity with glucose

intolerance on the myocardial response to sevoflurane, and further elaborated

whether lowering caloric intake can modulate this effect.

Methods

Male Wistar rats were exposed to a western diet (WD) or control diet (CD) for 8

weeks. A third group of WD-fed rats reversed after 4 weeks to CD for 4 consecutive

weeks. Study parameters included an oral glucose tolerance test, echocardiography

and protein analyses before and after exposure to 2% sevoflurane.

Results

Eight weeks of WD-feeding resulted in a prediabetic phenotype with obesity, glucose

intolerance, mild hyperglycemia, hyperinsulinemia, dyslipidemia and reduced

myocardial systolic and diastolic function. While sevoflurane did not alter myocardial

contractile function in healthy control animals, systolic function in WD-fed rats was

further impaired after sevoflurane exposure. Reversion of WD to control diet

normalized the prediabetic phenotype, and restored myocardial function during

baseline and after sevoflurane exposure to control values. Western diet and diet

reversal exerted distinct effects on myocardial calcium handling proteins, while

changes in proteins related to substrate metabolism were only minimal.

Conclusions

Sevoflurane is a stronger cardiodepressant in prediabetic than in control rats, which

could be restored by lowering caloric intake. These results suggest that normalization

of the cardiometabolic profile by dietary changes are of direct influence on the

myocardial response to sevoflurane in rats.

5

88 Chapter 5

Introduction

Patients with obesity and type 2 diabetes mellitus (T2DM) due to excessive caloric

intake may exert a different response to anesthesia and surgery than healthy

subjects. In healthy subjects, volatile anesthetics like sevoflurane exhibit negative

inotropic effects as shown by depressed myocardial contractility,1;2 lusitropic effects

as reflected by early diastolic dysfunction1;3 and decreased systemic vascular

resistance.3 While it may be expected that cardiometabolic alterations induced by

obesity and/or diabetes mellitus alter the response of the heart to volatile

anesthetics, most studies focusing on this relationship are limited by their in vitro

nature and the use of models for type 1 diabetes mellitus. Moreover, the results are

conflicting, showing either augmentation4 or suppression5 of the cardiodepressive

effects of volatile anesthetics in papillary muscles of type 1 diabetic rats. A third

study suggested that the inotropic effects of several volatile anesthetics were not

altered in single ventricular myocytes from type 1 diabetic rats.6

These findings warrant further exploration of the effects of obesity and/or diabetes

mellitus on the interaction of volatile anesthetics with the heart. Moreover,

increasing evidence suggests that preoperative alterations in dietary composition and

intake may alter the susceptibility of the cardiovascular system to surgical and

anesthetic stress.7 It has indeed been shown that diet-induced obesity8-11 and

T2DM12;13 in small rodents is reversible by reducing caloric intake, which resulted in

weight loss and improved insulin sensitivity. In obese and T2DM patients, a low

caloric diet decreased myocardial fatty acid uptake14 and improved diastolic

function,15 respectively. Additionally, preoperative dietary restriction exerted

favorable effects on surgery-related inflammation, oxidative stress and ischemia.7;16

It is however unknown whether the interaction of volatile anesthesia with myocardial

function can be altered by dietary intake. In the present study we therefore

investigated whether western diet-induced obesity with glucose intolerance alters the

effects of the volatile anesthetic sevoflurane on myocardial function and calcium

handling proteins in rats, and whether changing diet composition can modulate these

alterations.


Methods


All animal experiments were approved by the Institutional Animal Care and Use

Committee of the VU University, and were conducted following the European

Convention for the Protection of Vertebrate Animals used for Experimental and Other

Scientific Purposes, and the guide for the Care and Use of Laboratory Animals. The

performed research is in compliance with the modern ARRIVE guidelines on animal

research.17

The first part of the study was performed in a group of 24 male Wistar rats

(baseline body weight: 264±5 g; Charles River Laboratories, France). Rats were

exposed to a western diet in combination with sucrose water (20%) (WD, n=16) or

control diet (CD, n=8) for a period of 8 weeks. Four weeks after the start of the diet

exposure, WD (n=8) exposed rats reversed to CD for 4 consecutive weeks. Rats

were housed in a temperature-controlled room (20-23°C; 40-60% humidity) under a

12/12h light/dark cycle starting at 6.00 am. Body weight and caloric intake were

determined on a weekly basis. After 4 and 8 weeks of diet exposure, rats underwent

an oral glucose tolerance test and echocardiography. After a 6h fasting period, rats

were sacrificed by decapitation, trunk blood was collected for plasma determinations

and hearts were removed, rinsed in saline, weighted, snap-frozen in liquid nitrogen

and stored at -80°C until further analysis.

The second part of the study included 30 male Wistar rats (baseline body weight:

262±6 g; Charles River Laboratories, France) that were either exposed to the control

diet (CD, n=10), western diet in combination with sucrose water (20%) (WD, n=10)

or the western diet with reversal to control diet (REV, n=10) as described above.

After 8 weeks, rats were exposed to sevoflurane (AbbVie, the Netherlands). Rats

were sacrificed by decapitation without fasting, trunk blood was collected for plasma

determinations and hearts were removed, rinsed in saline, weighted, snap-frozen in

liquid nitrogen and stored at -80°C until further analysis.

Diets

Control diet (CD; Teklad 2016, Harlan, Horst, the Netherlands) consisted of 20 %kcal


sugars), whereas western diet (WD; D12451, Research Diets, New Brunswick, NJ)

consisted of 20 %kcal protein, 45 %kcal fat and 35 %kcal carbohydrates (291

kcal/kg starch, 691 kcal/kg sugars) with 20% sucrose water (800 kcal/kg), totally

containing 3300 kcal/kg and 4857 kcal/kg for CD and WD with sucrose water,

respectively.

5

90 Chapter 5

Oral glucose tolerance test

Awake rats fasted overnight received an oral glucose load (2 g/kg of body weight).

Blood glucose was measured from tail bleeds with a Precision Xceed Blood Glucose

monitoring system (MediSense, UK) before and 15, 30, 60, 90 and 120 min after

glucose ingestion. At similar time points, plasma insulin (LINCO research, St.

Charles, Missouri) levels were measured as described previously.18;19

Echocardiography

Echocardiography (ALOKA ProSound SSD 4000, Aloka, Tokyo, Japan) using a 13-MHz

linear interfaced array transducer was performed after 4 and 8 weeks as described

previously.18-21 Briefly, rats received S-Ketamine (Ketanest®, 75 mg/kg, Pfizer, the

Netherlands) and diazepam (4 mg/kg, Centrafarm, the Netherlands) anesthesia

intraperitoneally, allowed spontaneous respiration and placed on a underbody

heating pad. Left ventricular (LV) dimensions during end-systole (ES) and end-

diastole (ED) were determined in the M- (motion) mode of the parasternal short-axis

view at the level of the papillary muscles. LV systolic function is represented by

fractional shortening (FS) and fractional area change (FAC), which were calculated

by the equations: FS = (EDD- 2-ESD2)/EDD2 100).

Diastolic function was measured in the apical four-chamber view and shown as E

flow, E deceleration time and isovolumic relaxation time (IVRT; except 8 weeks

data). LV mass was calculated as described previously.18 All parameters were

averaged over at least three cardiac cycles. Analyses were performed off-line

(Image-Arena 2.9.1, TomTec Imaging Systems, Unterschleissheim/Munich,

Germany).

Sevoflurane intervention

The second group of rats was anesthetized with 125 mg/kg S-ketamine (Ketanest®,

Pfizer, the Netherlands) and 4 mg/kg diazepam (Centrafarm, the Netherlands)

intraperitoneally and maintenance was performed with 40 mg/kg/h S-ketamine and

1 mg/kg/h diazepam intravenously in the tail vein and allowed spontaneous

(AbbVie,

the Netherlands) in 40% O2/60% N2, whereas control rats received only 40%

O2/60% N2 for the same time period (baseline, n=4). Measurement of systolic

performed as described above.


Blood and plasma measurements

Plasma hematocrit levels were determined using microcentrifugation. Plasma glucose

levels (Abcam, Cambridge, MA), plasma insulin (LINCO research, St. Charles,

Missouri), plasma free fatty acids (WAKO NEFA-HR, Wako Pure Chemical Industries,

Osaka, Japan), plasma triglyceride (Sigma, Saint Louis, Missouri), and plasma HDL

and LDL/VLDL cholesterol (Abcam, Cambridge, MA) levels were measured from trunk

blood as described previously.18-21

Protein analysis

LV tissues were homogenized to obtain cytoplasmic protein fractions for western blot

analysis as previously described.18;20;21 Phosphorylation and/or expression of

signaling intermediates were analyzed using the following primary antibodies: Akt,

phospho-Akt- - -Ser9,

- -Thr172 (all Cell signaling

Technology, Beverly, MA), pyruvate dehydrogenase kinase 4 (PDK4, Santa Cruz

Biotechnology, Santa Cruz, CA), glucose transporter 4 (GLUT4; Abcam, Cambridge,

MA), phospholamban (PLB), phospho-PLB-Ser16, (Upstate, Lake Placid, NY),

sarcoplasmic reticulum calcium ATPase 2a (SERCA2a)21 and fatty acid transporter

(FAT)/CD36 (MO25).18;21 All signals were normalized to total protein expression or

actin (Sigma, Saint Louis, Missouri).

Myofilament protein phosphorylation was determined using Pro-Q Diamond

Phosphoprotein Stain as described previously.22 LV tissues were separated on

gradient gels (Criterion tris-HCl 4-15% gel, BioRad) and proteins were stained with

Pro-Q Diamond Phosphoprotein Stain. Subsequently gels were stained overnight with

SYPRO Ruby stain (Molecular Probes). Phosphorylation status of myosin binding

protein-C (MyBP-C), troponin T (TnT) and cardiac troponin I (cTnI) were expressed

relative to SYPRO-stained protein bands to correct for differences in sample loading.

Staining was visualized using the LAS-3000 Image Reader (FUJI; 460 nm/605 nm

Ex/Em) and immunoblots were quantified by densitometric analysis of films (AIDA,

4.21.033, Raytest, Strau-benhardt Germany).

Histology

Sections (4 μm) of left ventricular tissue from the free wall at the level of the

papillary muscles were cut in the direction of the fibers and mounted on 3-

aminopropyltriethoxisilane-coated slides (Superfrost® Plus, Menzal, Darmstadt,

Germany). After deparaffinization and rehydration, cardiomyocyte cross-sectional

area was determined in randomly chosen fields of hematoxylin-eosin stained sections

for 20-30 cells per heart and normalized to sarcomere length.18;20;21

5

92 Chapter 5


Data were analyzed using Graphpad Prism 5.0 (La Jolla, USA) and are presented as

mean±SD. Statistical analyses were performed using a student t-test after 4 weeks,

one-way ANOVA with Bonferroni post-hoc analysis after 8 weeks, two-way ANOVA

with repeated measures and Bonferroni post-hoc analysis for the oral glucose

tolerance test and two-way ANOVA with Bonferroni post-hoc analysis for data after 8

weeks with sevoflurane intervention. p<0.05 was considered as statistically

significant.


Results

Animal model characteristics

Western diet (WD) feeding resulted in a prediabetic phenotype with obesity, mild

hyperglycemia, hyperinsulinemia, hyperlipidemia and glucose intolerance at 4 and 8

weeks after initiation of the diet (Table 5.1 and Figure 5.1 A-F). Reversion to a

control diet (REV) after 4 weeks of western diet feeding resulted in normalization of

caloric intake and body weight, and reversed the prediabetic phenotype in western

diet-fed rats.

Heart weight and the cross sectional area of individual cardiomyocytes were

significantly increased in WD-fed rats compared to controls, and these values

normalized after diet reversal. Additionally, diet reversion following western diet

feeding resulted in a decrease in liver weight and epididymal and perirenal fat pads

(Table 5.1).

At 4 and 8 weeks, western diet feeding decreased myocardial lumen diameter and

increased wall thickness during diastole when compared to control rats (Table 5.2).

Moreover, end-systolic lumen diameter was increased at 4 and 8 weeks of western

diet feeding, while systolic wall thickness was slightly reduced after 4 weeks of diet

exposure. Reversal from western to control diet resulted in normalization of systolic

lumen diameter and myocardial diastolic and systolic wall thickness, while the

diastolic lumen diameter was not affected by lowering caloric intake (Table 5.2).

Western diet feeding induced a reduction in myocardial fractional shortening (Figure

5.2A) and fractional area change (Figure 5.2B), which could be restored by diet

reversal. While E flow was unchanged by WD-feeding, the deceleration time of the E

peak and isovolumic relaxation time at 4 weeks were prolonged in rats fed a WD

when compared to control rats (Table 5.2). After 8 weeks of diet exposure, there

was a trend towards impaired left ventricular relaxation in WD-fed rats. Diet reversal

improved diastolic function as shown by a shortening of the E deceleration time when

compared to WD-fed rats (Table 5.2).

5

94 Chapter 5 Tab

le 5

.1:

Ch

ara

cteri

stic

s aft

er

4 a

nd

8 w

eeks

of

die

t in

terv

en

tion

wit

hou

t se

vofl

ura

ne e

xp

osu

re

4

weeks

8

weeks

C

on

trol

die

t W

est

ern

die

t C

on

trol

die

t

West

ern

die

t R

evers

ion

Cal

ori

c in

take

(kc

al/1

00gBW

) 138±

8

155±

17 *

124±

6

129±

7

114±

6 #

Blo

od

/p

lasm

a c

hara

cteri

stic

s

6h fas

ting p

lasm

a g

luco

se (

mm

ol/

L)

7.1

±0.6

7.8

±1.3

8.8

±0.7

10.7

±1.1

*

8.2

±1.2

#

6h fas

ting p

lasm

a in

sulin

(pm

ol/

L)

411±

135

726±

214 *

933±

383

1524±

353 *

813±

369 #

6h fas

ting p

lasm

a fr

ee fat

ty a

cids

(mm

ol/

L)

0.8

2±

0.2

0

0.9

6±

0.2

0

0.2

6±

0.1

0

0.4

6±

0.2

4 *

0.2

6±

0.1

1 #

6h fas

ting p

lasm

a tr

igly

cerides

(m

mol/

L)

0.9

2±

0.2

1

2.0

4±

0.7

2 *

0.6

8±

0.1

8

3.3

3±

1.1

9 *

0.7

8±

0.2

7 #

6h fas

ting p

lasm

a H

DL

chole

ster

ol (m

g/d

L)

71.0

±3.0

39.8

±8.3

*

112.3

±11.8

61.9

±6.7

*

108.9

±11.6

#

6h fas

ting p

lasm

a LD

L/VLD

L ch

ole

ster

ol (m

g/d

L)

12.4

±2.7

10.2

±3.9

15.6

±2.2

27.3

±7.0

*

17.8

±2.7

#

Hem

ato

crit (

%)

48.3

±2.8

47.4

±2.5

50.7

±3.0

47.9

±2.2

*

49.0

±1.3

Bod

y c

om

po

siti

on

Bodyw

eight

(g)

362±

23

397±

16 *

410±

27

478±

25 *

432±

30 #

Hea

rt w

eight

(g)

n.d

. n.d

. 1.1

9±

0.0

6

1.3

4±

0.1

5 *

1.2

8±

0.0

6

Live

r w

eight

(g)

n.d

. n.d

. 10.7

±1.0

14.2

±1.0

*

11.3

±2.0

#

Epid

ydim

al fa

t w

eight

(g)

n.d

. n.d

. 6.0

±0.8

11.4

±2.1

*

7.3

±1.5

#

Peri

renal

fat

wei

ght

(g)

n.d

. n.d

. 8.0

±1.5

16.7

±3.8

*

9.5

±1.8

#

Tib

ia len

gth

(m

m)

n.d

. n.d

. 42.0

±0.5

41.8

±1.0

41.4

±1.0

Heart

com

posi

tio

n

Cro

ss s

ectional

are

a (

2)

n.d

. n.d

. 372±

54

625±

105 *

389±

53 #

Dat

a ar

e m

ean±

SD

, n=

8-1

6,

4 w

eeks

: st

uden

t t-

test

, * p

<0.0

5 v

s. c

ontr

ol

die

t, 8

wee

ks:

one-w

ay A

NO

VA w

ith B

onfe

rroni post

-hoc

test

, * p

<0.0

5 v

s.

contr

ol die

t, #

p<

0.0

5 v

s. w

este

rn d

iet.

n.d

., n

on d

eter

min

ed.


Figure 5.1: Oral glucose tolerance test after 8 weeks of diet feeding without sevoflurane

exposure

Blood glucose (A, C), plasma insulin (B, D) and area under the curve (AUC; E, F) during an oral

glucose tolerance test in rats after 4 and 8 weeks of control diet (CD), western diet (WD) or diet

reversion (REV) feeding. Data are mean±SD, n=6, t-test, one- and two-way ANOVA with repeated

measures and Bonferroni post-hoc analyses, * p<0.05 vs. CD, # p<0.05 vs. WD.

5

96 Chapter 5 Tab

le 5

.2:

Myoca

rdia

l fu

nct

ion

aft

er

4 a

nd

8 w

eeks

of

die

t in

terv

en

tion

wit

ho

ut

sevofl

ura

ne e

xp

osu

re

4

weeks

8

weeks

C

on

trol

die

t W

est

ern

die

t C

on

trol

die

t W

est

ern

die

t R

evers

ion

Hea

rt r

ate

(bpm

) 465±

36

488±

23

443±

31

491±

16 *

474±

16

LV m

ass

(mg)

786±

95

820±

57

868±

118

955±

87

805±

62 #

LV

dim

en

sion

s

Dia

stol

ic lum

en d

iam

eter

(m

m)

6.6

±0.4

5.9

±0.5

*

6.7

±0.4

6.0

±0.5

*

6.1

±0.5

Sys

tolic

lum

en d

iam

eter

(m

m)

1.7

±0.2

2.7

±0.4

*

2.0

±0.3

2.6

±0.4

*

1.7

±0.3

#

Dia

stol

ic w

all th

ickn

ess

(mm

) 1.7

±0.1

2.0

±0.2

*

1.7

±0.1

2.3

±0.2

*

1.9

±0.2

#

Sys

tolic

wal

l th

ickn

ess

(mm

) 3.7

±0.2

3.4

±0.2

*

3.7

±0.1

3.6

±0.1

3.8

±0.2

#

LV

dia

stoli

c fu

nct

ion

E flo

w (

cm/s

) 99.8

±15.5

109.3

±17.0

98.3

±17.3

110.9

±10.9

115.0

±9.5

E d

ecel

erat

ion t

ime

(ms)

23.9

±7.4

31.1

±4.2

*

24.5

±2.4

29.1

±5.1

23.4

±6.3

#

Isovo

lum

ic r

elaxa

tion t

ime

(ms)

13.8

±2.3

20.3

±4.2

*

n.d

. n.d

. n.d

.

Dat

a ar

e m

ean±

SD

, n=

6-1

0,

4 w

eeks

: st

uden

t t-

test

, * p

<0.0

5 v

s. c

ontr

ol die

t, 8

wee

ks:

one-w

ay A

NO

VA w

ith B

onfe

rroni post

-hoc

anal

yses

, * p

<0.0

5

vs.

contr

ol die

t, #

p<

0.0

5 v

s. w

este

rn d

iet.

LV,

left

ven

tric

ula

r.


Sevoflurane-induced cardiodepression is only present in western diet-fed

rats

Exposure to sevoflurane did not alter plasma levels of glucose, insulin, free fatty

acids, triglycerides, LDL/VLDL cholesterol and HDL cholesterol in all 3 diet groups

(Figure 5.3A-F). After 8 weeks of diet exposure, sevoflurane exerted no

cardiodepressive effects in control rats as indicated by maintained factional

shortening and fractional area change. However, in rats fed a western diet,

sevoflurane induced an additional reduction in myocardial contractility, and reducing

caloric intake could restore this (Figure 5.2C and D). These findings suggest that diet

composition and/or the cardiometabolic phenotype of the rat modulate the

interaction of sevoflurane with myocardial function.

Figure 5.2: Systolic function after diet feeding with and without sevoflurane exposure

Fractional shortening (A) and fractional area change (B) in rats after 4 and 8 weeks of control diet

(CD), western diet (WD) or diet reversion (REV) feeding. Fractional shortening (C) and fractional

area change (D) during baseline and sevoflurane in rats after 8 weeks of control diet (CD), western

diet (WD) or diet reversion (REV) feeding. Data are mean±SD, n=4-6, two-way ANOVA with

Bonferroni post-hoc analyses, * p<0.05 vs. CD, # p<0.05 vs. WD, $ p<0.05 vs. baseline.

5

98 Chapter 5

Figure 5.3: Plasma levels after sevoflurane exposure

Plasma glucose (A), insulin (B), free fatty acid (C), triglyceride (D), LDL/VLDL cholesterol (E) and

HDL cholesterol (F) levels during baseline and sevoflurane in rats after 8 weeks of control diet

(CD), western diet (WD) or diet reversion (REV) feeding. Data are mean±SD, n=4-6, two-way

ANOVA with Bonferroni post-hoc analyses, * p<0.05 vs. CD, # p<0.05 vs. WD.


Sevoflurane induced molecular alterations

Figure 5.4 shows the effects of sevoflurane and type of diet on phosphorylation

D) and the phosphorylation of two proteins involved in myocardial calcium handling

(PLB (panel E) and cTnI (panel F)).

The absent effect of sevoflurane exposure on myocardial function in control animals

protein expression of GLUT4, phospholamban or cTnI. Sevoflurane did not alter

SERCA2a protein expression or phosphorylation of myosin binding protein-C and

troponin T (data not shown).

(p=0.05), which tended to reduce towards control levels after diet reversion. Akt and

western diet-

phosphorylation in control rats compared to baseline, which was blunted in western

diet-fed rats and restored after diet reversal, however, these alterations failed to

reach statistical significance (Figure 5.4A and 4C). Sevoflurane induced a non-

on in western diet-fed rats only.

WD-feeding significantly increased phosphorylation of phospholamban (PLB) and

increased phosphorylation of cardiac troponin I (cTnI). PLB phosphorylation was

normalized after diet reversal, while cTnI phosphorylation was still high in the diet

reversal group. Compared to baseline, sevoflurane significantly reduced PLB

phosphorylation in WD-fed rats, which was restored after diet reversal. Sevoflurane

also significantly reduced phosphorylation of cTnI in WD-fed rats (p=0.05), and a

similar pattern was observed in the diet reversion group (Figure 5.4F). Diet or

sevoflurane did not alter SERCA2a protein expression or phosphorylation of myosin

binding protein-C and troponin T after western diet exposure (data not shown).

5

100 Chapter 5

Figure 5.4: Molecular alterations in substrate metabolism and calcium handling after

sevoflurane exposure

Phosphorylation of Akt (A), glycogen synthase kinase 3 B), AMP activated protein kinase

C), protein expression of glucose transporter 4 (GLUT4, D), phosphorylation of

phospholamban (PLB, E) and Pro-Q Diamond Phosphoprotein Stain determined cardiac troponin I

(cTnI, F) during baseline and sevoflurane in rats after 8 weeks of control diet (CD), western diet

(WD) or diet reversion (REV) feeding. Data are mean±SD, n=4-6, two-way ANOVA with Bonferroni

post-hoc analyses, * p<0.05 vs. CD, # p<0.05 vs. WD, $ p<0.05 vs. baseline.

Akt

Akt-Ser473p

CD W D REV

b s b s b s

Akt

Akt-Ser473p

CD W D REV

b s b s b s

G SK3

GSK3 -Ser9p

CD W D REV

b s b s b s

G SK3

GSK3 -Ser9p

CD W D REV

b s b s b s

AM PK

AM PK -Thr172p

CD W D REV

b s b s b s

AM PK

AM PK -Thr172p

CD W D REV

b s b s b s

Actin

GLUT4

CD W D REV

b s b s b s

Actin

GLUT4

CD W D REV

b s b s b s

PLB

PLB-Ser16p

CD W D REV

b s b s b s

PLB

PLB-Ser16p

CD W D REV

b s b s b s

cTnI

cTnI-p

CD W D REV

b s b s b s

cTnI

cTnI-p

CD W D REV

b s b s b s


Discussion

Sevoflurane exerts cardiodepressive properties in healthy subjects, therefore its

perioperative use may lead to hemodynamic alterations. Although it has been

suggested that the interaction of sevoflurane with the heart is additionally altered by

cardiometabolic diseases, most available studies are restricted to in vitro

observations and experimental models for type I diabetes mellitus. With the growing

epidemic of obesity and type 2 diabetes mellitus it might be interesting to

understand the influence of this condition on the interaction of sevoflurane with

myocardial function.

Unexpectedly, in an experimental rat model we could not show a direct effect of

sevoflurane on myocardial contractility. However, in western diet-induced obesity,

which was accompanied by glucose intolerance, hyperglycemia, hyperinsulinemia,

dyslipidemia and myocardial dysfunction, sevoflurane exerted cardiodepressive

effects. Interestingly, lowering caloric intake following western diet exposure could

reverse these effects of sevoflurane on myocardial function. These results suggest

that sevoflurane has negative inotropic effects on myocardial function in

metabolically altered hearts, and that a reduction of caloric intake may improve the

effects of sevoflurane on myocardial function. Our results therefore implicate that

diet modification may be explored as an intervention to preserve perioperative

myocardial function in subjects with cardiometabolic disease.

The present study showed that western diet feeding resulted in a prediabetic

phenotype accompanied by myocardial dysfunction. Our results confirm previous

findings showing impaired systolic function after 8 weeks of diet feeding.18

Interestingly, the present study showed that lowering caloric intake completely

normalized diet-induced T2DM. In small rodents, reducing dietary intake has been

shown to reverse diet-induced obesity8-11 and T2DM.12;13 However, it is also shown

that diet-induced obesity was not reversed by withdrawal of an energy dense

diet.23;24 Importantly, our study showed that lowering caloric intake also improved

diet-induced systolic as well as diastolic dysfunction, which is in agreement with the

clinical observation that in obese and T2DM patients a low caloric diet decreased

myocardial fatty acid uptake14 and improved diastolic function,15 respectively. Taken

together, lowering caloric intake normalized the prediabetic phenotype induced by

short-term western diet feeding and improved myocardial function.

Exposure to sevoflurane further impaired systolic function in diet-induced

prediabetes, but not in healthy rats. In contrast, sevoflurane caused a reduction in

left ventricular systolic function at concentrations of 2% and higher compared to

isoflurane in healthy mice.25 An important difference with previous findings is that in

the present study all rats were primary sedated with S-ketamine and diazepam. As

S-ketamine and diazepam may have intrinsic cardiodepressive effects,26;27 this might

5

102 Chapter 5

have blurred the additional effect of sevoflurane on the heart in control animals.

Limited data are available on the effects of sevoflurane in metabolic altered hearts.

In papillary muscles from type 1 diabetic rats, decreased sensitivity to the negative

inotropic interaction of halothane, enflurane and isoflurane was found,5 whereas

sevoflurane resulted in greater negative inotropic effects compared to controls.4 In

contrast, it has been shown that in single ventricular myocytes from type 1 diabetic

rats the inotropic effects of halothane, isoflurane, desflurane and sevoflurane were

not altered compared to controls.6 As far as we know we are the first to show the

negative inotropic effects of sevoflurane on hearts of diet-induced prediabetic rats.

A suggested underlying mechanism in the effect of sevoflurane on myocardial

function is substrate availability and metabolism,28 which is known to be altered by

T2DM.18;20;21 Unexpectedly, sevoflurane did not affect plasma glucose, insulin and

lipid levels. In humans, there is indirect evidence that volatile anesthesia increases

blood glucose; however, these observations may be biased by confounding factors

such as surgical stress.29 Compared to non-anesthetized rats without surgical stress,

sevoflurane increased plasma glucose levels, but not plasma insulin levels.30 It is

possible that glucose levels did not increase in the present study due to short

duration and low concentration of sevoflurane used, but also differences in

nutritional status may exist. Moreover, in the present study rats were sedated with

S-ketamine and diazepam. Ketamine31 and diazepam32 are known to increase blood

glucose levels, which could have abolished the additional effect of sevoflurane on

blood glucose levels.

Sevoflurane may also affect proteins related to myocardial substrate metabolism.

Akt, which regulates translocation of glucose transporter 4 (GLUT4) to the

sarcolemma for glucose uptake, is increased during sevoflurane in the isolated

ischemic rat heart.33 Moreover, sevoflurane enhances GLUT4 expression in lipid rafts,

increases glucose oxidation and decreases fatty acid oxidation after ischemia and

reperfusion injury in isolated working rat hearts.34 Although not in ischemic and

reperfused hearts, the present study showed no significant differences in proteins

related to myocardial substrate metabolism.

Another possible mechanism is altered calcium handling, which also seems a

common target in T2DM and sevoflurane. Sevoflurane reduces myocardial calcium

availability, but increases sarcoplasmic reticulum calcium content.35 In contrast,

sevoflurane decreased fractional calcium release, but maintained sarcoplasmic

reticulum calcium content.36 In papillary muscles of type 1 diabetic rats, sevoflurane

decreased myofilament calcium sensitivity to a greater extent than control rats.4 In

the present study, western diet feeding blunted phosphorylation of phospholamban

and cardiac troponin I during sevoflurane, but not SERCA2a protein expression. In

preconditioned hearts, phosphorylation of phospholamban and SERCA2a protein

expression were unaltered after ischemia and reperfusion injury;37 however, no data

were reported for non-ischemic hearts. Our data indicate that phospholamban and


cardiac troponin I might play a role in impaired systolic function in prediabetic rats

during sevoflurane.

In conclusion, sevoflurane induced cardiodepression in prediabetic rats, whereas

lowering caloric intake restored myocardial function during sevoflurane. These

results suggest that a change in dietary intake could be a potential intervention to

support the preservation of myocardial function during sevoflurane in subjects with

obesity and type 2 diabetes mellitus.

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104 Chapter 5

References

1. Harkin CP, Pagel PS, Kersten JR, Hettrick DA, Warltier DC: Direct negative inotropic and

lusitropic effects of sevoflurane. Anesthesiology 1994, 81:156-167.

2. Pagel PS, Kampine JP, Schmeling WT, Warltier DC: Influence of volatile anesthetics on

myocardial contractility in vivo: desflurane versus isoflurane. Anesthesiology 1991, 74:900-907.

3. Malan TP, Jr., DiNardo JA, Isner RJ, Frink EJ, Jr., Goldberg M, Fenster PE, Brown EA, Depa R,



4. David JS, Tavernier B, Amour J, Vivien B, Coriat P, Riou B: Myocardial effects of halothane and

sevoflurane in diabetic rats. Anesthesiology 2004, 100:1179-1187.

5. Hattori Y, Azuma M, Gotoh Y, Kanno M: Negative inotropic effects of halothane, enflurane, and

isoflurane in papillary muscles from diabetic rats. Anesth Analg 1987, 66:23-28.

6. Graham M, Qureshi A, Noueihed R, Harrison S, Howarth FC: Effects of halothane, isoflurane,

sevoflurane and desflurane on contraction of ventricular myocytes from streptozotocin-induced

diabetic rats. Mol Cell Biochem 2004, 261:209-215.

7. Mitchell JR, Beckman JA, Nguyen LL, Ozaki CK: Reducing elective vascular surgery perioperative


8. Hill JO, Dorton J, Sykes MN, DiGirolamo M: Reversal of dietary obesity is influenced by its

duration and severity. Int J Obes 1989, 13:711-722.

9. Rogers PJ: Returning 'cafeteria-fed' rats to a chow diet: negative contrast and effects of obesity

on feeding behaviour. Physiol Behav 1985, 35:493-499.

10.Bartness TJ, Polk DR, McGriff WR, Youngstrom TG, DiGirolamo M: Reversal of high-fat diet-

induced obesity in female rats. Am J Physiol 1992, 263:R790-R797.

11.Walks D, Lavau M, Presta E, Yang MU, Bjorntorp P: Refeeding after fasting in the rat: effects of

dietary-induced obesity on energy balance regulation. Am J Clin Nutr 1983, 37:387-395.

12.Parekh PI, Petro AE, Tiller JM, Feinglos MN, Surwit RS: Reversal of diet-induced obesity and

diabetes in C57BL/6J mice. Metabolism 1998, 47:1089-1096.

13.Harris RB, Martin RJ: Changes in lipogenesis and lipolysis associated with recovery from

reversible obesity in mature female rats. Proc Soc Exp Biol Med 1989, 191:82-89.

14.Viljanen AP, Karmi A, Borra R, Parkka JP, Lepomaki V, Parkkola R, Lautamaki R, Jarvisalo M,

Taittonen M, Ronnemaa T, Iozzo P, Knuuti J, Nuutila P, Raitakari OT: Effect of caloric restriction

on myocardial fatty acid uptake, left ventricular mass, and cardiac work in obese adults. Am J

Cardiol 2009, 103:1721-1726.

15.Hammer S, Snel M, Lamb HJ, Jazet IM, van der Meer RW, Pijl H, Meinders EA, Romijn JA, de

Roos A, Smit JW: Prolonged caloric restriction in obese patients with type 2 diabetes mellitus

decreases myocardial triglyceride content and improves myocardial function. J Am Coll Cardiol

2008, 52:1006-1012.

16.Mitchell JR, Verweij M, Brand K, van d, V, Goemaere N, van den Engel S, Chu T, Forrer F, Muller

C, de JM, van IW, IJzermans JN, Hoeijmakers JH, de Bruin RW: Short-term dietary restriction

and fasting precondition against ischemia reperfusion injury in mice. Aging Cell 2010, 9:40-53.

17.Kilkenny C, Browne WJ, Cuthill IC, Emerson M, Altman DG: Improving bioscience research

reporting: the ARRIVE guidelines for reporting animal research. PLoS Biol 2010, 8:e1000412.
















8:39.

22.Zaremba R, Merkus D, Hamdani N, Lamers JML, Paulus WJ, Remedios C, Duncker DJ, Stienen

GJM, Van der Velden J: Quantitative analysis of myofilament protein phosphorylation in small

cardiac biopsies. proteomics Clin Appl 2007, 1:1285-1290.

23.Llado I, Proenza AM, Serra F, Palou A, Pons A: Dietary-induced permanent changes in brown

and white adipose tissue composition in rats. Int J Obes 1991, 15:415-419.

24.Rolls BJ, Rowe EA, Turner RC: Persistent obesity in rats following a period of consumption of a

mixed, high energy diet. J Physiol 1980, 298:415-427.

25.Gentry-Smetana S, Redford D, Moore D, Larson DF: Direct effects of volatile anesthetics on

cardiac function. Perfusion 2008, 23:43-47.

26.Plante E, Lachance D, Roussel E, Drolet MC, Arsenault M, Couet J: Impact of anesthesia on

echocardiographic evaluation of systolic and diastolic function in rats. J Am Soc Echocardiogr

2006, 19:1520-1525.

27.Stein AB, Tiwari S, Thomas P, Hunt G, Levent C, Stoddard MF, Tang XL, Bolli R, Dawn B: Effects

of anesthesia on echocardiographic assessment of left ventricular structure and function in rats.

Basic Res Cardiol 2007, 102:28-41.

28.van den Brom CE, Bulte CSE, Loer SA, Bouwman RA, Boer C: Diabetes, perioperative ischaemia

and volatile anaesthetics: Consequences of derangements in myocardial substrate metabolism.

Cardiovasc Diabetol 2013, In press.

29.Lattermann R, Schricker T, Wachter U, Georgieff M, Goertz A: Understanding the mechanisms

by which isoflurane modifies the hyperglycemic response to surgery. Anesth Analg 2001,

93:121-127.

30.Zuurbier CJ, Keijzers PJ, Koeman A, Van Wezel HB, Hollmann MW: Anesthesia's effects on

plasma glucose and insulin and cardiac hexokinase at similar hemodynamics and without major

surgical stress in fed rats. Anesth Analg 2008, 106:135-42.

31.Saha JK, Xia J, Grondin JM, Engle SK, Jakubowski JA: Acute hyperglycemia induced by

ketamine/xylazine anesthesia in rats: mechanisms and implications for preclinical models. Exp

Biol Med (Maywood ) 2005, 230:777-784.

32.Yamada J, Sugimoto Y, Noma T: Involvement of adrenaline in diazepam-induced hyperglycemia

in mice. Life Sci 2000, 66:1213-1221.




32:1854-1861.




Anaesth 2011, 106:792-800.

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35.Hannon JD, Cody MJ: Effects of volatile anesthetics on sarcolemmal calcium transport and


1464.

36.Davies LA, Gibson CN, Boyett MR, Hopkins PM, Harrison SM: Effects of isoflurane, sevoflurane,

and halothane on myofilament Ca2+ sensitivity and sarcoplasmic reticulum Ca2+ release in rat

ventricular myocytes. Anesthesiology 2000, 93:1034-1044.

37.An J, Rhodes SS, Jiang MT, Bosnjak ZJ, Tian M, Stowe DF: Anesthetic preconditioning enhances

Ca2+ handling and mechanical and metabolic function elicited by Na+-Ca2+ exchange inhibition

in isolated hearts. Anesthesiology 2006, 105:541-549.

CE van den Brom, C Boer, RPF van den Akker, SA Loer,

RA Bouwman

6Western diet modulates the susceptibility

of the heart to ischemic injury and

sevoflurane-induced cardioprotection in

rats

Effect of diet and sevoflurane on ischemic injury 111

Abstract

Introduction

Sevoflurane is known for its cardioprotective effects during myocardial ischemia and

reperfusion, which however seems blunted in the presence of type 2 diabetes

mellitus. In this study we investigated whether a reduction in caloric intake reverses

the impact of western diet on the cardioprotective potency of sevoflurane in rats

subjected to ischemic injury.

Methods

Male Wistar rats were exposed to a western diet (WD) or control diet (CD). A third

group of WD fed rats reversed after four weeks to CD for 4 consecutive weeks. After

8 weeks, rats underwent 40 minutes of coronary occlusion followed by 120 minutes

of reperfusion with or without 3x 5 min sevoflurane (2 vol%) preconditioning. An

extra group of CD-fed rats underwent myocardial infarction during hyperinsulinemic

euglycemic clamping.

Results

WD feeding resulted in a prediabetic phenotype with obesity and hyperinsulinemia.

Sevoflurane exerted cardioprotective effects in control rats, resulting in a reduction

of infarct size by 59% (p<0.001). Unexpectedly, WD feeding itself reduced infarct

size by 43% (p<0.05) compared to control rats, while sevoflurane even enlarged

infarct size in WD fed rats (31%, p<0.05). Reversion of WD to CD resulted in

normalization of obesity and hyperinsulinemia, but did not affect infarct size and the

protective effect of sevoflurane when compared to WD-fed rats. Interestingly,

sevoflurane prior to myocardial infarction tended to increase insulin levels in control

rats, while sevoflurane exposure in WD-fed rats undergoing myocardial infarction

resulted in a decrease of plasma insulin levels. Our data suggested that the

protective effects of diet feeding may be mediated by insulin. Indeed, exposure of

rats to a hyperinsulinemic intervention also reduced infarct size after myocardial

ischemia.

Conclusion

Unexpectedly, western diet itself had a protective effect against ischemic injury, and

lowering caloric intake did not alter this effect. Sevoflurane anesthesia exerted

cardioprotective effects in control rats, but this effect was blunted in prediabetic rats.

Our data further suggest that the protective effects of sevoflurane in control rats or

western diet are mediated by insulin.

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112 Chapter 6

Introduction

Cardiometabolic alterations due to excessive dietary intake, insulin resistance and

type 2 diabetes mellitus (T2DM) increase the risk for perioperative myocardial

ischemia.1 In particular, patients with T2DM are more likely to develop coronary

artery disease and myocardial ischemia2 and have an increased cardiovascular

complication rate after major non-cardiac surgery.3

The volatile anesthetic sevoflurane exerts protective effects that enhance

perioperative preservation of the heart.4;5 We previously showed that sevoflurane

reduced the impact of ischemic myocardial injury.6;7 However, there is growing

evidence that these cardioprotective properties are blunted in case of obesity and/or

T2DM. Sevoflurane-induced postconditioning was blocked in hyperglycemic8 and

obese/insulin resistant Zucker rats9 after myocardial ischemia and reperfusion.

Moreover, obesity suppressed sevoflurane preconditioning-induced

cardioprotection.10

Preoperative lifestyle interventions such as lowering caloric intake to induce weight

loss and improve insulin sensitivity may be an attractive approach to improve

perioperative myocardial function in obese or diabetic subjects. We previously

showed that lowering caloric intake restored the cardiodepressive effects of

sevoflurane in western diet-induced prediabetic rats (van den Brom et al.,

unpublished results). This suggests that the myocardial response to sevoflurane

depends on the cardiometabolic state that may be altered by changes in dietary

intake. It remains unknown whether these beneficial effects of caloric restriction also

affect the cardioprotective effect of sevoflurane in the cardiometabolic altered heart.

In this study we investigated whether reversal of western diet-induced prediabetes

by caloric restriction restores the cardioprotective potency of sevoflurane in rats

subjected to ischemic injury.


Material and methods


All animal experiments were approved by the Institutional Animal Care and Use

Committee of the VU University, and were conducted following the European

Convention for the Protection of Vertebrate Animals used for Experimental and Other

Scientific Purposes, and the guide for the Care and Use of Laboratory Animals.

The first part of the study was performed in male Wistar rats (n=144, baseline

body weight: 263±1 g; Charles River). Rats were exposed to a western diet in

combination with sucrose water (20%) (WD, n=98) or control diet (CD, n=46) for a

period of 8 weeks. Four weeks after the start of diet exposure, 48 WD-fed rats

reversed to CD for 4 weeks (Figure 6.1A).

Rats were housed in a temperature-controlled room (20-23°C; 40-60% humidity)

under a 12/12h light/dark cycle starting at 6.00 am. Body weight was determined on

a weekly basis. After 8 weeks of diet exposure, rats underwent myocardial infarction.

The second part of the study included 14 male Wistar rats (263±3 g) that were

exposed to control diet. After 8 weeks, these rats underwent a myocardial infarction

during hyperinsulinemia. After sacrifice, arterial blood was collected for plasma

determinations and hearts were removed and stored at -20°C until further analysis.

Diets

Control diet (Teklad 2016, Harlan, Horst, the Netherlands) consisted of 20 %kcal


sugars), whereas western diet (D12451, Research Diets, New Brunswick, NJ)

consisted of 20 %kcal protein, 45 %kcal fat and 35 %kcal carbohydrates (291

kcal/kg starch, 691 kcal/kg sugars) with 20% sucrose water (800 kcal/kg), totally

containing 3300 kcal/kg and 4857 kcal/kg for CD and WD with sucrose water,

respectively.

Surgery

After 8 weeks of diet exposure, rats were anesthetized with 125 mg/kg S-ketamine

(Ketanest®, Pfizer, the Netherlands) and 4 mg/kg diazepam (Centrafarm, the

Netherlands) intraperitoneally and were intubated and mechanically ventilated (UNO,

the Netherlands; positive end-expiratory pressure, 1-2 cm H2O; respiratory rate,

~65 breaths/min; tidal volume, ~10 ml/kg) with oxygen-enriched air (40% O2/60%

N2). Anesthesia was maintained by continuous infusion of 40 mg/kg/h S-ketamine

and 1 mg/kg/h diazepam intravenously via the tail vein or the right jugular vein.

The respiratory rate was adjusted to maintain pH and carbon dioxide within

physiological limits. Body temperature was maintained stabile (37.1±0.05°C) by

using a warm water underbody heating pad.

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114 Chapter 6

The left carotid artery was cannulated for blood sampling for blood gas analyses

(ABL50, radiometer, Copenhagen, Denmark) and for measurements of arterial blood

pressure (Safedraw Transducer Blood Sampling Set, Argon Medical Devices, Texas,

USA). Arterial blood pressure, ECG and heart rate were continuously recorded using

PowerLab software (PowerLab 8/35, Chart 7.0; ADInstruments Pty, Ltd., Castle Hill,

Australia). Mean arterial blood pressure was calculated according the following

formula: 2/3*diastolic blood pressure + 1/3* systolic blood pressure.

Myocardial infarction

Left thoracotomy was performed between the fourth and fifth rib. A ligature

(Prolene® 6-0; Ethicon LLC, San Lorenzo, Puerto Rico) was placed around the left

anterior descending coronary artery (LAD) with a special ligation device that allowed

to pull the suture in one simple movement in order to reduce the chance of

preconditioning.11 Successful coronary occlusion was verified by ECG changes and

ischemia was maintained for 40 min and followed by 120 min of reperfusion (MI)

(Figure 6.1B). Rats in the control (CON) group received a sham surgery were the

LAD was not occluded. Sevoflurane (AbbVie, the Netherlands) intervention was

induced before ischemia and reperfusion by three periods of 5 minutes exposure to

2% (v/v) sevoflurane, interspersed with washout periods of 5 minutes (MI+SEVO).

In total, n=144 rats were included, from which n=122 rats survived the surgery

(84.7 %). Per diet group the survival rate was 87.0 %, 80.0 % and 85.4 % for CD-,

WD- and REV-fed rats, respectively.

Hyperinsulinemic euglycemic clamp

Rats in the second part of the study were subjected to the same ischemia and

reperfusion protocol during a hyperinsulinemic euglycemic clamp. In total, n=14 rats

were included, from which n=11 rats survived the surgery (78.6 %). Surgery was

performed similarly as described above; only anesthesia was infused intravenously

via the femoral vein due to blood glucose measurements via the tail vein. The

hyperinsulinemic euglycemic clamp was initiated by 3 minutes insulin priming at an

infusion rate of 120 mU/kg/min (Novorapid®, Novo Nordisk, Denmark), followed by

constant infusion of insulin at a rate of 12 mU/kg/min via the jugular vein as

described before.12 Blood glucose was measured every 5 minutes from tail bleeds

with a Precision Xceed Blood Glucose monitoring system (MediSense, UK).

Simultaneously, a 20% glucose solution was infused at a variable rate to maintain

euglycemia. After ± 90 minutes, glucose disposal rate (M-value, mg/kg/min) was

calculated as the average glucose infusion rate during 30 minutes. To prove

hyperinsulinemia, plasma insulin levels were determined during the steady state,

ischemia and reperfusion phase. The M/I index, which is a measure for insulin

sensitivity, was calculated by the amount of glucose metabolized per unit of plasma

insulin.


Figure 6.1: Experimental protocol

A) Rats were fed a control diet (CD) or western diet with sucrose water (WD) for 8 weeks. A third

group of WD fed rats reversed after 4 weeks to CD for 4 consecutive weeks (REV). B) After 8

weeks of diet exposure, animals underwent left coronary artery occlusion for 40 min followed by

120 min of reperfusion without (MI) or with 3x 5 min sevoflurane (s) exposure (MI+SEVO). C)

After 8 weeks of exposure to CD, animals underwent left coronary artery occlusion for 40 min

followed by 120 min of reperfusion during hyperinsulinemia (MI+INS).

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116 Chapter 6

Evans blue and TTC staining

After 120 min of reperfusion, the hearts were excised and the aorta was cannulated.

The LAD was re-occluded and 0.2 % Evans Blue (Sigma, St. louis, MO) was infused

to stain the non-ischemic myocardium and leaving the area at risk unstained. After

rinsing with 0.9% NaCl, hearts were stored at -20°C.

For determination of infarct size, frozen hearts were cut in slices of 1 mm,

incubated for 15 min in 2,3,5-triphenyl tetrazolium chloride (TTC; Sigma, St. Louis,

MO) solution at 37°C, followed by fixation in 4% formaldehyde (Klinipath, Duiven,

the Netherlands). Slices were scanned and the area at risk and infarct size were

determined in each slice using ImageJ (1.42q, National Institue of Health). The

infarct size is presented as the percentage of the area at risk.

Plasma measurements

Plasma glucose (Abcam, Cambridge, MA), insulin (Millipore, St. Charles, MO), free

fatty acid (WAKO NEFA-HR, Wako Pure Chemical Industries, Osaka, Japan),

triglyceride (TG; Sigma, St. Louis, MO) and HDL and LDL/VLDL cholesterol (Abcam,

Cambridge, MA) levels were measured from arterial blood as described previously.12-

15


Data were analyzed using Graphpad Prism 5.0 (La Jolla, USA) and presented as

mean±SD. Statistical analyses were performed using one-way ANOVA with

Bonferroni post-hoc analysis and two-way ANOVA with Bonferroni post-hoc analysis

(with repeated measurements) for additional interventions, where p<0.05 was

considered as statistically significant.


Results

Lowering caloric intake normalized obesity and hyperinsulinemia

After 8 weeks of western diet (WD) feeding, bodyweight and plasma insulin were

significantly increased compared to control diet (CD)-fed rats (Table 6.1). Plasma

glucose, triglycerides and free fatty acids tended to increase, whereas HDL

cholesterol tended to decrease in WD- compared to CD-fed rats (Table 6.1).

LDL/VLDL cholesterol remained unchanged (Table 6.1). Diastolic blood pressure,

systolic blood pressure, mean arterial pressure and heart rate were similar among

groups (Table 6.1). Reversion to control diet resulted in normalized bodyweight and

plasma levels of insulin, triglycerides and HDL cholesterol when compared to WD-fed

rats (Table 6.1).

Table 6.1: Characteristics of rats after 8 weeks of diet feeding

Control diet Western diet Reversion

Bodyweight (g) 421±22 450±27 * 431±26 #

Plasma insulin (pmol/L) 1274±621 2165±856 * 1006±559 #

Plasma glucose (mmol/L) 12.9±5.7 16.9±9.9 11.0±3.5

Plasma free fatty acids (mmol/L) 0.19±0.09 0.24±0.19 0.18±0.10

Plasma triglycerides (mmol/L) 1.76±0.66 2.23±0.70 1.46±0.44 #

Plasma HDL cholesterol (mg/dL) 49.4±18.2 38.5±10.5 63.5±7.7 #

Plasma LDL/VLDL cholesterol (mg/dL) 29.7±5.6 28.0±3.9 25.5±4.4

Systolic blood pressure (mmHg) 152±44 150±22 172±27

Diastolic blood pressure (mmHg) 110±39 111±23 130±18

Mean arterial pressure (mmHg) 124±40 124±22 144±21

Heart rate (bpm) 414±32 381±29 397±39

Data are mean±SD, n=5-11, two-way ANOVA with Bonferroni post-hoc analyses, * p<0.05 vs.

control diet, # p<0.05 vs. western diet.

Western diet feeding reduces myocardial ischemic injury

The area at risk after ischemia and reperfusion did not differ between groups (Figure

6.2A). Unexpectedly, WD-feeding reduced infarct size compared to control rats

(Figure 6.2B). Interestingly, these protective effects of western diet feeding on

ischemic injury persisted after lowering caloric intake following 4 weeks of WD-

feeding (REV group) (Figure 6.2B). Myocardial ischemia did not affect blood pressure

and heart rate in all diet groups (Figure 6.3A, C, E, G). During the reperfusion phase,

diastolic blood pressure and mean arterial pressure were only significantly reduced in

rats that underwent diet reversal (Figure 6.3B, D, F, H).

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118 Chapter 6

Figure 6.2: Myocardial ischemia and reperfusion injury

Area at risk (A), infarct size (B) and plasma insulin levels (C) after sham (CON), myocardial

infarction (MI) and myocardial infarction with sevoflurane (MI+SEVO) in rats after 8 weeks of

control diet (CD), western diet (WD) or reversion diet (REV) feeding. Data are mean±SD, n=5-11,

two-way ANOVA with Bonferroni post-hoc analyses, * p<0.05 diet effect, # p<0.05 sevoflurane


Figure 6.3: Hemodynamics during myocardial ischemia and reperfusion

Systolic blood pressure (A,B), diastolic blood pressure (C, D), mean arterial pressure (E, F) and

heart rate (G, H) after 40 minutes of ischemia (A, C, E, G) or 120 minutes of reperfusion (B, D, F,

H) in rats fed a control diet (CD), western diet (WD) or reversion diet (REV) for 8 weeks. Data are

mean±SD, one-way ANOVA with Bonferroni post-hoc analyses, * p<0.05 MI effect. CON, sham;

MI, myocardial infarction.

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Sevoflurane reduces ischemic injury in control rats

Sevoflurane-induced preconditioning reduced infarct size in CD-fed rats, but offered

no additional protection in WD- and REV-fed rats (Figure 6.2B). In particular, infarct

size was even higher in WD-fed rats compared to control rats after sevoflurane

exposure. Blood pressure and heart rate decreased during sevoflurane exposure, but

were partly restored during the washout periods without differences among groups

(Figure 6.4A-D).

Figure 6.4: Blood pressure and heart rate during sevoflurane exposure

Systolic blood pressure (B), diastolic blood pressure (B), mean arterial pressure (C) and heart rate

(D) in rats after 8 weeks of control diet (CD), western diet (WD) or reversion diet (REV) feeding.

Data are mean±SD, n=5-11.


Plasma insulin levels after surgery

Western diet feeding increased plasma insulin levels when compared to control rats,

while this hyperinsulinemic effect was absent in rats subjected to diet reversal. While

myocardial infarction had no effect on insulin levels in control rats, WD-fed rats

showed a hyperinsulinemic response upon cardiac ischemia and reperfusion. This

effect remained present in the diet reversal group, although to a lesser extent.

Sevoflurane prior to myocardial infarction tended to increase insulin levels in control

rats, while sevoflurane exposure in WD-fed rats undergoing myocardial infarction

decreased plasma insulin levels. In rats exposed to diet reversal the rise in insulin

levels during myocardial infarction was abolished after sevoflurane exposure (Figure

6.2C).

Myocardial infarction, but not MI+SEVO, significantly increased HDL cholesterol in

CD-fed rats, which was absent in WD- and REV-fed rats. MI and MI+SEVO had no

effect on plasma glucose, free fatty acids, triglycerides and LDL/VLDL cholesterol

(data not shown).

Hyperinsulinemia protects against ischemic injury in healthy rats

From the findings as shown in figure 6.2C it was hypothesized that the protective

effects of western diet feeding against myocardial ischemia may be the result of

increases in plasma insulin levels. In order to study the direct effects of

hyperinsulinemia on the cardioprotective effects of western diet, control diet-fed rats

were subjected to a hyperinsulinemic euglycemic clamp. Steady-state blood glucose

levels during hyperinsulinemic euglycemic clamping were 5.43±0.05 mmol/L,

whereas the glucose disposal rate (M-value) was 9.83±1.34 mg/kg/min in healthy

CD-fed rats. Plasma insulin levels were 9027±679 pmol/L and the insulin sensitivity

(M/I-index) was 1.19±0.22 in CD-fed rats during the steady state of the clamp.

Hyperinsulinemia combined with euglycemia resulted in a similar area at risk when

compared to control rats, but itself protected the heart against myocardial ischemia

(Figure 6.5A and B).

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122 Chapter 6

Figure 6.5: Myocardial ischemia and reperfusion injury during hyperinsulinemia

Area at risk (A) and infarct size (B) after sham (CON), myocardial infarction (MI) and myocardial

infarction during hyperinsulinemia (MI+INS) in rats after 8 weeks of control diet (CD) feeding. Data

are mean±SD, n=5-11, one-way ANOVA with Bonferroni post-hoc analyses, * p<0.05 vs. CON, #

p<0.05 vs. MI.


Discussion

The present study showed that short-term western diet feeding in rats resulted in a

prediabetic phenotype, and exerted an unexpected cardioprotective effect during

myocardial infarction, which was not abolished by lowering caloric intake.

Sevoflurane preconditioning protected the heart against myocardial ischemia in

healthy control rats. However, in diet-induced prediabetic rats, sevoflurane exposure

paradoxically increased infarct size. Sevoflurane prior to myocardial infarction tended

to increase insulin levels in control rats, while sevoflurane exposure in western diet-

fed rats undergoing myocardial infarction decreased plasma insulin levels. By

mimicking this hyperinsulinemic cardioprotective response using a hyperinsulinemic,

euglycemic clamp in control rats, we suspect a pivotal role for insulin underlying the

protective effects of sevoflurane or western diet against myocardial ischemia.

The present study demonstrated a protective effect of western diet feeding during

myocardial ischemia and reperfusion. We are not unique with respect to this

unexpected finding, as other groups also showed that high caloric diet feeding may

exert cardioprotective properties during ischemia and reperfusion.16-18 In contrast,

there are also reports available that show no effect10;19;20 or aggravation of ischemic

injury after diet feeding in rats.21-24 These conflicting findings may be due to the type

and severity of metabolic disease,25 the used ischemia and reperfusion protocol, the

duration of diet feeding and the composition of the diet.

Several mechanisms may explain diet-mediated cardioprotection, such as the

presence of a high percentage of carbohydrates.16-18 The present diet consisted

mainly of saturated fatty acids and a high percentage of sucrose. Jordan et al.

showed that 3 days or 4 weeks of fructose diet feeding both reduced infarct size,

while only the 4-week fructose intervention was associated with insulin resistance.

These findings suggest a protective effect of fructose.17 It is suggested that

carbohydrates, such as fructose or sucrose, may protect the heart against ischemia

and reperfusion injury via antioxidant mechanisms26,27 or via the response of the

heart to injury by increasing myocardial glucose metabolism to improve its energetic

efficiency.28,29 Glycolysis becomes an important source of energy due to its ATP-

generating ability in the absence of oxygen and is reported to be cardioprotective

during ischemia and reperfusion.30 Sucrose feeding may further stimulate myocardial

substrate metabolism. On the other hand, the high fat content of diets may also be

cardioprotective.31

Another explanatory mechanism is the presence of metabolic alterations, such as

hyperinsulinemia, during ischemic stress. Several studies showed a cardioprotective

effect of insulin in healthy rats during myocardial ischemia and reperfusion32-35 and

positive effects glucose, insulin and potassium (GIK) treatment in patients with

myocardial infarction.36;37 Moreover, infarct size was decreased in diet-induced obese

6

124 Chapter 6

compared to healthy rat hearts in absence of insulin, whereas the presence of insulin

in the coronary perfusate was cardioprotective in healthy and obese hearts,

suggesting the importance of insulin.22 In accordance with the literature, we showed

a cardioprotective effect of insulin during myocardial ischemia and reperfusion using

a hyperinsulinemic euglycemic clamp. Moreover, two independent observations in

our study provide further support for the protective role of insulin. First, sevoflurane

exposure of control rats tended to increase insulin levels, while this was associated

with myocardial protection against ischemic injury. Secondly, while western diet-

associated cardioprotection was paralleled by hyperinsulinemia, the absent

cardioprotective effect of sevoflurane exposure in western diet fed rats undergoing

myocardial infarction resulted in a decrease of plasma insulin levels. However,

further research is needed to discriminate the underlying cardioprotective

mechanism in our diet-induced prediabetic rat model.

Beside the unexpected cardioprotective effect of western diet, the present study

also demonstrated that sevoflurane preconditioning reduced infarct size in healthy

rats, but did had no additive cardioprotective effect in western diet-induced

prediabetic rats nor was affected by lowering caloric intake. Previously, it is shown

that hyperglycemia8 and obesity/insulin resistance9 abolished the cardioprotective

effect of sevoflurane. However, the generalizability of the prediabetic phenotype in

these rat models was limited.8;9 Moreover, Song et al. demonstrated in high fat diet-

induced obese rats that sevoflurane preconditioning-induced cardioprotection is

suppressed.10 Interestingly, our study shows that sevoflurane even increases infarct

size in prediabetic rats when compared to control rats, suggesting that western diet

feeding has negative effects on sevoflurane preconditioning.

In conclusion, western diet feeding resulted in an unexpected cardioprotective

effect during myocardial infarction, which was not affected by lowering caloric intake.

Sevoflurane preconditioning had no additive cardioprotective effect in prediabetic

rats nor was affected by lowering caloric intake. However, the combination of diet

and sevoflurane was less cardioprotective. Moreover, hyperinsulinemia was

cardioprotective in healthy rats, which might be an explanatory mechanism of the

cardioprotective effect of the diet. Future studies will discriminate between the

possible cardioprotective mechanisms of the diet and sevoflurane anesthesia.


References












2. Preis SR, Pencina MJ, Hwang SJ, D'Agostino RB, Sr., Savage PJ, Levy D, Fox CS: Trends in






Circulation 1999, 100:1043-1049.

4. Frassdorf J, De HS, Schlack W: Anaesthesia and myocardial ischaemia/reperfusion injury. Br J

Anaesth 2009, 103:89-98.

5. De Hert SG, Preckel B, Hollmann MW, Schlack WS: Drugs mediating myocardial protection. Eur

J Anaesthesiol 2009, 26:985-995.

6. Bouwman RA, Vreden MJ, Hamdani N, Wassenaar LE, Smeding L, Loer SA, Stienen GJ,

Lamberts RR: Effect of bupivacaine on sevoflurane-induced preconditioning in isolated rat

hearts. Eur J Pharmacol 2010, 647:132-138.

7. Lamberts RR, Onderwater G, Hamdani N, Vreden MJ, Steenhuisen J, Eringa EC, Loer SA,

Stienen GJ, Bouwman RA: Reactive oxygen species-induced stimulation of 5'AMP-activated

protein kinase mediates sevoflurane-induced cardioprotection. Circulation 2009, 120:S10-S15.

8. Huhn R, Heinen A, Weber NC, Hollmann MW, Schlack W, Preckel B: Hyperglycaemia blocks

sevoflurane-induced postconditioning in the rat heart in vivo: cardioprotection can be restored

by blocking the mitochondrial permeability transition pore. Br J Anaesth 2008, 100:465-471.

9. Huhn R, Heinen A, Hollmann MW, Schlack W, Preckel B, Weber NC: Cyclosporine A

administered during reperfusion fails to restore cardioprotection in prediabetic Zucker obese

rats in vivo. Nutr Metab Cardiovasc Dis 2010, 20:706-712.

10.Song T, Lv LY, Xu J, Tian ZY, Cui WY, Wang QS, Qu G, Shi XM: Diet-induced obesity suppresses

sevoflurane preconditioning against myocardial ischemia-reperfusion injury: role of AMP-

activated protein kinase pathway. Exp Biol Med (Maywood ) 2011, 236:1427-1436.

11.van Dijk A, Krijnen PA, Vermond RA, Pronk A, Spreeuwenberg M, Visser FC, Berney R, Paulus

WJ, Hack CE, van Milligen FJ, Niessen HW: Inhibition of type 2A secretory phospholipase A2

reduces death of cardiomyocytes in acute myocardial infarction. Apoptosis 2009, 14:753-763.





8:39.

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126 Chapter 6











16.Ivanova M, Janega P, Matejikova J, Simoncikova P, Pancza D, Ravingerova T, Barancik M:

Activation of Akt kinase accompanies increased cardiac resistance to ischemia/reperfusion in

rats after short-term feeding with lard-based high-fat diet and increased sucrose intake. Nutr

Res 2011, 31:631-643.

17.Jordan JE, Simandle SA, Tulbert CD, Busija DW, Miller AW: Fructose-fed rats are protected

against ischemia/reperfusion injury. J Pharmacol Exp Ther 2003, 307:1007-1011.

18.Joyeux-Faure M, Rossini E, Ribuot C, Faure P: Fructose-fed rat hearts are protected against

ischemia-reperfusion injury. Exp Biol Med (Maywood ) 2006, 231:456-462.

19.Mozaffari MS, Schaffer SW: Myocardial ischemic-reperfusion injury in a rat model of metabolic

syndrome. Obesity (Silver Spring) 2008, 16:2253-2258.

20.Thim T, Bentzon JF, Kristiansen SB, Simonsen U, Andersen HL, Wassermann K, Falk E: Size of

myocardial infarction induced by ischaemia/reperfusion is unaltered in rats with metabolic

syndrome. Clin Sci (Lond) 2006, 110:665-671.

21.Axelsen LN, Lademann JB, Petersen JS, Holstein-Rathlou NH, Ploug T, Prats C, Pedersen HD,

Kjolbye AL: Cardiac and metabolic changes in long-term high fructose-fat fed rats with severe

obesity and extensive intramyocardial lipid accumulation. Am J Physiol Regul Integr Comp

Physiol 2010, 298:R1560-R1570.

22.du Toit EF, Smith W, Muller C, Strijdom H, Stouthammer B, Woodiwiss AJ, Norton GR, Lochner

A: Myocardial susceptibility to ischemic-reperfusion injury in a prediabetic model of dietary-

induced obesity. Am J Physiol Heart Circ Physiol 2008, 294:H2336-H2343.

23.Morel S, Berthonneche C, Tanguy S, Toufektsian MC, Foulon T, de LM, de LJ, Boucher F: Insulin

resistance modifies plasma fatty acid distribution and decreases cardiac tolerance to in vivo

ischaemia/reperfusion in rats. Clin Exp Pharmacol Physiol 2003, 30:446-451.

24.Gonsolin D, Couturier K, Garait B, Rondel S, Novel-Chate V, Peltier S, Faure P, Gachon P, Boirie

Y, Keriel C, Favier R, Pepe S, DeMaison L, Leverve X: High dietary sucrose triggers

hyperinsulinemia, increases myocardial beta-oxidation, reduces glycolytic flux and delays post-

ischemic contractile recovery. Mol Cell Biochem 2007, 295:217-228.

25.van den Brom CE, Bulte CSE, Loer SA, Bouwman RA, Boer C: Diabetes, perioperative ischaemia

and volatile anaesthetics: Consequences of derangements in myocardial substrate metabolism.

Cardiovasc Diabetol 2013, In press.

26.Yoshida T, Watanabe M, Engelman DT, Engelman RM, Schley JA, Maulik N, Ho YS, Oberley TD,

Das DK: Transgenic mice overexpressing glutathione peroxidase are resistant to myocardial

ischemia reperfusion injury. J Mol Cell Cardiol 1996, 28:1759-1767.

27.Dhalla NS, Elmoselhi AB, Hata T, Makino N: Status of myocardial antioxidants in ischemia-

reperfusion injury. Cardiovasc Res 2000, 47:446-456.

28.Stanley WC, Recchia FA, Lopaschuk GD: Myocardial substrate metabolism in the normal and

failing heart. Physiol Rev 2005, 85:1093-1129.

29.Lopaschuk GD, Ussher JR, Folmes CD, Jaswal JS, Stanley WC: Myocardial fatty acid metabolism


in health and disease. Physiol Rev 2010, 90:207-258.

30.Vanoverschelde JL, Janier MF, Bakke JE, Marshall DR, Bergmann SR: Rate of glycolysis during

ischemia determines extent of ischemic injury and functional recovery after reperfusion. Am J

Physiol 1994, 267:H1785-H1794.

31.Knapp M: Cardioprotective role of sphingosine-1-phosphate. J Physiol Pharmacol 2011, 62:601-

607.

32.Gao F, Gao E, Yue TL, Ohlstein EH, Lopez BL, Christopher TA, Ma XL: Nitric oxide mediates the

antiapoptotic effect of insulin in myocardial ischemia-reperfusion: the roles of PI3-kinase, Akt,

and endothelial nitric oxide synthase phosphorylation. Circulation 2002, 105:1497-1502.



Circ Res 2001, 89:1191-1198.

34.Jonassen AK, Aasum E, Riemersma RA, Mjos OD, Larsen TS: Glucose-insulin-potassium reduces

infarct size when administered during reperfusion. Cardiovasc Drugs Ther 2000, 14:615-623.



67:1682-1688.



119:37-44.




6

7Conclusions & General discussion

Conclusions & General discussion 131

Patients undergoing surgery and anesthesia are at risk for perioperative cardiac

complications, especially in the presence of comorbidities like obesity and type 2

diabetes mellitus. The inhalational anesthetic sevoflurane has been shown to have

cardioprotective as well as cardiodepressive properties, which might be affected by

metabolic alterations in the heart. While the cardioprotective effects are reduction of

ischemia and reperfusion injury, the cardiodepressive effects are negative inotropic

and lusitropic effects. In the present thesis we investigated the impact of dietary

intake on perioperative myocardial perfusion, function and ischemia and reperfusion

injury, thereby focusing on the interaction of dietary intake and sevoflurane

anesthesia. We specifically hypothesized that anesthesia-induced changes in

perioperative myocardial function are influenced by preoperative dietary alterations.

Critique of methods

We tested our hypotheses in instrumented, anesthetized rats. These rats received a

standardized diet with a high concentration of saturated fatty acids and different

percentages of simple carbohydrates to induce an obese/type 2 diabetic phenotype

as a surrogate model for the human setting. This standardized diet improved the

comparability of diet interventions within our studies. Overall, 4 weeks of high fat

diet (HFD) feeding resulted in glucose intolerance with decreased HDL cholesterol

(chapter 3), while 4 weeks of western diet feeding, a diet consisting of HFD with

additional sucrose, resulted in obesity, hyperglycemia, hyperinsulinemia and

hyperlipidemia, going from a glucose intolerant to a prediabetic rat model (chapter

5). Furthermore, these characteristics were extravagated when the duration of

feeding was doubled from 4 to 8 weeks (chapter 4, 5 and 6). From these data we

can conclude that western diet feeding simulates the human phenotype better than

high fat diet feeding in rats.

Diet-induced models of obesity and diabetes are more human-like than other

models, because obesity is based on excessive caloric intake.1-3 Models such as the

genetic Zucker or Zucker Diabetic Fatty rat are not representative for the human

pathogenesis of obesity and type 2 diabetes mellitus due to a deficient leptin system,

which is uncommon in humans.4 Besides, it is important to realize that untreated

type 2 diabetic patients are rare, and that most patients receive medication leading

to mild or absence of hyperglycemia. The translational character of diet feeding in

rats to humans is still under discussion. There is a large variation in content of the

diet, time of feeding and strain used. Diets can vary in the source and percentages of

fat and carbohydrates. However, in the present thesis western diet feeding

represents a prediabetic phenotype in rats and closely mimics the human diet.

Our studies were performed in instrumented rats. Rats were anesthetized,

endotracheally intubated and mechanically ventilated. During the experiments, ECG

was recorded and arterial blood pressure was measured invasively. Moreover,

7

132 Chapter 7

interventional techniques such as an oral glucose tolerance test to determine glucose

tolerance, hyperinsulinemic euglycemic clamping to induce hyperinsulinemia,

coronary occlusion to induce ischemia and reperfusion injury, and (contrast)

echocardiography to determine myocardial perfusion and diastolic and systolic

function have been performed. These techniques allowed standardized study

conditions such as preservation of hypoglycemia during hyperinsulinemia.

All our results were obtained under strictly standardized conditions with respect to

timing and composition of diet feeding. Nevertheless, as with all animal experiments

extrapolation to the human setting has to be done with caution.

Major findings

Sevoflurane induces cardiodepression without altering myocardial perfusion

Under physiological conditions, myocardial blood flow and systolic function are in

balance.5 The supply of oxygen to the heart depends on the oxygen content in the

blood and coronary blood flow. While oxygen content remains mostly constant,

regulation of coronary blood flow is responsible for matching oxygen supply with

metabolic demands. Major determinants of myocardial blood flow are perfusion

pressure and coronary vascular resistance.

Under pathophysiological conditions, such as obesity and type 2 diabetes mellitus,

the balance between energy supply and demand could be altered. The present thesis

focused on the evaluation of dietary alterations on myocardial systolic function and

blood flow. In high fat diet-induced glucose intolerant rats, myocardial perfusion and

systolic function are not affected (chapter 3), whereas in western diet-induced

prediabetic rats, myocardial perfusion and function are both declined when compared

to control animals (chapter 4). This clearly suggests that the stage of diabetes plays

a role in the development of cardiovascular alterations and that a reduction in

myocardial contractility coincides with a reduction in coronary blood flow. In the

context of these findings the question remains whether these myocardial alterations

in prediabetic rats are additionally associated with changes in myocardial oxygen

demand and supply. It is known that substrate metabolism of diabetic hearts shifts

toward fatty acid utilization, which is paralleled by a decrease in glucose

metabolism.6 However, the consequence of increased use of fatty acids is a higher

demand of ATP which requires more oxygen. In addition to possible alterations in

oxygen supply and demand in the heart of prediabetic rats there are other

pathophysiological mechanisms that may underlie the reduction in myocardial

perfusion and function in our animal model. At first, cardiometabolic alterations may

affect myocardial calcium handling.7-10 The consequent reduction in contractility of

the heart may also lead to alterations in myocardial blood flow. Secondly, myocardial

relaxation is impaired in prediabetic rats, which might result in impairment of

coronary perfusion. As shown in patients undergoing biventricular pacing, coronary


perfusion is dependent on a suction like effect during diastole, the so-called

backward-traveling decompression effect.11

The present thesis showed that sevoflurane did not affect myocardial perfusion,

while systolic function was decreased in healthy and prediabetic animals (chapter 4).

This suggests that sevoflurane uncouples myocardial perfusion and function

irrespective of the metabolic state of the heart. The uncoupling of perfusion and

function is also observed after prolonged ischemia, which has been describes as a

perfusion-contraction mismatch.5 Moreover, sevoflurane exerts vasodilating

properties, and is known to reduce coronary vascular resistance and perfusion

pressure in addition to myocardial depression.12-14 In addition, the vasodilating

properties of sevoflurane are influenced by vasoactive mediators, such as nitric

oxide.15 Sevoflurane activates nitric oxide; however, altered nitric oxide availability

can significantly impair myocardial perfusion-contraction matching.16 Thus,

sevoflurane seems to uncouple myocardial perfusion and contraction, however, up to

now the mechanisms are unclear.

The cardiodepressive effects of sevoflurane are modulated by dietary composition

Sevoflurane exerts cardiodepressive properties in healthy subjects, therefore its

perioperative use may cause hemodynamic alterations. It has been suggested that

the interaction of sevoflurane with the heart is additionally altered by

cardiometabolic diseases. With the growing epidemic of obesity and type 2 diabetes

mellitus it is important to understand the influence of this condition on the

interaction of sevoflurane with myocardial function. We found that high fat diet-

induced glucose intolerance did not affect myocardial function during baseline

conditions, whereas myocardial function was impaired during conditions of

hyperemia (chapter 3). When prediabetes was induced in rats exposed to a diet high

in saturated fatty acids and simple carbohydrates, a so-called western diet, we

observed impairment of myocardial systolic and diastolic function (Chapter 4 and 5).

Moreover, we found that sevoflurane is a stronger cardiodepressant in prediabetic

rats than in control rats (chapter 4 and 5), which could be restored by lowering

caloric intake (chapter 5). Suggested mechanisms are myocardial substrate

metabolism and calcium handling. Proteins related to myocardial substrate

metabolism, such as the protein kinase Akt17 and glucose transporter 418 are

increased by sevoflurane after ischemia and reperfusion, resulting in increased

glucose and decreased fatty acid oxidation.18 Moreover, sevoflurane reduces

myocardial calcium availability7 and affects sarcoplasmic reticulum calcium

content.7;19 These results suggest that normalization of the cardiometabolic profile

by dietary changes are of direct influence on the myocardial response to sevoflurane

in rats.

7

134 Chapter 7

The cardioprotective effects of sevoflurane during myocardial ischemia are altered by

dietary intake

Western diet, in combination with a sedentary lifestyle, is an important cause for

overweight, obesity and type 2 diabetes mellitus.1 Interestingly, while the overall

negative consequences of western diet feeding for the development of

cardiometabolic diseases are well acknowledged, there is increasing evidence that

diets containing a high percentage of saturated fatty acids and simple carbohydrates

may also be protective during stressful conditions, like surgery and anesthesia.20

In the present thesis we observed that western diet feeding itself exerted

cardioprotective effects in the ischemic heart (Chapter 6). In animals, feeding a diet

high in saturated fatty acids after myocardial ischemia showed preserved myocardial

function even in the presence of insulin resistance, suggesting a possible protective

effect of high fat diet feeding.21;22 Interestingly, it has been suggested that

overweight and obese patients exert a more appropriate inflammatory and immune

response to stress, and have a better outcome that their lean or morbid obese

counterparts.20 However, it should be kept in mind that, despite macronutrient

excess, obese patients remain at risk for perioperative nutritional deficits, such as

iron deficiency, and the development of cardiovascular diseases.20

Moreover, we showed that sevoflurane exerted cardioprotective effects during

myocardial ischemia and reperfusion in healthy rats. In agreement with our

expectations, western diet feeding blunted the protective effects of sevoflurane

during ischemia and reperfusion (Chapter 6). Western diet-induced cardioprotection

was associated with a rise in plasma insulin levels, suggesting that insulin levels may

be involved in the protective response of this diet (chapter 6). Besides a more

appropriate inflammatory and immune response to stress, a possible mechanism

might be hyperinsulinemia. Hyperinsulinemia is cardioprotective during ischemic

stress.23-28 Moreover, high fat diet feeding induces hyperinsulinemia as a

compensatory mechanism. This hyperinsulinemia in the early phase of diabetes may

therefore be a protective mechanisms during conditions of stress. Our experiments

showed that hyperinsulinemia induced by a hyperinsulinemic euglycemic clamp

mimicked the cardioprotective response as observed after western diet feeding

(Chapter 6). These exciting and potentially also clinically relevant findings warrant

future studies in humans.

Normalizing the caloric intake reverses the cardiodepressive effects, but not the

protective characteristics of sevoflurane

Perioperative cardiac complications are an economical, medical and social burden

that warrants optimization of perioperative health and cardiovascular care to

improve patient outcome and reduce health care costs. Preoperative treatment of

obesity and type 2 diabetes mellitus may reduce perioperative cardiac complications.

Current therapies directed at reducing these risk factors focus on weight loss. Weight


loss is induced by a negative energy balance, such as dietary interventions, physical

activity, pharmacotherapy and surgery.29 Lifestyle interventions, such as changing

dietary intake and increasing physical activity to improve insulin sensitivity and

glucose control in obese and type 2 diabetic patients is an important part of these

treatments.30;31 The evidence for their benefits is nowadays growing and might

reduce perioperative complications and improve short and long-term outcome.

Hyperglycemia, which may be triggered by the stress condition of anesthesia and

surgery, is a strong predictor for morbidity and mortality after non-cardiac

procedures, while high perioperative glucose variability enhances this risk even

further.32-34 Lowering of the incidence of perioperative hyperglycemia in obese and

type 2 diabetic patients may therefore contribute to improved patient outcome and

reduced health care costs after surgery.35 Although the benefits of lifestyle

interventions to improve the incidence of intraoperative hyperglycemia are

increasingly acknowledged, it may be expected that these interventions also

modulate anesthesia-related physiological alterations.

The present thesis showed that lowering caloric intake improved myocardial

systolic and diastolic function (chapter 5 and 6) and that the cardioprotective effect

of western diet feeding during ischemia and reperfusion is sustained (chapter 6).

Further, sevoflurane induced cardiodepression was normalized (chapter 5), whereas

sevoflurane had no additional cardioprotective effect on ischemia and reperfusion

injury (chapter 6). Although studies focusing on preoperative dietary changes in

humans are limited,36 the overall idea is that weight loss reduces risk factors such as

diabetes and cardiovascular disease and extends lifespan and reduces mortality.

Besides the possibility of the sustained cardioprotective effect of western diet

feeding, it is also known that caloric restriction protects against ischemia and

reperfusion injury.36 Mitchell et al. reviewed the feasibility and benefits of caloric

restriction and concluded that caloric restriction has a wide range of benefits against

surgical stress in the experimental setting, which might also account in the human

setting.36 One of the suggested underlying mechanisms is reduced insulin signaling.

Improvement of insulin signaling by caloric restriction results in improved insulin

sensitivity. This might also be an underlying mechanism in improved cardiometabolic

state of western diet-induced prediabetic rats. Overall, dietary modulation by

lowering caloric intake may have a wide range of benefits including improved insulin

sensitivity, altered cardiometabolic state and reduced injury during stress.

7

136 Chapter 7

Future directions

Laboratory investigations already studied the cardioprotective properties of volatile

anesthesia for many years.37 However, the clinical applicability in modulating the risk

of perioperative cardiovascular complications by volatile anesthetics is still an

ongoing debate.38 Several experimental studies showed the protective effects of

volatile anesthetics in human heart tissue. However, recent clinical studies suggest

that propofol and volatile anesthetics are associated with a comparable incidence of

cardiac events. Therefore further research based on large clinical trials is necessary

to conclude on the possible beneficial effects of volatile anesthesia on the heart and

outcome. Moreover, clinical studies need to be performed to investigate the effects

of volatile anesthetics during myocardial infarction in obese and/or type 2 diabetic

patients.

Secondly, drugs might modulate the sensitivity of the heart for ischemic injury,

such as drugs for improvement of insulin sensitivity by glucagon-like peptide 1 (GLP-

1) or metformin. GLP1 is a gut incretin hormone that is released in response to

nutrient intake, stimulates insulin secretion and exerts insulinotropic and

insulinomimetic properties. Metformin is a biguanide with antihyperglycemic

properties and exerts its actions by enhancing insulin sensitivity. Improvement of the

diabetic phenotype might improve the cardioprotective effects of volatile anesthesia

during ischemia and reperfusion.

Thirdly, we found the protective effects of western diet feeding on ischemia and

reperfusion injury. One of the suggested mechanisms is the presence of metabolic

alterations, such as hyperinsulinemia, during ischemic stress. Several studies

showed a cardioprotective effect of insulin in healthy animals and patients with

myocardial infarction. However, the question remains if insulin administration has

protective effects in the diabetic heart, due to the presence of insulin resistance.

Future studies using insulin blockers need to be performed to prove the beneficial

effects of hyperinsulinemia during ischemia and reperfusion in the prediabetic heart.


Conclusions

Patients with obesity and/or diabetes are at increased risk for perioperative

cardiovascular complications. Moreover, the choice of anesthetic may modulate the

risk of these perioperative cardiovascular complications. In the present thesis we

focused on the modulation of dietary intake on sevoflurane-induced alterations in

myocardial perfusion and function in rats. Moreover, we investigated whether the

cardioprotective effect of sevoflurane is altered by diet composition, and whether

changing the dietary balance by lowering fat and sucrose intake could be useful as

an intervention to influence the effects of sevoflurane on the heart.

Sevoflurane induces cardiodepression without altering myocardial perfusion

In healthy control rats, sevoflurane resulted in reduced systolic function without

altering myocardial blood flow, leading to uncoupling of cardiac systolic function and

perfusion (Chapter 4).

The cardioprotective effects of sevoflurane are modulated by dietary composition

Rats exposed to a diet high in saturated fatty acids (HFD) developed glucose

intolerance, however this phenotype was not associated with alterations in

myocardial perfusion and function (Chapter 3). Though, after induction of hyperemia,

we observed a reduction in myocardial systolic function in rats fed a high fat diet

(HFD) without alterations in myocardial perfusion when compared to control animals

(Chapter 3). When rats were exposed to a diet high in saturated fatty acids and

simple carbohydrates, a so-called western diet (WD), we observed glucose

intolerance and impairment of myocardial perfusion and function at baseline when

compared to control animals (Chapter 4 and 5). In the presence of sevoflurane, the

reduction in myocardial systolic function in rats fed a western diet (WD) was even

more pronounced, while sevoflurane did not alter myocardial perfusion (Chapter 4

and 5).

The cardioprotective effects of sevoflurane during myocardial ischemia are altered by

dietary intake

Sevoflurane exerted cardioprotective effects during myocardial infarction and

reperfusion in healthy rats. In agreement with our expectations, western diet feeding

blunted the protective effects of sevoflurane during ischemia and reperfusion

(Chapter 6). More interestingly, western diet feeding itself exerted cardioprotective

effects in the ischemic heart (Chapter 6). Western diet-induced cardioprotection was

associated with a rise in plasma insulin levels, suggesting that insulin levels may be

involved in the protective response of this diet. Separate experiments showed that

hyperinsulinemia induced by a hyperinsulinemic euglycemic clamp mimicked the

cardioprotective response as observed after western diet feeding (Chapter 6).

7

138 Chapter 7

Normalizing the caloric intake reverses the cardiodepressive effects, but not the

protective characteristics of sevoflurane

Rats subjected to diet reversal, which comprised a period of normal healthy diet

following western diet feeding, showed normalization of their prediabetic phenotype.

(Chapter 5) This was associated with improvement of cardiac systolic as well as

diastolic function (Chapter 5) and a reduction in the cardiodepressive effects of

sevoflurane (Chapter 5). While western diet had a protective effect against ischemia

and reperfusion injury, lowering caloric intake did not alter the cardioprotective

effect of western diet feeding during ischemia and reperfusion (Chapter 6).

Moreover, the absence of sevoflurane-induced cardioprotection during myocardial

infarction in western diet fed rats could not be restored by diet reversal (Chapter 6).


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and halothane on myofilament Ca2+ sensitivity and sarcoplasmic reticulum Ca2+ release in rat

ventricular myocytes. Anesthesiology 2000, 93:1034-1044.

20.Valentijn TM, Galal W, Tjeertes EK, Hoeks SE, Verhagen HJ, Stolker RJ: The obesity paradox in

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21.Rennison JH, McElfresh TA, Okere IC, Vazquez EJ, Patel HV, Foster AB, Patel KK, Chen Q, Hoit

BD, Tserng KY, Hassan MO, Hoppel CL, Chandler MP: High-fat diet postinfarction enhances

mitochondrial function and does not exacerbate left ventricular dysfunction. Am J Physiol Heart

Circ Physiol 2007, 292:H1498-H1506.

22.Rennison JH, McElfresh TA, Chen X, Anand VR, Hoit BD, Hoppel CL, Chandler MP: Prolonged

exposure to high dietary lipids is not associated with lipotoxicity in heart failure. J Mol Cell

Cardiol 2009, 46:883-890.

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Taittonen M, Ronnemaa T, Iozzo P, Knuuti J, Nuutila P, Raitakari OT: Effect of caloric restriction

on myocardial fatty acid uptake, left ventricular mass, and cardiac work in obese adults. Am J

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Roos A, Smit JW: Prolonged caloric restriction in obese patients with type 2 diabetes mellitus

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Poldermans D: Increased preoperative glucose levels are associated with perioperative mortality

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7

8Summary

Summary 145

In the present thesis we investigated the impact of preoperative dietary composition

on sevoflurane anesthesia-induced changes in myocardial perfusion and function.

Moreover, we studied the effects of high intake of saturated fatty acids in

combination with simple carbohydrates on the cardioprotective effects of sevoflurane

in case of myocardial ischemia. We hypothesized that preoperative dietary intake

influence sevoflurane-induced changes in perioperative myocardial function. To

investigate this we studied myocardial perfusion, function and ischemic injury in diet-

induced glucose intolerant or prediabetic rats.

Patients undergoing anesthesia and surgery are at risk of perioperative cardiac

complications, especially in the presence of comorbidities like obesity and type 2

diabetes mellitus. A general introduction is given in Chapter 1 on perioperative

complications, myocardial performance and ischemia during anesthesia and the

combination with dietary interventions and lifestyle changes.

A broader introduction on diabetes, perioperative ischemia and volatile anesthetics

is given in Chapter 2. Several mechanisms involved in anesthesia-induced

cardioprotection have been evaluated in the experimental setting. However, the

existing evidence suggests that the obese and type 2 diabetic heart is less adaptable

to cardioprotective interventions and that anesthesia-induced cardioprotection is just

substrate metabolism is one of the underlying protective mechanisms; therefore we

focused on the consequences of derangements in myocardial substrate metabolism.

This overview of recent literature reveals the importance of interventional options

focusing on recovery of the metabolic flexibility of the heart, especially by improving

insulin sensitivity.

Myocardial perfusion during diet exposure and sevoflurane anesthesia

This thesis focused on the effect of diet-induced glucose intolerance and prediabetes

on myocardial perfusion. Chapter 3 describes the effect of high fat diet-induced

glucose intolerance on myocardial perfusion and function during baseline and

hyperemic conditions. High fat diet-induced glucose intolerance did not affect

myocardial perfusion and function during baseline conditions. During hyperemia, we

found that diet-induced glucose intolerance is associated with impaired myocardial

function, but myocardial perfusion is maintained. These findings result in new

insights into the effect of glucose intolerance on myocardial function and perfusion

during hyperemia.

Sevoflurane is associated with myocardial depression and these effects seem more

abundant during obesity and type 2 diabetes mellitus. Chapter 4 focused on the

impact of a more severe western, or cafeteria, diet on myocardial perfusion and

function and the interaction with sevoflurane exposure. Western diet-induced

prediabetes impaired myocardial perfusion and function. Exposure to sevoflurane did

8

146 Chapter 8

not affect myocardial perfusion, but impaired systolic function in healthy and

prediabetic conditions. Our findings therefore suggest that sevoflurane leads to

uncoupling of myocardial perfusion and function, irrespective of the metabolic state.

Effect of changing the dietary balance

Furthermore, this thesis investigated the effect of altering the dietary balance on

myocardial function and ischemic injury by lowering the caloric intake. In Chapter 5

we investigated whether lowering the caloric intake can restore the effects of

sevoflurane on myocardial performance in prediabetes. Western diet-induced

prediabetes impaired myocardial systolic and diastolic function, whereas sevoflurane

even further impaired systolic function in prediabetes. Lowering caloric intake

normalized the prediabetic phenotype, improved myocardial function and restored

systolic function during sevoflurane exposure. Therefore, sevoflurane is a stronger

cardiodepressant in prediabetes than in healthy conditions, which could be restored

by lowering caloric intake. These results suggest that dietary changes-related

normalization of the cardiometabolic profile is of direct influence on the myocardial

response to sevoflurane.

Sevoflurane is known for its cardioprotective effects during myocardial ischemia

and reperfusion, which seems blunted in the presence of obesity or type 2 diabetes

mellitus. In Chapter 6 we investigated whether a reduction in caloric intake reverses

the deteriorating impact of western diet feeding on the cardioprotective potency of

sevoflurane during ischemia and reperfusion. In contrast to our hypothesis, western

diet itself protected the heart against ischemic injury, and this effect was maintained

even after lowering caloric intake. Exposure to sevoflurane exerted cardioprotective

effects in healthy conditions, but this effect was blunted in prediabetes. Plasma

insulin levels suggested that the protective effects of diet feeding may be induced by

insulin. Hyperinsulinemia was cardioprotective in healthy conditions, suggesting that

the protective effects of western diet feeding or sevoflurane are mediated by insulin.

The main conclusions of this thesis are 1) sevoflurane induces cardiodepression

without altering myocardial perfusion, 2) the cardiodepressive effects of sevoflurane

are modulated by dietary composition, 3) the cardioprotective effects of sevoflurane

during myocardial ischemia are altered by dietary intake, and 4) normalizing caloric

intake reverses the cardiodepressive effects, but not the protective characteristics of

sevoflurane. In Chapter 7, the conclusions are discussed and this chapter ends with

future directions.

9Samenvatting

Samenvatting 151

Dit proefschrift beschrijft de invloed van dieetsamenstelling op de effecten van het

dampvormige anestheticum sevofluraan op de doorbloeding en pompfunctie van het

hart, en de beschermende werking van sevofluraan op het hart tijdens

zuurstoftekort. Om dit te onderzoeken is de doorbloeding, functie en schade ten

gevolge van zuurstoftekort in het hart bestudeerd in ratten. Deze ratten hebben door

blootstelling aan een vetrijk dieet met of zonder toegevoegde suikers glucose

intolerantie of prediabetes ontwikkeld.

Het is een bekend gegeven dat patiënten die een operatie onder anesthesie

ondergaan een verhoogd risico hebben op het ontwikkelen van complicaties aan het

hart. Aandoeningen zoals ernstig overgewicht (obesitas) en type 2 diabetes mellitus

vergroten dit risico op complicaties. Hoofdstuk 1 geeft een algemene introductie

over deze complicaties, het functioneren van het hart en de schade ten gevolge van

zuurstoftekort tijdens anesthesie in combinatie met dieet-interventies en levensstijl

veranderingen.

Dampvormige anesthetica hebben een beschermend effect op het hart tijdens

zuurstoftekort. Recente literatuur toont aan dat deze beschermende effecten

verminderd zijn in het obese en/of type 2 diabetische hart. Het lijkt er daarbij op dat

deze beschermende effecten alleen worden waargenomen in het gezonde hart. Eén

van de verklaringen hiervoor is dat de beschermende effecten van dampvormige

anesthetica worden beïnvloed door het glucose en vetzuur metabolisme in het hart,

en hier wordt uitgebreid op ingegaan in Hoofdstuk 2. Daarnaast wordt ook het

belang van interventiemogelijkheden beschreven, waarbij de focus ligt op het herstel

van de metabole flexibiliteit van het hart door het verbeteren van de insuline

gevoeligheid. Dit kan bijdragen aan het verminderen van het perioperatieve risico op

de complicaties van zuurstoftekort.

Doorbloeding van het hart tijdens dieetblootstelling en sevofluraan

anesthesie

Dit proefschrift richt zich onder andere op het effect van glucose intolerantie en

prediabetes op de doorbloeding van het hart. Hoofdstuk 3 beschrijft het effect van

een hoog vet dieet op de doorbloeding van het hart in rust en tijdens maximale

vaatverwijding. Inname van een hoog vet dieet resulteerde in glucose intolerantie,

maar had geen effect op de doorbloeding en pompfunctie van het hart. Tijdens

maximale vaatverwijding tonen wij aan dat glucose intolerantie geassocieerd is met

een verstoorde hartfunctie, maar dat de doorbloeding van het hart behouden blijft.

Deze resultaten geven nieuwe inzichten in het effect van glucose intolerantie op de

doorbloeding en functie van het hart tijdens maximale vaatverwijding.

Sevofluraan wordt geassocieerd met een verslechterde functie van het hart en deze

effecten lijken versterkt aanwezig te zijn in obese en type 2 diabetische harten.

Hoofdstuk 4 focust op de invloed van een vet-

9

152 Chapter 9

(westers dieet) op de doorbloeding en functie van het hart in combinatie met

blootstelling aan sevofluraan. Inname van een westers dieet resulteerde in

prediabetes en verslechterde de doorbloeding en functie van het hart. Blootstelling

aan sevofluraan had geen effect op de doorbloeding, maar verslechterde wel de

functie in gezonde en prediabetische harten. Deze bevindingen suggereren dat

sevofluraan de doorbloeding en functie in het hart ontkoppelt, onafhankelijk van de

metabole staat van het hart.

Effect van veranderingen in dieetsamenstelling

Dit proefschrift beschrijft het effect van dieetsamenstelling op de functie en op de

schade ten gevolge van zuurstoftekort in het hart. De resultaten van Hoofdstuk 5

tonen aan dat een westers dieet resulteerde in een prediabetisch fenotype en

verslechtering van de hartfunctie. Daarnaast bleken de ongunstige effecten van

sevofluraan op de functie van het hart versterkt te worden door prediabetes. Het

verlagen van de calorie-inname verbeterde het prediabetische fenotype, de functie

van het hart en verminderde de ongunstige effecten van sevofluraan op het hart.

Hieruit concluderen we dat de negatieve effecten van sevofluraan op het hart

versterkt worden in prediabetische harten. Verlaging van de calorie inname kunnen

deze negatieve effecten herstellen. Deze resultaten suggereren dat veranderingen in

dieet samenstelling van invloed is op de respons van het hart op sevofluraan.

Sevofluraan staat bekend om de beschermende werking op het hart tijdens

zuurstoftekort. Obesitas en type 2 diabetes mellitus lijken deze beschermende

effecten te verstoren. Hoofdstuk 6 beschrijft of een verlaging van de calorie inname

de effecten van sevofluraan op de schade van het prediabetische hart ten gevolge

van zuurstoftekort beïnvloedt. Het hoog vet- en suikerrijke westers dieet

beschermde het hart tegen de schade ten gevolge van zuurstoftekort en dit

beschermende effect bleef behouden na verlaging van de calorie inname.

Blootstelling aan sevofluraan resulteerde in bescherming van het gezonde hart

tijdens zuurstoftekort, maar dit effect was verminderd in prediabetische harten.

Insuline levels in het plasma suggereerden dat de beschermende effecten van het

westers dieet en sevofluraan geïnduceerd worden door insuline. Hoge concentraties

van insuline werkten beschermend op het gezonde hart tijdens zuurstoftekort. Dit

suggereert dat insuline mogelijke een rol speelt bij de beschermende effecten van

het westers dieet en sevofluraan.

De belangrijkste conclusies in dit proefschrift zijn 1) het dampvormige anestheticum

sevofluraan verslechtert de functie, maar niet de doorbloeding van het hart; 2) de

samenstelling van het dieet beïnvloedt de effecten van sevofluraan op de

doorbloeding en functie van het hart; 3) een hoog vet- en suikerrijk dieet heeft een

ongunstige invloed op de beschermende effecten van sevofluraan tijdens

zuurstoftekort in het hart en 4) verlaging van de calorie inname herstelt de

Samenvatting 153

verminderde functie van het prediabetische hart, maar niet de beschermende

kenmerken van sevofluraan. In Hoofdstuk 7 worden deze conclusies bediscussieerd

en staat de toekomstige richting van dit onderzoek beschreven.

9

List of abbreviations


A

A microvascular blood volume

AMPK AMP-activated protein kinase

ATP adenosine triphosphate

AUC area under the curve

B

microvascular filling velocity

C

Ca2+ calcium

CABG coronary artery bypass grafting

CD control diet

CON control

CPT-1 carnitine palmitoyl transferase 1

cTnI cardiac troponin I

D

DCA dichloroacetate

DPP4 dipeptidyl peptidase IV

DSPC 1,2-distearoyl-sn-glycero-3-phosphocholine

E

echo echocardiography

eNOS endothelial nitric oxide synthase

ED end-diastole

ES end-systole

F

FABPpm plasma membrane fatty acid binding protein

FAC fractional area change

FAT/CD36 fatty acid translocase / CD36

FATP fatty acid transport protein

FS fractional shortening

G

GIK glucose-insulin-potassium

GLP1 glucagon-like peptide 1

GLUT glucose transporter

GSK3 glycogen synthase kinase

L

158 List of abbreviations

H

HDL high-density lipoprotein

HFD high fat diet

I

INS hyperinsulinemia

IVRT isovolumic relaxation time

L

LAD left anterior descending coronary artery

LDL low-density lipoprotein

LV left ventricle / left ventricular

M

MI mechanical index

MI myocardial infarction

mito K+ATP mitochondrial ATP-activated potassium channel

MyBP-C myosin binding protein-C

P

PDK4 pyruvate dehydrogenase kinase 4

PEG polyoxyethylene stearate

PLB phospholamban

PPAR peroxisome proliferator-activated receptor

R

REV reversion

RPP rate pressure product

S

SEVO sevoflurane

SERCA2a sarcoplasmic reticulum calcium ATPase 2a

T

T2DM type 2 diabetes mellitus

TCA tricarboxylic acid

TnT troponin T

TTC 2,3,5-triphenyl tetrazolium chloride


V

VLDL very-low-density lipoprotein

W

WD western diet

WT wall thickness

Y

Y0 signal intensity of first frame after microbubble destruction

L

Dankwoord

Dankwoord 163

Men zegt dat de laatste loodjes het zwaarst zijn. Maar waar komt dit spreekwoord

eigenlijk vandaan? Er zijn verschilende theorieë over de herkomst. De meest

plausible verklaring: het is ontleend aan het traditionele wegen waarbij de laatste

gewichtjes de l ) de doorslag geven om de weegschaal in balans te krijgen.

Bij mij heeft de weegschaal halverwege al flink geschommeld en alle gewichten eraf

halen en opnieuw plaatsen was op een gegeven moment de enige optie. Om door

deze fase heen te komen en mijn proefschrift af te ronden zijn er vele personen

belangrijk geweest, maar een aantal personen in het bijzonder en deze wil ik graag

bedanken.

Lieve Rob, meestal wordt het dankwoord geëindigd met de partner, maar ik wil heel

graag met jou beginnen, want zonder jouw onvoorwaardelijke steun was dit

proefschrift er nooit geweest. Jij bent mijn alles; mijn man, mijn beste vriend en een

fantastische vader! Op de momenten dat ik druk was regelde je naast je eigen

drukke werk alles; het huishouden, het eten, de zorg voor Thijs en daarnaast was je

er ook nog eens altijd voor mij. Eigenlijk onbeschrijf

Prof.dr. Loer, bedankt dat u mij de mogelijkheid heeft gegeven om bij u dit

proefschrift te maken tot wat het geworden is en daarnaast het onderzoek nog veel

verder uit te breiden. Ik kijk uit naar onze verdere samenwerking! Dr. Boer, lieve Chris. Jij hebt mij mijn verloren passie voor de wetenschap helpen

terug te vinden. Jij gaf mij de zelfverzekerheid en het enthousiasme om opnieuw aan

een proefschrift te beginnen. Zonder jouw passie voor onderzoek en altijd maar weer

motiverende woorden was dit proefschrift er nooit geweest. Zelfs van de kleinste

resultaten word je nog steeds enthousiast. Ik ben erg blij dat we ons onderzoek en

het delen van enthousiasme over nieuwe data blijven voortzetten!

Dr. Bouwman, beste Arthur. Dit project is ooit begonnen als een zijtakje van beide

promotie projecten en uiteindelijk gecombineerd en geëindigd in dit proefschrift. Wie

had dat ooit kunnen denken. Veel succes in Eindhoven!

Prof.dr. Westerhof, beste Nico. Heel erg bedankt voor uw ondersteuning bij mijn

.

Prof.dr. Tangelder, beste Geert Jan. Hartelijk dank voor uw gastvrijheid op de

afdeling fysiologie. Ik heb het er erg naar mijn zin gehad en me altijd thuis gevoeld.

Ik ben dan ook erg blij dat ik nog blijf. Geniet van uw pensioen en alle reizen die u

nog gaat maken!

D

164 Dankwoord

Graag wil ik mijn promotiecommissie, Prof.dr. Preckel, Prof.dr. van Royen, Prof.dr.

Glatz, Prof.dr. den Heijer, Prof.dr. Scheeren, Prof.dr. van der Velden en Prof.dr.

Serné bedanken voor het lezen van mijn proefschrift en het deelnemen aan mijn

verdediging.

Mijn paranimfen, Ester en Marianne. Lieve Ester, bijna gelijk zijn we bij de fysiologie

acht

vriendinnen. Ik vind het fijn d kheid naast me mag

hebben staan!

Lieve Marianne, lief schoonzusje, al 23 jaar ben je in de familie en je bent dan ook

echt mijn grote zus. Jij bent niet alleen mijn voorbeeld van hoe ik als moeder wil

zijn, maar je hebt ook de bijzondere eigenschap om oprecht blij voor me te zijn

zonder altijd exact te weten waarom. Ik ben er trots op dat je naast me wilt staan!

Ik ben van mening dat twee paranimfen te weinig zijn. Ondertussen zijn collega

vriendinnen geworden en hebben een belangrijke plek in mijn leven gekregen.

Carolien, drie jaar hebben we niet alleen een kamer gedeeld, maar ook de nodige

frustaties, irritaties, blijheden, borrels en nog veel meer. Succes met de laatste

loodjes, je bent er echt bijna en je hebt een prachtig proefschrift waar je trots op

mag zijn! Lonneke, jij was de nieuwe generatie, maar ondertussen ben je eerder

gepromoveerd dan ik ;-). Ik vond het geweldig dat ik jouw paranimf mocht zijn en

ben blij dat je nu een nieuw plekje hebt gevonden. Frances, ook bij jou mocht ik je

als paranimf ondersteunen en ik vond het erg bijzonder om dit moment met je te

mogen delen. Ik vind het super dat jij ook nog onderzoek blijft doen bij de fysiologie

en we weer roomies zijn. Sylvia, bedankt voor alle bakkies en onvoorwaardelijke

steun. Ik heb ongelooflijk veel respect voor jouw doorzettingsvermogen en

positivitieit!

In al die jaren heb ik toch heel wat roomies gehad. Louis, Melanie, Frances, Coen,

Carolien, Hein, Karolina, Floor, Bas, Noor, Chantal en Yoni. Bedankt voor de

gezelligheid, mentale steun, alle klaaguurtjes ;-

met bitterballen en natuurlijk de frikandellenlunch.

Na acht jaar werken

Hoe bijzonder is dat! -Lynn, Christine, Ester, Frances, Gerrina, Ingrid,

Lonneke, Nicky, Sylvia, en surrogaat oma Lynda, bedankt voor alle opa

dat we ons uitspreiden over de hele wereld (NY, Zanzibar, AMC), hoop ik dat we

onze opa jaarlijkse oma-de-luxe weekenden blijven voortzetten!

Dankwoord 165

Verder heb ik een mooi onderzoekteam om me heen. Rob, bedankt voor het uit

handen nemen van heel veel dierexperimenteel werk. Chantal, Nick, Rob en

Carolien, samen gaan we het ELVIS lab op de kaart zetten.

en studenten (te veel om op te noemen) op de afdelingen

anesthesiologie, fysiologie, secretariaat, werkplaats, electronica, interne, en UPC:

bedankt voor de ondersteuning op alle vlakken en gezelligheid!

(zijn) super!

Verder wil ik ook graag mijn familie, vrienden en iedereen die mij dierbaar is

bedanken. Ieder heeft op zijn eigen wijze interesse getoond of een bijdrage geleverd

(in de vorm van een wijntje of welke andere vorm van ondersteuning dan ook ):

Michael, Anja, Xander & Justin, Gaby, Marianne Ilse & Maud, Wim, Ton & Lia, Mark,

Lianne, Tim, Chris & Julia, Ronald Vlasblom, Klaas Kramer, Robert-Jan, Silvia,

Xander & Timon, Andu, Maurice & Benji, Kees, Mariska, Lianne & Feline, Jennie, Ivo

& Feline, Diederik, Rik, Connie & Edwin, Tilly & Chelso en nog vele anderen .

Lieve mama en papa. Eindelijk ga ik dan ik niet van jullie

kon verwachten helemaal te begrijpen wat promoveren precies inhoudt, hebben jullie

altijd 200% achter mij gestaan! Papa, jouw doorzettingsvermogen is onbeschrijfbaar

en voor mij een belangrijk voorbeeld. Daarom is mijn proefschrift voor jou.

Lieve Rob, Thijs en onze nieuwe aanwinst, het werk zal er niet minder op worden nu

ik klaar ben , maar zal er wel altijd voor jullie zijn. Jullie maken het leven bijzonder

en promoveren relatief. Ik hou van jullie.

Liefs Charissa

D

List of publications


Full papers

van den Brom CE, Boly CA, Bulte CSE, Boer C. Perioperative organ perfusion in

obesity and diabetes.

Submitted

van den Brom CE, Boly CA, Bulte CSE, van den Akker RFP, Kwekkeboom RFJ, Loer

SA, Boer C, Bouwman RA. Sevoflurane impairs myocardial systolic function, but not

myocardial perfusion in diet-induced prediabetic rats.

Submitted

van den Brom CE, Boer C, van den Akker RFP, van der Velden J, Loer SA, Bouwman

RA. Diet composition modulates sevoflurane-induced myocardial depression in rats.

Submitted

Bulte CSE, van den Brom CE, SA Loer, Boer C, Bouwman RA. Myocardial blood flow

under sevoflurane anaesthesia in type 2 diabetic patients: a pilot study.

Submitted

Dekker SE, Viersen VA, Duvekot A, de Jong M, van den Brom CE, Schober P, Boer

C. Lysis Onset Time as Diagnostic Rotational Thromboelastometry Parameter for

Fast Detection of Hyperfibrinolysis.

Submitted

Vonk ABA, Veerhoek D, van den Brom CE, van Barneveld LJM, Boer C.

Individualized heparin and protamine management improves rotational

thromboelastometric parameters and postoperative hemostasis in valve surgery.

Accepted at Journal of Cardiothoracic and Vascular Anesthesia

van den Brom CE, Bulte CSE, Loer SA, Bouwman RA, Boer C. Diabetes,

perioperative ischaemia and volatile anaesthetics: Consequences of derangements

in myocardial substrate metabolism.

Cardiovascular Diabetology (2013), 12:42

Koning NJ, Vonk AB, Verkaik M, van Barneveld LJ, Beishuizen A, Atasever B,

Baufreton C, van den Brom CE, Boer C. Pulsatile flow during cardiopulmonary

bypass preserves postoperative microcirculatory perfusion irrespective of systemic

hemodynamics.

Journal of Applied Physiology (2012), 112(10): 1727-1734

L

170 List of publications

van den Brom CE, Bulte CSE, Kloeze BM, Loer SA, Boer C, Bouwman RA. High-fat

diet-induced glucose intolerance impairs myocardial function, but not myocardial

perfusion during hyperaemia: a pilot study.


van den Brom CE, Bosmans JWAM, Vlasblom R, Huisman MC, Lubberink M, Molthoff

CFM, Lammertsma AA, Boer C, Ouwens DM, Diamant M. Diabetic cardiomyopathy in

Zucker diabetic fatty rats: the forgotten right ventricle.


van den Brom CE, Huisman MC, Vlasblom R, Boontje NM, Duijst S, Lubberink M,

Molthoff CFM, Lammertsma AA, van der Velden J, Boer C, Ouwens DM, Diamant M.

Altered myocardial substrate metabolism is associated with myocardial dysfunction

in early diabetic cardiomyopathy in rats: studies using positron emission

tomography.

Cardiovascular diabetology (2009), 8(1):39

de Snoo MW, van den Brom CE, Jeneson JAL, Everts ME. The effect of wheel

running exercise on plasma IL-6 and muscle IL-6 mRNA levels in mice.

Snoo (2009) chapter 6

Ouwens DM, Diamant M, Fodor M, Habets DDJ, Pelsers MMAL, El Hasnaoui M, Dang

ZC, van den Brom CE, Vlasblom R, Rietdijk A, Boer C, Coort SLM, Glatz JCF,

Luiken LLFP. Cardiac contractile dysfunction in insulin resistant high-fat diet fed rats

associates with elevated CD36-mediated fatty acid uptake and esterification.

Diabetologia (2007), 50: 1938-1948

Van Schothorst EM, Keijer J, Pennings JL, Opperhuizen A, van den Brom CE, Kohl

T, Franssen-van Hal NL, Hoebee B. Adipose gene expression response of lean and

obese mice to short-term dietary restriction.

Obesity (2006), 14(6): 974-979


Papers in preparation

Koning NJ, de Lange F, van den Brom CE, Shelep I, Bogaards SJ, Niessen HW,

Baufreton C, Boer C. Hemodilution and extracorporeal circulation induce distinct

alterations in microcirculatory perfusion and heterogeneity in a rat model of

cardiopulmonary bypass.

Duvekot A, Viersen VA, Dekker SE, van den Brom CE, Geeraedts jr. LMG, Loer SA,

Schober P, de Waard MC, Spoelstra A, Boer C. Hyperfibrinolysis in out-of-hospital

cardiopulmonary arrest is more prevalent in patients with low cerebral tissue

oxygenation during cardiopulmonary resuscitation.

van den Brom CE, Boer C, van den Akker RFP, Loer SA, Bouwman RA. Western diet

modulates the susceptibility of the heart to ischemic injury and sevoflurane-induced

cardioprotection in rats.

Lamberts RR*, van den Brom CE*, Vlasblom R, Duijst S, Loer SA, Boer C, Diamant

M, Bouwman RA. Diet composition modulates resistance to ischemia and

sevoflurane induced preconditioning in the rat heart.

Overmars MAH, Koning NJ, van den Brom CE, van Bezu J, Vonk ABA, van Nieuw

Amerongen GP, Boer C. In vitro endothelial cell barrier function decreases after

exposure to plasma from patients subjected to cardiopulmonary bypass.

L


Published abstracts


modulates the susceptibility of the heart to ischemic injury and sevoflurane-induced

cardioprotection in rats.

Nederlands tijdschrift voor Anesthesiologie (2013)

van den Brom CE, Boly CA, Bulte CSE, van den Akker RFP, Loer SA, Boer C,

Bouwman RA. Sevoflurane additionally impairs myocardial function, but not

myocardial perfusion in diet-induced prediabetic rats.



feeding protects against myocardial ischaemic injury, but abolishes sevoflurane-

induced cardioprotection in rats.

European Journal of Anaesthesia (2013)

van den Brom CE, Boer C, van den Akker RFP, Loer SA, Bouwman RA. Changing

diet composition normalizes sevoflurane-induced impaired cardiac function in

diabetic rats.


van den Brom CE, Bulte CSE, Voogdt C, Kloeze BM, Loer SA, Boer C, Bouwman RA.

Short-term reduction of saturated fatty acids improves glucose tolerance,

myocardial structure and function in western diet-induced obesity.

Anesthesiology (2011)

Bouwman RA, Bulte CSE, Loer SA, Boer C, van den Brom CE. High fat diet feeding

impairs myocardial function during stress, but not during sevoflurane anesthesia.


van den Brom CE, Bulte CSE, Loer SA, Boer C, Bouwman RA. Western diet feeding

impairs myocardial function during stress, but not during sevoflurane anesthesia.


van den Brom CE, Bouwman RA, Gräler MH, Ouwens DM, Diamant M, Boer C.

Reversion of high-fat diet normalizes myocardial function and sphingolipid levels.



Bouwman RA, van den Brom CE, Loer SA, Boer C, Lamberts RR. Diet composition

modulates sevoflurane induced cardioprotection in the rat heart.


Huisman MC, van den Brom CE, Buijs FL, Molthoff CFM, Boellaard R, Lammertsma

AA. Implementation of Physiological Gating on the High Resolution Research

Tomograph.

Nuclear Science Symposium Conference Record (2008): 4767-4769

Bouwman RA, van den Brom CE, Loer SA, Boer C, Lamberts RR. Sevoflurane-

induced cardioprotection in a rat model of high-fat diet-induced type 2 diabetes

mellitus.

European Journal of Anaesthesia (2008); 25 [Supl 44]: 4AP6-3

Bouwman RA, van den Brom CE, Vlasblom R, Loer SA, Boer C, Diamant M,

Lamberts RR. Cardioprotection and dietary intake in a rat model of diet-induced

type 2 diabetes mellitus.


Ouwens DM, van den Brom CE, Kriek J, Schaart G, Hesselink MK, Schrauwen P,

Vlasblom R, Diamant M. Dietary fish-oil preserves cardiac contractile function by

upregulation of UCP3 in a rat model of high-fat diet-induced glucose tolerance and

cardiomyopathy.

Diabetologia (2007); 50 [Suppl1]: S90 (P-0206)

van den Brom CE, Vlasblom R, Kriek J, Rietdijk A, Duijst S, Salic K, Ouwens DM,

Diamant M. High fat diet-induced relocation of CD36 to the sarcolemma precedes

cardiac dysfunction and associates with activated PKB/AKT.

Nederlands tijdschrift voor diabetes (2007)

Vlasblom R, van den Brom CE, Salic K, Kriek J, Boer C, Ouwens DM, Diamant M.

Western-type diet-induced cardiac contractile dysfunction is associated with

nutrient-specific cardiomyocyte remodeling and activation of the Akt-regulated

transcription factor GATA-4 in mice.


Salic K, van den Brom CE, Vlasblom R, Kriek J, Schaart G, Hesselink MK,

Schrauwen P,Ouwens DM, Diamant M. Myocardial contractile function is preserved

by dietary fish-oil supplementation through upregulation of UCP3 in a rat model of

high-fat diet-induced impaired glucose tolerance and cardiomyopathy.


L


van den Brom CE, Vlasblom R, Fodor M, Boer C, Ouwens DM, Diamant M. Short-

term exposure to polyunsaturated high fat diet preserves cardiac contractile

function in rats.

Diabetologia (2006) 49: [Suppl1] 326(P-0534)

Vlasblom R, Ouwens DM, van den Brom CE, Boer C, Diamant M. In wild type mice

long-term exposure to high-fat diet induces systemic insulin resistance without

affecting cardiac function.

Diabetologia (2006) 49: [Suppl1] 327

Vlasblom R, Ouwens DM, van den Brom CE, Boer C, Diamant M. Long-Term

Exposure to High-Fat Diet Induces Systemic Insulin Resistance without Affecting

Cardiac Function in Wild Type Mice.

Diabetes (2006) 55: [Suppl 1] A338

Vlasblom R, Ouwens DM, van den Brom CE, Fodor M, Boer C, Diamant M. Short

Term Exposure to a High-Fat Diet Results in Impaired Glucose Tolerance, Cardiac

Dysfunction and Epicardial Fat Accumulation in Rats.

Diabetes (2006) 55: [Suppl 1] A338

van den Brom CE, Vlasblom R, Ouwens DM, Fodor M, Boer C, Diamant M. Short

Term Exposure to a High-Fat Diet Results in Impaired Glucose Tolerance, Cardiac

Dysfunction and Epicardial Fat Accumulation in Rats.


Curriculum Vitae

Curriculum Vitae 179

Charissa Esmé van den Brom was born on July 8th 1982 in Nijkerk, the

Netherlands. After graduating from secondary school (havo, Amersfoort) in 1999 she

started the study Biochemistry at the University of Applied Sciences in Utrecht. She

did a research apprenticeship at the Department of Toxicology, Pathology and

started her master Medical Biology at the VU University in Amsterdam. During her

master she did a research apprenticeship at the Institute of Food Research in

Norwich, England and a research apprenticeship within the Department of Anatomy

and Physiology at Utrecht University. She obtained her Master degree in August

2005 and subsequently started as PhD student at the Department of Internal

Medicine and Laboratory for Physiology at the VU University Medical Center in

Amsterdam. In July 2010 she started her PhD-research at the Department of

Anesthesiology and Laboratory for Physiology at the VU University Medical Center in

Amsterdam. The results of this research are presented in this dissertation. She is

two-time runner-up and in 2011 winner of the best presentation at the Dutch Society

of Anesthesiologists conference. She is co-founder and coordinator of the

Experimental Laboratory for VItal Signs (ELVIS). From January 2013 she is employed

as a postdoctoral researcher at the Department of Anesthesiology of the VU

University Medical Center in Amsterdam. Charissa is married to Rob Morren. They

are the proud parents of Thijs (2012). In May 2014 they are expecting their second

child.

Charissa Esmé van den Brom werd geboren op 8 juli 1982 te Nijkerk. Na het

behalen van haar havo diploma startte zij in 1999 met de studie Biochemie aan de

hogeschool van Utrecht. Haar afstudeerstage heeft zij gelopen op de afdeling

Toxicologie, Pathologie en Genetica van het RIVM te Bilthoven. Na het behalen van

haar ingenieurs titel begon zij in 2003 met de master Medische Biologie aan de Vrije

Universiteit te Amsterdam. Tijdens deze studie verbleef ze voor een half jaar in

Norwich (Verenigd Koninkrijk) voor een onderzoeksstage bij het Institute for Food

Research. Haar afstudeerstage volbracht zij op de afdeling Anatomie en Fysiologie

aan de faculteit Diergeneeskunde van de Universiteit van Utrecht. In augustus 2005

behaalde zij haar doctoraalexamen. In januari 2006 startte zij als promovenda aan

de afdelingen Interne Geneeskunde en Fysiologie van het VU medisch centrum. In

juli 2010 begon zij als promovenda aan de afdelingen Anesthesiologie en Fysiologie

aan het VU medisch centrum te Amsterdam. De resultaten van dit onderzoek staan

in dit proefschrift beschreven. Ze is tweevoudig runner-up en de 2011 winnaar van

de beste presentatie op het congres van de Nederlandse Vereniging voor

Anesthesiologie. Ze is mede oprichtster en coördinator van het Experimental

Laboratory for VItal Signs (ELVIS). Per januari 2013 is zij werkzaam als post-

doctorale onderzoeker op de afdeling Anesthesiologie aan het VU medisch centrum

CV

180 Curriculum Vitae

te Amsterdam. Charissa is getrouwd met Rob Morren. Samen zijn ze de trotse

ouders van hun zoon Thijs (2012). In mei 2014 verwachten ze hun tweede kindje.


Dietary modulation of the effects of sevoflurane on myocardial perfusion, function and ischemic injuryin rats

Dietary m

odulation of the effects of sevoflurane on myocardial perfusion, function and ischem

ic injury in rats Charissa Esm

é van den Brom

UITNODIGING

voor het bijwonen van de openbare verdediging van

het proefschrift

Dietary modulation of the effects of sevoflurane on

myocardial perfusion, function and ischemic

injury in rats

door Charissa van den Brom

Donderdag 30 januari 2014 om 15.45 uur

In de aula van het Hoofdgebouw

Vrije UniversiteitDe Boelelaan 1105

te Amsterdam

Receptie na afloop van de promotie

Paranimfen

Ester [email protected]

Marianne de Gruil-van [email protected]

Proefschrift van den Brom

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