ANALYSIS OF THE PHYSIOLOGICAL AND MOLECULAR ROLES OF DIABETES IN CARDIAC REMODELING EVENTS
MEGUMIEGUCHI
A DISSERTATION SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
GRADUATE PROGRAM IN HIGHER EDUCATION YORK UNIVERSITY, TORONTO, ONTARIO
SEPTEMBER 2011 Library and Archives Bibliotheque et Canada Archives Canada
Published Heritage Direction du Branch Patrimoine de I'edition
395 Wellington Street 395, rue Wellington Ottawa ON K1A0N4 Ottawa ON K1A 0N4 Canada Canada Your file Votre reference ISBN: 978-0-494-88665-6
Our file Notre reference ISBN: 978-0-494-88665-6
NOTICE: AVIS: The author has granted a non L'auteur a accorde une licence non exclusive exclusive license allowing Library and permettant a la Bibliotheque et Archives Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par I'lnternet, preter, telecommunication or on the Internet, distribuer et vendre des theses partout dans le loan, distrbute and sell theses monde, a des fins commerciales ou autres, sur worldwide, for commercial or non support microforme, papier, electronique et/ou commercial purposes, in microform, autres formats. paper, electronic and/or any other formats.
The author retains copyright L'auteur conserve la propriete du droit d'auteur ownership and moral rights in this et des droits moraux qui protege cette these. Ni thesis. Neither the thesis nor la these ni des extraits substantiels de celle-ci substantial extracts from it may be ne doivent etre imprimes ou autrement printed or otherwise reproduced reproduits sans son autorisation. without the author's permission.
In compliance with the Canadian Conformement a la loi canadienne sur la Privacy Act some supporting forms protection de la vie privee, quelques may have been removed from this formulaires secondaires ont ete enleves de thesis. cette these.
While these forms may be included Bien que ces formulaires aient inclus dans in the document page count, their la pagination, il n'y aura aucun contenu removal does not represent any loss manquant. of content from the thesis. Canada ANALYSIS OF PHYSIOLOGICAL AND MOLECULAR ROLES OF DIABETES ON CARDIAC REMODELING EVENTS
by Megumi Eguchi
By virtue of submitting this document electronically, the author certifies that this is a true electronic equivalent of the copy of the dissertation approved by York University for the award of the degree. No alteration of the content has occurred and if there are any minor variations in formatting, they are as a result of the conversion to Adobe Acrobat format (or similar software application).
Examining Committee members:
1. John McDermott 2. Michael Scheid 3. Robert Tsushima 4. Caroline Davis 5. Eric Thorin
iii ABSTRACT
The development of heart failure (HF) is associated with molecular and physiological changes in the heart which are termed cardiac remodeling. Cardiac remodeling events include an increase in cardiomyocyte death, remodeling of cardiac extracellular matrix (ECM), myofibroblast proliferation, cardiomyocyte hypertrophy, and changes in cardiac metabolism. Diabetes is an independent risk factor in the development of heart failure and diabetic patients suffer from higher incidence of morbidity and mortality after myocardial infarction. However, the exact physiological and molecular changes associated with cardiac remodeling in the presence versus absence of diabetes are still not clearly defined.
In this PhD project, I investigated the influence of diabetes on cardiac remodeling components under several physiologically relevant cardiac stimuli. Three major topics were covered in the project: 1) in vivo characterization of diabetes effects on various cardiac remodeling events after ischemia reperfusion injury showed that the diabetic heart exhibits a smaller infarct area with an upregulation of autophagy; however it shows a reduction in glucose metabolism and an excessive development of interstitial fibrosis. Whereas non-diabetic heart develops mild left ventricular (LV) dilation, the diabetic heart develops concentric hypertrophy; 2) in vivo analysis of diabetes role in ECM remodeling post-myocardial infarction revealed that ECM remodelling is aggravated in the presence of diabetes with an excessive activation of matrix metalloproteinases (MMPs) which may be associated with dilation of the LV and higher mortality. Insulin is able to inhibit the release and activation of MMP-2 and -9 in cardiac fibroblasts, and the absence of insulin in diabetic animals may be one mechanism responsible for the excessive activation of MMPs; 3) in vitro analysis iv of adiponectin's effect on hyperglycemia-induced cardiomyocyte apoptosis showed that hyperglycemia-induced apoptosis is inhibited by adiponectin via activation of p38, possibly through the upregulation of autophagy.
Each of the findings in this project further deepens our understanding of both the physiological and the molecular effects of diabetes in the progression of cardiac remodeling, ultimately leading to heart failure. Overall, these findings will help in finding possible therapeutic targets to treat or prevent the adverse development of heart failure in diabetic patients.
v ACKNOWLEDGEMENTS
Firstly I would like to thank my supervisor, Dr. Gary Sweeney, for giving me the opportunity to work in his lab for the past six years. Indeed, I am thankful for his hospitality, advice, and espeically in light of my thesis, the initial inducement to continue my research in the PhD program. The discussions, arguments, frustrations, and fun of these last six years have made the completion of this project possible. I would also like to thank Dr. Sweeney for giving me the opportunity to work in Hong
Kong for a few months and to move to South Korea and work at Institut Pasteur
Korea. Both experiences were eye opening. Acquainting myself with the researchers from various scientific and ethnic backgrounds and exposing myself to a famed institute with a vital drug discovery program has been vital to my growth as a researcher myself.
I must thank the Heart and Stroke Foundation of Canada for supporting me for the last 3 years of my PhD studies with a Doctoral Research Award, allowing me the freedom to concentrate on my research.
I would like to thank all the former and the present members of the Sweeney lab at York University and the Diabetes lab at Institut Pasteur Korea. I am very grateful for all their support, not only in scientific areas, but also in life outside of lab.
Many thanks to all the collaborators and technical assistants involved in this
PhD project, who made it possible to conduct experiments and analyze data. Without their help, this work wouldn't have been possible.
I would especially like to thank my husband, who has always trusted in me.
His support and encouragement of my work and life as a researcher have allowed me to complete this project. vi And finally, I would like to thank my parents who have proudly supported and encouraged my research endeavours.
vii TABLE OF CONTENTS
ABSTRACT iii
ACKNOWLEDGEMENTS vi
TABLE OF CONTENTS viii
LIST OF TABLES xiii
LIST OF FIGURES xiv
LIST OF ABBREVIATIONS xvi
1 CHAPTER 1: INTRODUCTION AND RESEARCH OBJECTIVES 1
1.1 PREVALENCE OF CARDIOVASCULAR DISEASES 2
1.2 PREVALENCE AND CHARACTERISTICS OF DIABETES 2
1.3 DIABETES IS AN INDEPENDENT RISK FACTOR FOR CVD 3
1.4 PATHOPHYSIOLOGY AND CARDIAC REMODELING IN HEART FAILURE 3
1.5 CARDIOMYOCYTE DEATH IN CARDIAC REMODELING 7
1.5.1 Apoptosis 8
1.5.2 Autophagy 15
1.6 CARDIAC ECM REMODELING 29
1.6.1 Role of ECM in the Healthy Heart 29
1.6.2 ECM Structure 30
1.6.3 Matrix Metalloproteinases 32
1.6.4 Tissue Inhibitors of MMPs 38
1.6.5 Regulation of ECM Remodeling by MMPs and TIMPs in the Development of Heart Failure 38
1.7 ADIPONECTIN, A CARDIOPROTECTIVE ADIPOKINE 40
viii 1.7.1 A diponectin Receptors and Signaling 41
1.7.2 Adiponectin and Cardiac Remodeling 42
1.8 RESEARCH OBJECTIVES 43
2 CHAPTER 2: ISCHEMIA-REPERFUSION INJURY LEADS TO DISTINCT TEMPORAL CARDIAC REMODELING IN NORMAL VERSUS DIABETIC MICE 45
2.1 ABSTRACT 46
2.2 INTRODUCTION 47
2.3 MATERIALS AND METHODS 48
2.3.1 Generation of Diabetic Animals 48
2.3.2 Induction of Cardiac lschemia-Reperfusion Injury 49
2.3.3 Determination of Apoptotic Cells in Cardiac Tissue 50
2.3.4 Protein Expression Determination Using Western Blotting 50
2.3.5 Echocardiography 50
2.3.6 PET Imaging and Analysis 51
2.3.7 Histology 52
2.3.8 Statistical Analysis 52
2.4 RESULTS 53
2.4.1 Overall Cardiac Function 24h after IR Injury in Diabetic Mice 53
2.4.2 Cardiac Glucose Uptake after IR is Reduced in Diabetic Mice 55
2.4.3 Infarct Size Induced by IR is Smaller in Diabetic Mice 56
2.4.4 A utophagy is Upregulated in Diabetic Mice Hearts 57
2.4.5 lR-Induced Macrophage Infiltration is Not Affected by Diabetes 59
2.4.6 Diabetes Leads to Distinct Progressive Changes in Cardiac Function Over Time 61
ix 2.4.7 Diabetes Enhances Interstitial Collagen Deposition after IR Injury 63
2.5 DISCUSSION 64
2.6 ACKNOWLEDGEMENTS 70
3 CHAPTER 3: EFFECTS OF DIABETES ON CARDIAC REMODELING AFTER MYOCARDIAL INFARCTION AND THE SUBSEQUENT DEVELOPMENT OF CARDIAC DYSFUNCTION 71
3.1 ABSTRACT 72
3.2 INTRODUCTION 73
3.3 MATERIALS AND METHODS 74
3.3.1 Generation of Diabetic Mice and Induction of MI. 74
3.3.2 Masson Trichrome Staining an Infarct Area Calculation 75
3.3.3 Echocardiography Analysis 75
3.3.4 MMP Activity Analysis Using Fluorescent Molecular Tomography 76
3.3.5 Scanning Electron Microscopy Analysis of Collagen Structure 76
3.3.6 Primary Cardiac Fibroblasts Isolation 77
3.3.7 Gelatin Zymography 77
3.3.8 Collagen Content Assay 78
3.3.9 Statistical Analysis 79
3.4 RESULTS 79
3.4.1 Survival and Infarct Area Size of VEH and STZ Mice after MI 79
3.4.2 MMP Activation and Collagen Degradation is Greater in STZ Mice after MI 81
3.4.3 STZ Mice Develop Severe L V Dilation and Dyssynchronicity 84
3.4.4 Collagen Deposition in the Remote Area Post-Mi Injury 89
x 3.4.5 Regulation of MMP and Collagen Secretion by Insulin 91
3.5 DISCUSSION 93
3.6 ACKNOWLEDGEMENT 99
4 CHAPTER 4: ADIPONECTIN PROTECTS H9C2 CELLS FROM HYPERGLYCEMIA-INDUCED APOPTOSIS THROUGH THE ACTIVATION OF P38 100
4.1 ABSTRACT 101
4.2 INTRODUCTION 102
4.3 MATERIALS AND METHODS 104
4.3.1 Cell Culture and Induction of Apoptosis 104
4.3.2 Production of fAdandgAd. 105
4.3.3 Cell Viability Assay 105
4.3.4 Detection of Apoptosis 105
4.3.5 Western Blot 105
4.3.6 Statistical Analysis 106
4.4 RESULTS 106
4.4.1 Hyperglycemia Induces Apoptosis in H9c2 Cells 106
4.4.2 Adiponectin Protects Cells from Hyperglycemia-Induced Apoptosis.... 108
4.4.3 Adiponectin's Anti-Apoptotic Action is Mediated Through the Activation ofp38 109
4.4.4 Hyperglycemia Inhibits Autophagy 112
4.4.5 Adiponectin Prevents the Suppression of Autophagy Induced by Hyperglycemia 114
4.5 DISCUSSION 116
4.6 ACKNOWLEDGEMENT 122
xi 5 CHAPTER 5: CONCLUSION 123
6 REFERENCES 128
xii LIST OF TABLES
Table 1.1: MMPs and Their Substrates 35
Table 2.1: Echocardiography measurements 24 hours after IR 55
Table 3.1: Blood glucose level prior to STZ injection 75
Table 3.2: Echocardiography measurements on Day 1 post-MI 85
xiii LIST OF FIGURES
Figure 1.1: Progression of cardiac remodeling post-MI 6
Figure 1.2: Three distinct patterns of cardiac remodeling based on LVMi and RWT...7
Figure 1.3: Mechanism of extrinsic and intrinsic apoptotic pathways 9
Figure 1.4: The three types of autophagy described in mammalian cells 16
Figure 1.5: Role of Atg proteins required for autophagosome assembly 21
Figure 1.6: Ubiquitin-like conjugation system of the macroautophagy machinery 23
Figure 1.7: Signaling pathways controlling autophagy 26
Figure 1.8: Cardiac ECM organization 31
Figure 1.9: MMP Structure 34
Figure 1.10: MMP activation 37
Figure 1.11: Different isoforms and oligomeric forms of adiponectin 41
Figure 2.1: Echocardiography analysis performed to examine the cardiac function after IR injury 54
Figure 2.2 Glucose uptake at 24hr reperfusion 56
Figure 2.3 Effects of IR injury on cardiac cell death 58
Figure 2.4 Macrophage infiltration into the heart 60
Figure 2.5: Echocardiography analysis followed up to 6 weeks of reperfusion 62
Figure 2.6: Fibrosis is enhanced in STZ heart at 6 weeks of reperfusion 63
Figure 2.7: Summary schematic diagram 64
xiv Figure 3.1: Survival rate of VEH and STZ mice up to Day 28 post-MI 80
Figure 3.2: Change in infarct area over time after MI 81
Figure 3.3: Changes in cardiac MMP activity and collagen structure post-MI 83
Figure 3.4: Changes in cardiac structure and functions post-MI compared to sham control 86
Figure 3.5: Cardiac function measured on Day 14-post-MI 88
Figure 3.6: Collagen deposition in the remote area after MI 90
Figure 3.7: Effects of insulin on MMP activation and fibrillar collagen secretion in primary cardiac fibroblasts 92
Figure 3.8: Summary of results 93
Figure 4.1: Hyperglycemia induces apoptosis in H9c2 cells 107
Figure 4.2: Adiponectin treatment attenuates the effect of hyperglycemia 108
Figure 4.3: Adiponectin protects H9c2 from HG-induced apoptosis 109
Figure 4.4: Adiponectin stimulates phosphorylation of p38 MAPK at T180/Y18... 110
Figure 4.5: Activation p38 is essential for adiponectin's ability to protect H9c2 cells from hyperglycemia-induced apoptosis Ill
Figure 4.6: HG treatment of H9c2 cells induces autophagy 113
Figure 4.7: Adiponectin induces phosphorylation of AMPK and reverts the effect of HG on autophagy 115
Figure 4.8: Adiponectin's action against HG-induced apoptosis: summary schematic diagram 122
xv LIST OF ABBREVIATIONS
ACCORD Action to Control Cardiovascular Risk in Diabetes ADVANCE Action in Diabetes and Vascular Disease AMP Adenosine monophosphate AMPK 5' AMP-activated protein kinase ANOVA Analysis of Variance Apaf-1 apoptosis activating factor-1 APMA N-(4-Anilinophenyl)methacrylamide APPL1 adaptor protein containing PH domain, PTB domain and LZ motif Atg autophagy-related gene ATP adenosine triphosphate BafAl bafilomycin A1 Bcl-2 B-cell leukemia/lymphoma 2 BH Bcl-2 homology BW body weight CARD caspase recruitment domain CIRKO cardiomyocyte-selective insulin receptor knockout CMA chaperone-mediated autophagy CO cardiac output CVD cardiovascular disease eye c cytochrome c DAPK death-associated protein kinase DCCT Diabetes Control and Complications Trial DD death domain DED death effector domain DISC death-inducting signalling complex DMEM Dulbecco's modified Eagle medium DN dominant negative ECM extracellular matrix EDLW endocardial diastolic LV volume EDTA ethylene diamine tetraacetic acid EF ejection fraction EGTA ethylene glycol tetraacetic acid ESLW endocardial systolic LV volume fAd full-length adiponectin FDG fluoro-deoxy-glucose FIP200 focal adhesion kinase family interacting protein of 200kDa FITC fluorescein isothiocyanate FMT fluorescent molecular tomography FRET fluorescence resonance energy transfer FS fractional shortening gAd globular adiponectin HF heart failure xvi HG hyperglycemia HMW high molecular weight HR heart rate IAP inhibitor of apoptosis IR ischemia reperfusion KH Krebs-Henseleit LAD left anterior descending LAMP-2A lysosome-associated membrane protein-2A LC3 II PE-conjugated LC3 LKB1 large kinase B1 LV left ventricular LVMi LV mass index LZ leucine zipper MAPK mitogen-activated protein kinase MI myocardial infarction MKK3 mitogen-activated protein kinase kinase 3 MMP matrix metalloproteinase MPTP mitochondrial permeability transition pore MT-MMP membrane-type MMP mTOR mammalian TOR MV E/A mitral valve early filling/atrial filling NG normoglycemia OSEM ordered subset expectation maximization PARP poly (ADP-ribose) polymerase PE phophatidylethanolamine PET-CT positron emission tomography-computed tomography PH pleckstrin homology PI3 phosphatidylinositol-3 pNA p-nitroaniline PS phophatidylserine PTB phosphotyrosine binding RFU relative fluorescence unit ROI region of interest ROS reactive oxygen species RWT relative wall thickness SDS sodium dodecyl sulfate SEM standard error of mean STZ streptozotocin SUV standard uptake value SV stroke volume TAKl transforming growth factor-P-activated kinase 1 tBid truncated Bid TEMP tissue inhibitor of MMP TNF tumor necrosis factor TOR target of rapamycin TSC tuberous sclerosis complex xvii TTC 2,3,5-triphenyltetrazolium chloride TUNEL terminal deoxynucleotidyl transferase dUTP nick end labelling ULK1 uncordinated-51-like kinase 1 VADT Veterans Affairs Diabetes Trial VALIANT Valsartan in Acute Myocardial Infarction VEGF vascular endothelial growth factor VEH vehicle solution treated Vps vacuolar protein sorting gene XIAP X-chromosome-linked IAP
xviii 1 Chapter 1
Introduction and Research Objectives
1 1.1 Prevalence of Cardiovascular Diseases
Cardiovascular diseases (CVD) are among the leading causes of death and account for over 17.5 million deaths worldwide annually [1]. In 2006, CVD accounted for the deaths of 69,019 Canadians. That number correlates to 30% of all deaths in 2006 and is one of the top three causes of mortality in the country. Of all cardiovascular deaths, 54% were due to ischemic heart disease and 23% to myocardial infarction (heart attack). There are an estimated 70,000 myocardial infarctions each year in Canada, thus at a rate of one myocardial infarction every 7 minutes. CVD is also the major cause of hospitalization, posing an economic burden of over $22.2 billion every year to the Canadian government [2],
1.2 Prevalence and Characteristics of Diabetes
Diabetes mellitus (hereafter referred to as diabetes) is defined by the elevation of glucose concentrations in circulation and the increase in circulating glucose level is caused by the failure of the pancreatic P-cell to secrete enough insulin, the development of bodily insulin resistance, or both [3]. Diabetes, affecting over 285 million people worldwide and 3 million Canadians, is known to be associated with many medical complications which include macro and microvascular diseases, nephropathy, and neuropathy [3]. Diabetes is classically divided into two main types.
Type 1 diabetes, also known as insulin dependent diabetes, is caused by lack of enough insulin secretion due to the destruction of the pancreatic P-cell. The destruction of the insulin secreting cells is thought to be caused by the T-lymphocyte- dependent autoimmune response. Although the exact cause initiating this response is still unknown, genetic element is known to be a major factor [4]. Patients develop this type of diabetes most often during their childhood or their teens, and approximately
10% of diabetic patients have this type of diabetes. Type 2 diabetes, affecting the remaining 90% of diabetic patients, is usually diagnosed in adults over 40 years old and is associated with the development of bodily insulin resistance [4]. Insulin resistance development is accelerated in the presence of sedentary lifestyle, aging, and genetic predisposition. In the late, type 2 diabetes is often seen in combination with a decreased production of insulin from the pancreas [3].
1.3 Diabetes is an Independent Risk Factor for CVD
Diabetes is classified as an independent risk factor for the development of CVD.
In women, over 54.8% of diabetic individuals have CVD and more than 78.6% of men with diabetes have some form of CVD [5]. The presence of diabetes is also known to significantly increase the risk of dying from heart disease [6]. Several studies [7-9] indicate that that both dyslipidemia and hyperglycemia are associated with a higher risk of development of, and mortality due to, heart failure. Indeed, it has been reported that at least 68% of people with diabetes die of some form of heart disease [10].
1.4 Pathophysiology and Cardiac Remodeling in Heart Failure
Heart failure is the most common CVD and occurs when the heart is unable to pump sufficient blood to meet the demands of the body [11]. The development of heart failure is associated with changes in cardiac function coupled with alterations in left ventricular structure and chamber configuration [12, 13]. Heart failure is accompanied by complex and progressive left ventricular (LV) remodeling which is 3 characterized by an elevated level of cardiomyocyte death, abnormal extracellular matrix (ECM) composition, hypertrophy of the myocytes, impaired vascularization in the heart, proliferation of myofibroblasts and changes in cardiac metabolism [13, 14],
Although some of the initial changes that occur during cardiac remodeling may be beneficial, prolonged progression of cardiac remodeling events ultimately leads to a decline in overall cardiac function [13]. Greater insult given to the heart is associated with a greater degree of cardiac remodeling [15] and a greater degree of cardiac remodeling is coupled with a higher incidence of the development of cardiac dysfunction and mortality from heart failure [16]. VALIANT (Valsartan in Acute
Myocardial Infarction), a large scale clinical echocardiographic study of 15,000 patients with myocardial infarction (MI), clearly indicated that the existence of any form of cardiac remodeling was associated with a greater risk of death from MI, heart failure, or resuscitated cardiac arrest [17]. Echocardiography is the most commonly used technique to identify LV remodeling in clinical settings for its wide availability and noninvasive process [12]. LV ejection fraction (EF), which is a measurement of the amount of blood ejected out of the LV per systolic stroke, is often used in clinical practices to measure cardiac performance along with the LV chamber size determined from the LV end diastolic and systolic volumes (EDLW and ESLW) [12].
MI accounts for 23% of all cardiovascular deaths in Canada [2]. MI occurs when a coronary artery is blocked and cannot supply the nutrients and oxygen required by the heart. This state of low nutrient and blood supply is called "ischemia" and is accompanied by an accumulation of lactate, low pH, and a decreased ability to produce adenosine triphosphate (ATP). Following ischemia, the heart needs to be
"reperfused" (restart blood flow) however, reperfiision itself may cause "reperfusion 4 injury" due to the robust production of reactive oxygen species (ROS) [18]. Early reperfusion after acute MI is known to be beneficial and decrease mortality. However, whether reperfusion after a longer MI over the time range that allows myocardial salvage is beneficial is not entirely clear [18,19].
Although it is now known that cardiac remodeling is provoked by a diverse cardiac injury, including pressure overload and cardiomyopathies induced by factors such as hyperglycemia and pathogenic infection [12,20], it was first described as a set of responsive events that occur in the heart after MI [12]. Early response after MI includes cell death in the infarct area, which stimulates the inflammatory response and recruitment of inflammatory cells to the site of injury, fibroblast proliferation, and fibrotic repair of the infarct area. Mature scar formation is required to prevent infarct expansion caused by myocyte slippage and cardiac wall rupture [21]. Cardiac remodeling also induces changes to the non-infarcted remote region of the heart, causing the elongation of the myocytes and the entire LV to dilate, and a shift from an elliptical to a more spherical chamber shape [22]. By increasing the LV volume, this adaptive response to volume overload may initially be beneficial in maintaining normal stroke volume and cardiac output, but this will eventually become maladaptive over an extended time period and lead to a decline in LV function [12]. Interstitial fibrosis is enhanced in the remote area of the heart as well, making the LV stiff and difficult to relax during the pumping of blood [12]. Figure 1.1 displays a schematic representation of the progression of geometric cardiac remodeling over time post-MI.
5 POST-MI REMODELING
nnwR npvi w_J •» ImuflWant Saw FMnwtfan• MM Rupture
Figure 1.1: Progression of cardiac remodeling post-MI. The initial remodeling phase exhibits infarct area thinning caused by cell death and elongation of remaining cardiomyocytes. The formation of fibrous scar tissue leads to strong mature scars. These scars inhibit further myocyte slippage, infarct area expansion, and cardiac wall rupture. Over time, myocytes in the remote area are hypertrophied and elongated, resulting in the dilation of LV chamber. Accumulation of collagen between cells, termed interstitial fibrosis, is also enhanced in the remote area. Figure reprinted with permission with a slight modification from [12],
Three distinct patterns of LV remodeling based on measurement of the LV mass index (LVMi) and relative wall thickness (RWT) and cardiac geometry have been identified: concentric remodeling (normal LVMi and increased RWT), eccentric hypertrophy (increased LVMi and normal RWT) and concentric hypertrophy
(increased LVMi and RWT) (Figure 1.2). Each pattern is associated with a high degree of morbidity and mortality; concentric hypertrophy carrying the worst prognosis, followed by eccentric hypertrophy, and finally, concentric remodelling
[17].
6 95 (?) >95 (?) "5(cf) >115 (cf) (.aft Ventricular Mas* Index (jjm/m2)
Figure 1.2: Three distinct patterns of cardiac remodeling based on LVMi and RWT. These are: concentric remodeling (normal LVMi and increased RWT), eccentric hypertrophy (increased LVMi and normal RWT) and concentric hypertrophy (increased LVMi and RWT). Figure reprinted with permission from [12].
1.5 Cardiomyocyte Death in Cardiac Remodeling
Cardiomyocyte cell death has been reported in a number of myocardial pathologies including myocardial infarction and heart failure [23-27]. As cardiomyocytes have a very limited capacity to proliferate, a small reduction in the number of cardiomyocytes is detrimental to cardiac function [28]. Necrosis was initially considered as a major mechanism through which cardiomyocytes are lost during cardiac remodeling, although it is now believed that apoptosis may have more important pathophysiological consequences in the development and progression of heart failure [29, 30]. Altered cardiomyocyte autophagy has also been detected in the failing heart, although whether the upregulation of autophagy is protective or detrimental to the survival of cardiomyocytes is not entirely clear. Nevertheless,
7 autophagy is thought to play an extremely important role in the pathogenesis of heart failure and has recently acquired a great deal of attention [31, 32].
1.5.1 Apoptosis
1.5.1.1 Caspases
Apoptotic pathway is largely regulated by a family of caspases of which 14 members have been identified in mammals. These include apoptosis initiator caspases, caspase-2, -9, -8, -10 and -12, and effector caspases, caspase-3, -6 and -7 [33].
Caspases are a class of cysteine proteases that hydrolyses protein peptide bonds following an aspartic acid residue [34]. Caspases are synthesized as zymogens and require proteolytic cleavage after the aspartic acid residue within the small linker regions between the N-terminal prodomain and the C-terminal catalytic domain consisting of p20 and plO subunits for activation [35],
Initiator caspases contain a caspase recruitment domain (CARD) or a death effector domain (DED) which interacts with adaptor proteins to aid in the autoproteolysis of caspase for its activation. Activated initiator caspase then cleaves and activates downstream effector caspases and caspase activation cascade is initiated.
Aside from cleaving its downstream target protein, activated effector caspase can also cleave more effector caspases to amplify the apoptotic signal [33]. It is believed that the completion of the apoptotic process is extremely rapid once the caspases are activated [36].
8 1.5.1.2 Types of Apoptosis
There are two principal apoptotic pathways that have been described to date: the extrinsic pathway, which involves the activation of cell surface death receptors by specific ligands; and the intrinsic pathway, which is driven by the mitochondria [33].
A schematic diagram briefly describing each pathway is found in Figure 1.3.
Extrinsic pathway ) Intrinsic pathway
Death ligand Extracellular stimuli Receptor Cell Membrai
Intracellular stimuli
Pro-caspase 8
Caspase 8
Pro-caspase 9 Caspase 3 activation
Mitochondria t f " Apaf-l X » • Caspase 9 activation Apoptosorne | dATP Cell Death
Figure 1.3: Mechanism of extrinsic and intrinsic apoptotic pathways. Extrinsic pathway involves the activation of caspase 8 by the activation of the death receptor. Intrinsic pathway is activated both via extracellular and intracellular stimuli and involve mitochondria. Figure redrawn from [37].
(A) Extrinsic Pathway
The extrinsic pathway is initiated by the binding of a death ligand to its cell surface death receptor. The ligand in a trimeric form is thought to bind to the receptor, which also exists in a trimer [38]. The "death" ligand can be both membrane bound 9 proteins such as the Fas ligand or an extracellular soluble protein such as TNF (tumor necrosis factor)-alpha. Death receptors contain an intracellular motif called the death domain (DD). Upon ligand binding to the receptor, formation of a large protein complex at the receptor named death-inducing signaling complex (DISC) composed of the homotypic protein interactions between DD- and DED-containing proteins, is initiated [33, 37]. Taking Fas death receptor as an example, Fas ligand binding to Fas receptor leads to the recruitment of an adaptor protein FADD (Fas-associated via death domain). FADD binds to the DD of the receptor through its complementary C- terminal DD. It contains a DED on its N-terminal region and to this DED, procaspase
8, which also contains a DED, is recruited. Once recruited, procaspase 8 dimerizes and this dimerization allows autoproteolysis and activation of the caspase. Activated caspase 8 participates in the cleavage of downstream effector caspase 3 and Bid, a proapoptotic Bcl-2 protein. The cleaved product of Bid at the C-terminal end is known as tBid (truncated Bid) and it serves as a link between the extrinsic and the intrinsic apoptotic pathway by translocating to the outer mitochondrial membrane after being myristoylated [26,39],
(B) Intrinsic Pathway
In contrast to the extrinsic pathway, which may only be induced by a set of specialized stimuli, the intrinsic pathway can be activated by a variety of both extracellular and intracellular stimuli such as nutrient deprivation, irradiation, and oxidative stress [26]. The intrinsic apoptotic pathway involves the loss of mitochondrial membrane potential (A*Pm) and the release of cytochrome c (cyt c) caused by opening of the mitochondrial permeability transition pore (MPTP) in the
10 outer mitochondria membrane. After the release of cyt c into the cytosol, it interacts with an adapter protein, apoptosis-activating factor (Apaf)-1. The binding of cyt c to
Apaf-1 releases the autoinhibition of Apaf-1 to increase its affinity to dATP. The binding of dATP promotes the oligomerization of this complex into a stable wheel like structure composed to 7 Apaf-1-cyt c-dATP monomers, termed apoptosome [40].
Apoptosome recruits caspase 9 in dimers through the interaction of Apaf-1 CARD and procaspase 9 CARD and facilitates the activation of the procaspase [41]. Similar to the activation mechanism for caspase 8, caspase 9 is activated through autocleavage and further participates in the activation of downstream effector caspase 3 by recruiting it to the complex for activation [42]. In order to inhibit the accidental activation of caspases, the activation of these apoptosis executing proteins is also regulated at another level with the existence of the inhibitor of apoptosis (IAP) proteins. The most well characterized IAP protein is X-chromosome-linked IAP,
(XIAP), which binds to the activated form of caspases in the active site to directly prevent substrate binding. It also possesses E3 ubiquitin ligase activity and can target the bound caspase for proteosomal degradation [43]. For example, XIAP binds to caspase-9 after its first cleavage at Asp315 and keeps the caspase inactive. However, further cleavage of caspase-9 at Asp330 will remove the XIAP binding site and allows its full activation [33]. Upon activation of apoptosis, IAP inhibitors Smac
(second mitochondria-derived activator of caspases) and Diablo (direct LAP-binding protein with low pi) are also released from the mitochondria and bind to XIAP to disrupt the interaction of XIAP with caspase-9 and release the inhibitory effect of
XIAP [33]. Once activated, caspase-3 cleaves a variety of structural and regulatory proteins to complete the apoptotic process. For example, caspase 3 causes cell 11 shrinkage and nuclear damage such as DNA fragmentation and chromatin condensation [44] which are characteristic apoptotic features, by cleaving cytokeratin
18 [45], actin [46] and PARP (Poly (ADP-ribose) polymerase) [47].
The key event that initiates intrinsic apoptotic pathway as well as significantly amplifies the extrinsic apoptotic signal is the release of pro-apoptotic factors from the
MPTP. The opening of the MPTP is largely regulated by two opposing classes of the
Bcl-2 protein family members.
1.5.1.3 Bcl-2 Family Proteins
Bcl-2 (B-cell leukemia/lymphoma 2) was first discovered as an oncogene which promotes the survival of cancer cells [48]. Further studies of this protein lead to the discovery of another family of proteins which shares up to 4 regions of homology with Bcl-2, the Bcl-2 homology (BH) domains [33]. Nonetheless, different family members of this large family of proteins may have distinct functions in the regulation of apoptosis and are divided into the following groups: the anti-apoptotic members, the pro-apoptotic multidomain members, and the pro-apoptotic BH3 domain only members.
The anti-apoptotic Bcl-2 family members, including Bcl-2 and Bcl-xL, possess four BH domains (BH1-4), and are thought to inhibit the MPTP opening by directly binding and sequestering the pro-apoptotic proteins. They have also been shown to stabilize the outer mitochondrial membrane which may be destabilized by the insertion of proapoptotic members. Bax and Bak are multidomain pro-apoptotic proteins [33]. Either Bax or Bak is absolutely required for the execution of the intrinsic apoptotic pathway and cells lacking both proteins are resistant to apoptosis
12 [49], Bax, a 25kDa pro-apoptotic cytosolic protein, undergoes a conformational change upon apoptotic stimulus leading to the exposure of the C-terminal transmembrane domain and allows it to be inserted into the mitochondrial membrane.
Translocation of Bax to the mitochondria is followed by the formation of high- molecular weight oligomers [50]. Bax oligomers possess channel-forming characteristic and may be able to form a pore alone or in association with the MPTP and the anti-apoptotic protein Bcl-2 [50]. The mechanism of Bax activation is still incompletely understood, however caspase 2 and several Bax-binding proteins are known to be involved [26, 51]. Removal of the inhibitory N-terminus of Bax by calpain as well as increase in Bax expression level are also seen during the activation of apoptosis [52, 53].
Although cyt c release from mitochondria may be directly induced by the formation of large pores by Bax, insertion of Bax into the mitochondria membrane is also believed to destabilize the membrane making it more permeable [33]. The localization and the interaction of Bax and Bcl-2 seem crucial in regulating apoptosis as overexpression of Bcl-2 inhibits apoptosis whereas overexpression of Bax induces apoptosis [54, 55].
BH3 domain-only Bcl-2 proteins show homology to other Bcl-2 family proteins only in the BH3 domain. BH3 pro-apoptotic proteins induce apoptosis in response to specific signals and transduce the cell death signals to Bax or Bak in order to activate intrinsic apoptotic pathway. For example, Bid is cleaved by caspase 8 and participates in the activation of apoptosis via extracellular death signal [26] by interacting with
Bax/Bak to facilitate oligomerization or by scavenging anti-apoptotic, anti-Bax proteins Bcl-2 or Bcl-xL [56]. 13 1.5.1.4 Molecular Targets Used to Identify Apoptosis
The classical definition used to define apoptosis was based on changes in cell morphology such as membrane blebbing, dark and dense cytoplasm, nuclear pyknosis, and the formation of apoptotic bodies [57, 58]. However, apoptosis is now mostly described by biochemical characteristics such as the externalization of phosphatidylserine (PS) to the outer cell membrane, activation of one or more of the caspases and DNA fragmentation. Apoptosis is also often characterized by investigating the expression and localization of Bcl-2 protein family members which can provide both anti- and pro-apoptotic effects [33].
1.5.1.5 Apoptosis and the Development of Heart Failure
It has been described that the apoptotic rate is significantly higher in a failing heart and it has been estimated that an increase from 0.001% to 0.08% occurs [59].
Cardiomyocyte apoptosis occurs under a variety of damaging stimuli including hypoxia, oxidative stress, metabolic alterations, and overt stretch[26]. In particular, ischemia-reperfusion in both rodent and human hearts results in a significant increase of apoptotic cells and the inhibition of apoptosis has proven to reduce infarct area and the development of cardiac dysfunction [26, 37, 60-62]. Cardiac chronic ischemia is also associated with a progressive loss of cardiomyocytes through apoptosis, triggering compensatory left ventricular hypertrophy and ultimately progression to heart failure [63]. An increased level of apoptosis is also observed in aged myocardium in both animal and human heart samples and it is considered a contributing factor to the development of heart failure in the elderly [64-66],
Inhibition of caspase activity has been shown to be cardioprotective, further
14 demonstrating the significance of the detrimental role of apoptosis in the development of heart failure. Diminished cardiac function accompanied by extensive hypertrophy caused by aortic stenosis was rescued by the inhibition of apoptosis with agents such as vascular endothelial growth factor (VEGF) [67] and statins [68]. Reduction of apoptosis by caspase 3 inhibition has also been demonstrated to improve cardiac function and reduce mortality in an animal model of hypertrophy-induced heart failure
[26].
1.5.2 Autophagy
Autophagy, literally meaning "self-eating", is an evolutionarily conserved process from yeast to mammals that involves the degradation and recycling of cytoplasmic components. It is initiated in response to starvation, reactive oxygen species (ROS) and accumulation of damaged organelles or proteins and plays important roles in survival, homeostasis and the development of organs [69].
1.5.2.1 Types of Autophagy
Three types of autophagy have been described in eukaryotic cells: microautophagy, chaperone-mediated autophagy and macroautophagy. These three are summarized in Figure 1.4 and the emphasis for my studies will be on macroautophagy.
15 t t
(b) -t »icrt •> °t>h, *8y
Figure 1.4: The three types of autophagy described in mammalian cells. These are (a) macroautophagy, (b) microautophagy, and (c) chaperone-mediated autophagy (CMA). Figure reprinted with permission from [70].
(A) Microautophagy
Microautophagy is a direct engulfment of cytoplasm at the lysosomal membrane. It is characterized by the formation of lysosomal membrane invaginations, which are believed to be pinched off and degraded inside the lysosome along with the content of the vesicles. Its exact molecular mechanisms and physiological roles have not been identified yet [71]. Research on microautophagy has mainly been done in yeast and its role in mammalian cells is still poorly understood.
16 (B) Chaperone-Mediated Autophagy
Chaperone-mediated autophagy (CMA) is a selective form of autophagy which is involved in the elimination and degradation of oxidized and misfolded proteins. Substrates for CMA are directly translocated across the lysosomal membrane. The substrates are first recognized by the cytosolic chaperone hsc70 (heat shock cognate of the heat shock protein 70 family) via a KFERQ-related motif within their amino acid sequence. The hsc70-substrate complex then attaches to the lysosomal membrane by binding to the lysosomal membrane protein, lysosome- associated membrane protein-2A (LAMP-2A). Upon substrate binding, LAMP-2A forms a translocation complex on the membrane and delivers the hsc70-substrate complex across the lysosomal membrane. The crossing of the substrate is further assisted by a luminal form of hsc70 and the substrate is rapidly degraded once it is delivered to the lysosome [70]. This type of autophagy has only been described in mammalian cells and its importance is implicated in the prevention of the age-related decline of organ homeostasis and function as well as neuronal survival, and kidney growth [72],
(C) Macroautophagy
Macroautophagy (hereafter, referred to simply as autophagy) is thought to be the most active form of autophagy [70] therefore it is the most studied and a principal focus in the pathophysiology of many diseases. It is characterized by the formation of the autophagosome, a double-membrane structure that encloses a portion of cytoplasm. The outer membrane of the autophagosome fuses with the lysosome to form autolysosome and its content is degraded by lysosomal proteases. Broken down macromolecules such as amino acids and fatty acids are transported back into the cytosol through membrane permeases and then recycled. Macroautophagy can selectively target and degrade certain organelles such as mitochondria (mitophagy) and ER (ER-phagy) and protein aggregates (aggrephagy), lipid droplets (lipophagy), and invading microorganisms (xenophagy) [72]. Selective mitochondrial degradation through autophagy, known as mitophagy, plays an essential role in the quality control of damaged mitochondria. Although specific mechanisms of how autophagosomes are able to recognize which mitochondria are to be degraded is not entirely clear, the
MPTP opening is suggested to serve as a signal to be recognized and induce mitophagy [73].
The existence of autophagy has been recognized since late 1950s, however a lack of molecular understanding of autophagy mechanisms made it extremely difficult to study autophagy until very recently. Our detailed understanding of the autophagy machinery only came within the last 15 years with the discovery of the autophagy- related proteins (Atg) first in yeast followed by the identification of homologues in eukaryotes [69],
Atg Proteins
Atg proteins are necessary for the formation and elongation of autophagosomal membrane, and to date, over 30 Atg proteins have been identified.
The ATG genes are highly evolutionarily conserved, and most of the mammalian
ATG genes have been identified simply through conventional BLAST search [74].
The Atg proteins required for autophagy are classified into the following independent functional categories: 1) the Atgl/ULK kinase complex (Atgl-13-17-29-
18 31); 2) the transmembrane proteins Atg9 and VMP1; 3) the class III phosphatidylinositol (PI) 3-kinase complex (Atg6-Atgl4-Vpsl5-Vps34); and 4) the ubiquitin-like Atgl2 (Atgl2-Atg5-Atgl6), and Atg8 conjugation (Atg8-PE) systems
[74].
Atgl/ULK complex
The activation of the Atgl/ULK complex is the most upstream event involved in autophagosome formation. Autophagosome formation is initiated by the activation of ULK complex and the primary regulator of this complex is the target of rapamycin
(TOR). Atgl is a serine threonine protein kinase required for autophagy which was first discovered in yeast. The mammalian homologues for this protein were later found, which are uncoordinated-51-like kinases 1 (ULK1) and 2. ULK1 complexes with mAtgl3, focal adhesion kinase family interacting protein of 200kDa (FIP200), and AtglOl [69, 74]. This complex is located in the cytoplasm and associates with the autophagosome pre-cursor membrane, phagophore, upon induction of autophagy. Its role is implicated in the recruitment of the downstream Atg proteins, however the exact mechanism of how the activation of the ULK complex leads to the generation of autophagosome is still under investigation [69,74].
Transmembrane Atg9 and VMP1
Atg 9 is a transmembrane protein which spans the membrane 6 times exposing both the C and the N-termini in the cytoplasmic side [69]. It is mainly located on the membrane of the trans-Golgi network and late endosomes and translocated to the autophagosomal membrane upon induction of autophagy. Its movement is dependent
19 on the ULK1 and Vsp34 activities [75] and its function in the induction of autophagy is implicated in the acquisition and delivery of membrane material to the shaping autophagosome [69,71].
VMP1 is only found in mammalian cells. It is localized on the plasma membrane but is moved to the autophagosomal membrane and colocalizes with LC3II and beclin-1 during autophagy [76]. VMP1 overexpression has been shown to force the induction of autophagy even under nutrient-rich condition, and knocking out this protein blocks autophagy [77]. The function of VMP1 is thought to be the recruitment of beclin-1 and other Class III phosphatidylinositol-3 (PI3) kinase complex members to the phagophore [76].
Class III PI3 Kinase
In mammals, two classes of PI3 kinase exits, class I and III [69]. Class III PI3 kinase vacuolar protein sorting gene 34 (Vps34) is essential for the induction of autophagy and forms a complex with pi50, its regulatory kinase, beclin-1 (Atg6), and
Atgl4. Atgl4 is implicated in the role of directing Class III PI3 kinase complex to phagophore to initiate autophagy and the nucleation and assembly of the initial phagophore membrane is controlled by the beclin-1:Class III PI3 kinase complex. beclin-1 is a BH3 protein which binds to a variety of proteins including VMP1 and
UVRAG. UVRAG is known to bind to Bif-1 and thought to provide machinery for the phagophore membrane to deform and bend during elongation [78]. UVRAG also interacts with the class C Vps/HOPS proteins to promote and accelerate autophagosome fusion with the lysosome [79].
20 Recruites Atg proteins
Uphagophorc Delivers membrane Recruites material ^ ^ W in-1 complex «* ** • • •
Recruited and initiates Phagophore assembly
Plasma Memrane
Figure 1.5: Role of Atg proteins required for autophagosome assembly. ULKl complex activation is the most upstream event required for autophagosome formation. It is involved in the recruitment of other Atg proteins to the phagophore. Atg 9 is translocated from the Golgi complex and believed to participate in the autophagosome formation by delivering membrane materials. VMP is also translocated from the plasma membrane and recruits Beclin-1 complex. Beclin-l:Class III PI3 kinase complex initiates phagophore assembly when recruited to the phagophore.
Ubiquitin-Like Conjugation Systems
The two ubiquitin-like conjugation systems of Atg12 and Atg8 are essential for the elongation of the autophagosomal membrane. Atgl2 is first activated similarly to ubiquitin by the El-like protein Atg7 and E2-like AgtlO protein. Atg 12 is then covalently conjugated to Atg5 on a lysine residue and this complex localizes with a 21 homodimer of Atgl6 which is then transferred to the phagophore. The definite origin of the phagophore is still unknown, however the endoplasmic reticulum is thought to be the source [80]. At the same time, LC3 (Atg8) is cleaved by Atg4 at the C-terminus to expose a glycine residue and to transform LC3 to what is known as LC3-I. LC3-I is activated by another El-like protein Atg7 which transfers LC3I to E2-like protein
Atg3 which conjugates LC3-I to phospatidylethanolamine (PE) to form LC3-II. LC3II is then recruited to the phagophore membrane to participate in the membrane elongation in Atg5-dependant manner [31, 81]. Atg5 is believed to be a rate-limiting step for autophagy as overexpression of this protein increases the rate of autophagy
[82]. The schematic diagram describing the macroautophagy ubiquitin-like conjugation system can be found in Figure 1.6.
22 Aig12-MgS cwtugMkm LC3-N foranHon Atg12 •5® 1 E; Ptmgophoim Autophtgoaonm Mtaphagototftosaum Figure 1.6: Ubiquitin-like conjugation system of the macroautophagy machinery. This system involves several Atg proteins and they participate in elongation and closure of the autophagosome. Figure reprinted with permission from [18]. 1.5.2.2 Crosstalk Between Autophagy and Apoptosis The molecular mechanisms and the proteins involved in the regulation of the two modes of self-destruction, apoptosis and autophagy, are clearly quite different. However, autophagy is now thought to be able to mediate apoptosis, either by opposing or enhancing it, or in fact representing an alternative form of cell death distinct from apoptosis [32, 83]. The two pathways merge at several points, however the most studied is the interaction between Beclin-1 and Bcl-2. Autophagy is inhibited by anti-apoptotic protein Bcl-2 by Bcl-2 directly binding to the BH3 domain of Beclin-1 to inhibit its association with Vsp34, thus inhibiting the activation of class III PI3 kinase complex. The association of Bcl-2 and Beclin-1 may be disrupted by 23 JNK1-mediated Bcl-2 phosphorylation at Y69, S70 and S87 and inhibition of JNK1 or expression of a non-phosphorylatable mutant Bcl-2 abolishes the dissociation of Bcl-2 and beclin-1 [84], Beclin-1 phosphorylation at T119 by death-associated protein kinase (DAPK) has also been shown to promote the dissociation of Beclin-1 from its inhibitor [85]. The binding of the two proteins can also be competitively disrupted by other BH3 protein binding to Bcl-2 [86]. Beclin-1 has also been demonstrated to be a caspase 3 substrate and cleavage of Beclin-1 by caspase 3 during apoptosis limits the possibility of autophagy oppose the apoptosis process [87]. It has also been shown that antiapoptotic protein FLIP, which is the inhibitor of caspase 8 activation, is able to act as an anti-autophagy molecule by binding to Atg3 and block the formation of LC3II [88]. 1.5.2.3 Autophagy Signaling Pathways (A) mTOR Mammalian TOR (mTOR) is an important signaling molecule involved in the regulation of autophagy in response to changes in the levels of nutrients and growth factors in mammals [69]. mTOR forms two distinct complexes: mTORCl which is composed of mTOR, raptor and GPL, and mTORC2 consisting of mTOR, rictor, mSinl and GPL. The activity of mTORC is regulated by tuberous sclerosis complex 1/2 (TSC1/TSC2), proteins that receive input from several signaling pathways such as the PI3 kinase-Akt, the ERK1/2, the p38MAPK and the LKB1-AMPK pathways. Phosphorylation of TSC2 by Akt or ERK1/2 leads to the disruption of the stable homodimer complex of TSC1/TSC2 and results in the loss of TSC2's GTPase- activating kinase activity toward Rheb. This in turn activates mTOR [89]. mTORCl is 24 I involved in the regulation of ULKl-mAtgl3-FIP200-Atgl01 complex by directly binding and inhibiting its activation. The interaction between the two complexes occurs between raptor and ULK1 and both in vitro and in vivo data also suggest that mTORCl phosphorylates ULK1 and inhibit its kinase activity [90]. (B) AMPK 5'AMP-activated protein kinase (AMPK) is another sensor of cellular metabolic stress and it is activated by an increase in AMP/ATP ratio through the upstream Large Kinase B1 (LKB1) kinase. AMPK regulates autophagy by directly modulating TSC2 activation. In contrast to phosphorylation by Akt, phosphorylation of TSC2 at SI345 residue by AMPK results in the activation of TSC2 and leads to the inactivation of mTOR. It has also been shown that AMPK can induce autophagy by directly phosphorylating raptor and inhibiting the activity of mTORCl [91]. AMPK is also demonstrated to phosphorylate p27kipl, a cell cycle inhibitor, at T198 and stabilize the protein. The stabilized p27kipl attracts cyclin-dependent kinase 4 (Cdk4) and the depletion of free Cdk4 is implicated in the induction of autophagy [92], Signaling pathways controlling autophagy discussed here are briefly summarized in Figure 1.7. 25 Akt AMPK ERK1/2 \ Hiptof m mTOR Figure 1.7: Signaling pathways controlling autophagy. mTOR regulates autophagy by altering the activity of the ULK1 complex, a complex required for the initiation of autophagosome formation. The activity of mTORCl can be regulated by various kinases such as AMPK and Akt. AMPK can activate autophagy by inhibiting mTORCl activity by either directly inhibiting Raptor or by activating the upstream TSC2. 1.5.2.4 Molecular Targets Used To Identify Autophagy Autophagy was first detected by electron microscopy to observe the characteristic double-membrane vesicles (autophagosomes) [71]. Until recent advances in our understanding of the autophagy machinery, such use of microscopy was the major method used to detect autophagy. More recently however, PE- conjugated LC3 (LC3II) is often used for a qualitative and quantitative analysis of autophagosome formation. As LC3II is directly incorporated into the autophagosomal membrane, the level of LC3II indicates the amount of autophagosome present in the cell [71]. When studying autophagy, it is extremely important to examine both the steady state and the accumulation state as autophagy is a dynamic process involving the constant production and degradation of autophagosome [93]. Steady state looks at 26 the induction of autophagy by examining the level of autophagosome at a certain time point whereas accumulation state allows us to examine whether the entire autophagy process was completed. Bafilomycin A1 (BafAl) is a vacuolar H+-ATPase inhibitor that inhibits autophagosome-lysosome fusion and is commonly used in combination with LC3 Western blot to examine the accumulation of autophagosome [93]. Blockage of autophagosome fusion with lysosome with BafAl will inhibit the degradation of LC3II. Even though LC3II levels may appear low at steady state, this could be because of either a downregulation of autophagosome formation or a rapid degradation of autophagosomes. At the same time, accumulation of LC3II at steady state could also be the result of inhibition of autophagosome degradation by lysosome or a robust increase in the activation of autophagy. Therefore the accumulation of LC3II during the inhibition of autophagosome-lysosome fusion needs to be examined in order to understand the absolute level of autophagosome formation [71]. p62 is another protein which is often used to detect the upregulation of autophagy. This protein has a high affinity to LC3 and serves as a link between LC3 and ubiquitinated proteins that are to be degraded through autophagy [94]. Since p62 becomes incorporated into autophagosome and degraded as autophagosomes are digested by the lysosome, the decrease in p62 protein is used as an indication of autophagosomal degradation. Cathepsin D expression is also often used to determine the level of autophagy in combination with other autophagy markers. It is among the most abundant lysosomal proteinases and its expression and activity are increased during autophagy [95,96]. 27 1.5.2.5 Autophagy and the Development of Heart Failure Autophagy exists in a healthy heart and plays an essential role in the quality check of mitochondria and the turnover of other organelles and long-lived proteins [31]. A certain level of autophagy is required for the maintenance of a healthy heart. For example, blockage of autophagosome degradation seen in patients with cardiomyopathic Danon disease, which results from the deficiency of LAMP-2, a protein needed for autophagosome docking with the lysosome, leads to intracellular accumulation of abnormal mitochondria which results in cardiomyopathy [97], While autophagy is an important part of the maintenance of a healthy heart, it is known to be upregulated in a failing heart [31, 81, 97]. It is controversial whether this is a cause of heart failure or an attempt to rescue the heart and we still do not understand the elements differentiating so-called "good" autophagy from "bad" autophagy. However, the stimulus triggering the autophagic event, the duration of autophagy, and the degree of autophagy seem to be extremely important in determining the role of autophagy in cell survival [97]. Currently, there is more evidence suggesting that the upregulation of autophagy is cardioprotective, particularly under ischemic stress. Autophagy has been implicated in the protective mechanism of cardiac preconditioning, and the upregulation of autophagy caused by repetitive ischemic events in swine hearts was associated with low levels of apoptosis [98]. Preconditioning agents such as statin, exercise, and starvation are also known to increase the level of autophagy [81], Upregulation of autophagy has also been shown to be protective against ischemia- reperfusion injury in both in vitro and in vivo models [99-101]. One potential mechanism whereby autophagy is considered to confer protection is sequestering leaky mitochondria that could otherwise trigger apoptosis [81,102]. In vitro, enhancement of autophagy by overexpressing beclin-1 inhibited the activation of Bax, and the inhibition of autophagy by treatment with pharmacological inhibitor of autophagy, 3-MA, or expression of a dominant negative form of Atg5 increased the level of apoptosis induced by ischemia-reperfusion [100]. In vivo experiment demonstrated that upregulation of autophagy by the moderate activation the AMPK pathway during ischemia was cardioprotective. However, excessive activation of autophagy by increased expression of beclin-1 during the reperfusion period was associated with enhanced ischemia reperfusion injury [101, 103] and it verified that fact the level and the mechanism of autophagy activation may be extremely important. Increased level of autophagy seen in aortic constriction-induced pressure-overload, however, has also been shown to be associated with the development of heart failure. The degree of autophagy correlates with the extent of cardiac hypertrophy and the pressure overload-induced left ventricular remodeling was minimized in genetically modified beclin-1 +/- mice [104]. 1.6 Cardiac ECM Remodeling 1.6.1 Role of ECM in the Healthy Heart ECM was classically viewed as an inert area which only provided structural support to hold cells together in place [105]. However more recently, it is appreciated as a dynamic interactive milieu which contains a large reservoir of signaling molecules to control the survival, proliferation, growth, development, migration, shape and function of the cells it surrounds [106], It is also an adaptive area which is 29 able to adjust its composition and organization in response to changes in the surrounding environment [105]. The ECM plays an essential role in the development, signaling, and remodeling of the heart, as well as providing structural integrity, and converting the shortening of individual cardiomyocytes into overall cardiac contraction [107], Continuity and interconnectivity of the matrix surrounding the myocytes is extremely important in the generation of powerful contraction. Although isolated cardiomyocytes are only capable of 15% linear shortening, the average ejection fraction during one cardiac cycle is >40% with fractional shortening of >20%, demonstrating the importance of matrix organization and integrity [108]. It is also known that the mammalian heart, which shows extensive matrix interconnection, can generate more force compared to the frog heart which shows poorly interconnected collagen fibers [109]. The cardiac ECM is also known to play a fundamental role in the adaptation to pathological stress and injury which facilitates cardiac remodeling [110] which will be explained in subsequent sections. 1.6.2 ECM Structure ECM is a complex organization of various secreted extracellular macromolecules of two main classes: 1) fibrous proteins including collagen, elastin, fibronectin, and laminin, and 2) the polysaccharide called proteoglycans [106]. The collagen fibers give tensile strength and aid in the organization of the matrix, elastin gives resilience, laminin and fibronectin serve as an adhesive between the ECM and the cell, and the proteoglycans form a gel-like structure to maintain hydration of the ECM and provide an environment for diffusion of nutrients, metabolites, and hormones [106]. The cardiac ECM is primarily composed of type I and III collagen. 30 Type I collagen has the tensile strength of steel and constitutes >85% of total collagen in the heart. The remainder is predominantly type III collagen which is less rigid and provides elasticity [107], It is postulated that both the quantity and the quality of collagen, as well as the level of collagen cross-linking, is important in maintenance of a healthy heart (Figure 1.8). Both the organization of the fibrillar collagen weave, and the stabilization of collagen through cross-linking are essential for maintaining alignment of the myofibrils and the heart geometry during each cardiac cycle [107, 110] as well as to coordinate cardiac movement and enhance function. Figure 1.8: Cardiac ECM organization. Electron scanning microscopic images demonstrating collagen cross-linking and organization in healthy hearts and in hearts stimulated with (A) chronic supraventricular tachycardia (SVT) or (B) MI. The collagen weave organization and structure are disturbed after the heart is challenged with such insults. Figures reprinted with permission from [111] (A) and [112] (B). Changes in collagen abundance, type, level of cross-linking, and the spatial organization of the collagen weave occur in response to cardiac injury and are significant factors involved in the alteration of cardiac mechanics and cardiac remodeling events [107, 113]. Many laboratories have reported a reduction in organization of collagen network formation and the level of collagen cross-linking within the infarct area of a heart damaged by myocardial infarction. Destruction of normal collagen architecture and cross-linking has also been observed in infectious dilated cardiomyopathies, and abnormal accumulation of collagen in the ECM space is observed in a pressure overload model of heart failure. In the healthy heart, ECM is slowly but constantly degraded and replaced with newly synthesized components. Collagen has a half-life of 80-120 days and is replaced at an approximate rate of 0.6% per day [114]. The degradation of ECM is carried out by the four families of proteinases: aspartic, cysteine, serine, and metal ion (metalloproteinase) proteinases. The serine and metalloproteinases are believed to be the most involved in the degradation of the cardiac ECM [115]. The primary role of the serine proteinases in the heart is to activate the metalloproteinases, which are known to play an important role in cardiac ECM remodeling during the development of heart failure [116]. Tight control of metalloproteinase activity is essential in the balanced turnover of the ECM components for the maintenance of a healthy heart. 1.6.3 Matrix Metalloproteinases Matrix metalloproteinases (MMPs) are zinc-dependant proteinases, which degrade a wide spectrum of ECM components. They play major roles in tissue morphogenesis and wound healing. First MMP was discovered in 1962 as an enzyme responsible for tadpole metamorphosis [117]. Over 20 isoforms of this proteinase have been identified since its first discovery [118, 119]. There are two types of MMPs: the first is secreted into the interstitial space and the second bound to cell membrane. MMP activity is tightly regulated under normal physiological conditions at the level of transcription, activity of precursor zymogen, interaction with the ECM components, and inhibition by endogenous inhibitors known as TIMPs. MMP isoforms share structural similarity with 4 distinguished domains: propeptide, catalytic, hinge region, and hemopexin domains [119]. All MMP enzymes possess the following characteristics: 1) they are able to degrade ECM components; 2) they are expressed as latent preforms and later activated through proteolytic cleavage; 3) they require Ca2+ for stability; 4) they are optimally active at neutral pH; 5) they are inhibited by TIMPs; 6) they have cysteine- switch motif PRCGXPD (except for MMP-23) in the propeptide domain which keeps the enzyme in the inactive proform; and 7) they contain Zn2+-binding motif HEXGHXXGXXH in the catalytic domain [119, 120], Classically MMPs are divided into 5 different groups depending on their substrate preference, domain organization, and sequence similarities (Figure 1.9). The summary of known MMPs, their classification and the known substrates is shown in Table 1.1. 33 Protein structure of MMPs t //// catalytic domain B —t- Geiatinases Stromelysins Mentxana-type E3E^iZ3*- MMPs MaMlysin Figure 1.9: MMP Structure. (A) The Protein structure of MMPs can be divided into several distinct domains. (B) MMPs divided into 5 different groups depending on their substrate preference share similar protein structures governed by the domains they possess. Figure reprinted with permission from [120], 34 Table 1.1: MMPs and Their Substrates Reprinted with permission from P201 MMP Enzyme Classification Substrate Collagenases Interstitial MMP-1 Collagens I, II, III, VII, and X, gelatin, entactin, collagenase aggrecan Neutrophil MMP-8 Collagens I, II, and III, aggrecan collagenase Collagenase-3 MMP-13 Collagens I, II, and III, gelatin, fibronectin, laminins, tenascin Gelatinases Gelatinase A MMP-2 Gelatin, collagens I, IV, V, VII, and X, fibronectin, laminins, aggrecan, tenascin-C, vitronectin Gelatinase B MMP-9 Gelatin, collagens IV, V, and XIV, aggrecan, elastin, entactin, vitronectin Stromelysins Stromelysin 1 MMP-3 Gelatin, fibronectin, laminins, collagens III, IV, IX, and X, tenascin-C, vitronectin Stromelysin 2 MMP-10 Collagen IV, fibronectin, aggrecan Stromelysin 3 MMP-11 Fibronectin, gelatin, laminins, collagen IV, aggrecan Membrane- type MMPs MT1-MMP MMP-14 Collagens I, II, and III, fibronectin, laminins, vitronectin, proteoglycans; activates proMMP-2 and proMMP-13 MT2-MMP MMP-15 Activates proMMP-2 MT3-MMP MMP-16 Activates proMMP-2 MT4-MMP MMP-17 Not known MT5-MMP80 MMP-24 Activates proMMP-2 MT6-MMP MMP-25 Others Matrilysin MMP-7 Gelatin, fibronectin, laminins, collagen IV, vitronectin, tenascin-C, elastin, aggrecan Metalloelastase MMP-12 Elastin Unnamed81 MMP-19 Enamelysin MMP-20 Aggrecan MMP-23 Endometase MMP-26 35 MMP-1, -8, -13, and -18 are collagenases. They are able to cleave fibrillar collagens types I, II and III at a specific cleavage site located at % distance from the C-terminal end, generating Vi and V* collagen fragments, which will eventually denature into gelatins [119, 120]. Gelatins are degraded by gelatinase MMP-2 and 9. Although MMP-2 is also able to degrade fibrillar collagen and type IV collagen in basement membranes [119,120], they cleave gelatin more efficiently [121]. MMP-3, - 10 and -11 are known as stomelysins. They are active against a broad spectrum of ECM components including proteoglycans, laminins, fibronectins, vitronectin and some types of collagens. MMP-3 also activates a number of proMMPs [119, 120], Matrilysin MMP-7 and -26 lack the hemopexin domain. Besides ECM components, MMP-7 can cleave cell surface molecules such as TNF-alpha and Fas-ligand to activate the molecule [119]. Transmembrane MMPs are called membrane-type MMPs (MT-MMPs) and they are different from other MMPs as they are embedded in the cell membrane and are active on the extracellular side of the membrane. Members of this group include MMP-14 (MT-1 MMP), -15, -16, -17, -24 and -25 and all MT-MMPs are capable of activating MMP-2 with the exception of MT-4 MMP. These enzymes also have a fiirin cleavage site and are unique in that they are activated intracellularly, not by other MMPs which reside within the ECM [110,119]. 1.6.3.1 MMP Activation All MMPs are expressed as proMMPs in their non-active forms. The inactive state is maintained by the interaction between the thiol of the conserved cysteine residue in the propeptide domain, and the Zn2+ ion in the catalytic site. For the full- activation of MMP to occur, this "cysteine switch" of thiol-Zn2+ interaction must be 36 disrupted. This disruption can be created by: 1) the cleavage of the propeptide domain by another proteases such as MMPs, furin, and plasmin in vivo or 2) the reduction of the free thiol by agents such as SDS and p-aminophenylmercuric acetate (APMA) or at low pH and treatment with heat or reactive oxygen species in vitro. It is currently believed that MMPs go through autolytic cleavage of the propeptide domain upon activation via allosteric disruption of the cysteine switch by chemical agents and the cleavage of the propeptide domain is the final required step for the activation of MMPs [119, 121]. A schematic diagram summarizing the activation of MMP is found in Figure 1.10. ion proteolytic Atostooc ROS SDS/APMA . Figure 1.10: MMP activation. Latent MMP is kept inactive by the "cysteine switch". Disruption of thiol in the conserved cysteine residue in the propeptide domain and the Zn2+ ion in the catalytic site leads to the activation of the enzyme by exposing the active site of MMP. Proteolytic cleavage of the prodomain is thought to be required for full activation. Figure reprinted with permission from [121]. 37 1.6.4 Tissue Inhibitors of MMPs Tissue inhibitors of MMPs (TIMPs) are endogenous inhibitors of MMPs which mediate inhibitory activity through tight non-covalent binding to the catalytic domain of the active MMPs in a 1:1 molar ratio [107, 122]. Four different isoforms of TIMPs have been identified to date and all four are expressed in the heart [107, 123]. Between TIMPs, there is some degree of specificity in binding to different MMP family members and in their expression pattern throughout the body. TIMP-1 is a highly inducible TIMP but its MMP inhibitory range is more restricted than the other three TIMPs. TIMP-2 is constitutively expressed in most tissues and it's known to inhibit all known MMPs. TIMP-3 is also capable of inhibiting all MMPs. TIMP-4, which is the most recently discovered TIMP, inhibits most MMPs including: MMP-1, -2, -3, -7, -8, -9, -12, -13, -19, -26 and MT-1, -2 and -3 MMPs [118, 124], TIMP-2 and 3 are highly expressed in the heart[123] and TIMP-4 shows a tissue-specific expression in the cardiovascular system, especially in the heart [107, 120, 123]. While the major known role for the TIMPs is the inhibition of active MMPs, there is conclusive evidence of TIMP-2 forming a tetrameric complex with proMMP-2 and dimeric MTl-MMPs to aid in the activation of proMMP-2 [119]. 1.6.5 Regulation of ECM Remodeling by MMPs and TIMPs in the Development of Heart Failure Significant alterations of the cardiac ECM composition are known to occur in the heart following MI and in various cardiomyopathies. Shortly after MI, the degree of collagen cross-linking and the normal collagen strut formation are reduced. This 38 degradation of the ECM following MI allows infiltration of the macrophages and proliferation and migration of the fibroblasts to form a mature scar [110, 119, 125, 126]. However, acceleration of ECM remodeling is known as an independent predictor of mortality and morbidity after MI since excessive or prolonged degradation of the ECM contributes to the slippage of viable cells and wall thinning. With very little ECM support, the heart is not able to resist the stress generated during pumping, and rupture of the cardiac wall will occur [122]. Various studies have shown that MMP activity and the development of left ventricular dysfunction following MI are closely related. In humans, activation of MMPs has been shown in the cardiac samples collected from patients suffering from cardiomyopathies and the upregulation of MMP activity is associated with a higher incidence of cardiac wall rupture following MI [127-131]. Close association between MMP activation after MI and the increased rate of morbidity and mortality has also been reported in animal studies [125, 126, 132, 133]. For example, MT-1 MMP expression is significantly increased in the infarct area and the overexpression of this protein is associated with an approximate 20% decrease in mortality rate after MI. Activity of MMP-2 is also increased in the infarct area following MI, which was associated with an increased level of ECM degradation. MMP-2 KO mice demonstrated a significant increase in survival rate after MI due to a decreased incident of cardiac wall rupture [125]. The significance of decreased regulation of MMP activation in ECM remodeling in the pathological development of heart failure following MI is well described in TIMP KO models. For example, Kandalam et al. showed that the expressions of TIMP-2, 3, and 4 are decreased in the infarct area after MI; this was 39 associated with the increased activity of various types of MMPs. TIMP-2 KO mice showed even higher activity of these MMPs and exhibited a greater degradation of the ECM, expansion of the infarcted area and decreased LV function [134]. In TIMP-4 KO mice, the organized linking of collagen and its content within the heart are decreased; as a result, the survival rate of TIMP-4 KO drops approximately 40% after MI due to an increased rate of cardiac wall rupture [124]. 1.7 Adiponectin, a Cardioprotective Adipokine Adiponectin is a 30kDa protein which is mainly secreted by the adipose tissue but also secreted by many other organs such as skeletal muscle, the heart, and the liver [135-137]. Adiponectin has been gaining great interest among scientists due to its anti-diabetic, anti-inflammatory and cardioprotective roles. It circulates in full-length form (fAd) at a high concentration of 5 - 30ng/ml [138] and this full-length form can be cleaved to produce the globular form of adiponectin (gAd), which also mediates physiological effects, by leukocyte elastase[139]. Moreover, fAd can exist in different orders of oligomeric forms: trimer, hexamer, and high molecular weight (HMW) [140] (Figure 1.11). The level of circulating adiponectin is altered in diabetic individuals and diabetic animal models. Either circulating total or circulating HMW adiponectin has been shown to be decreased in type 2 diabetic patients [141] as well as in type 1 and 2 diabetic animal models [140,142,143]. 40 Adiponectin monomer LMW CLE/IAGE Globular adiponectin ^r* AdipoR2 Figure 1.11: Different isoforms and oligomeric forms of adiponectin. Adiponectin is secreted as full-length (fAd) which can be proteolytically cleaved to form globular adiponectin (gAd). fAd can oligomerize to form the higher molecular complex of trimer (LMW), mid-molecular weight (MMW), and high molecular weight (HMW). Adiponectin receptor 1 (AdipoRl) has a higher binding affinity to gAd and adiponectin receptor 2 (AdipoR2) has an intermediate binding affinity to both gAd and fAd. Figure redrawn from [144]. 1.7.1 Adiponectin Receptors and Signaling Three receptors of adiponectin have been identified so far, AdipoRl, AdipoR2 and T-cadherin. AdipoRl and R2 were the first adiponectin receptors to be discovered. They have 7 transmembrane spanning domains but they have an inverted topology to conventional G protein-coupled receptors with the N terminus on the intracellular side of the plasma membrane [145]. Although both receptors are ubiquitously expressed throughout the body, each isoform shows a tissue-specific variability in the expression level. AdipoRl is highly expressed in skeletal muscle and has a higher binding affinity to gAd. AdipoR2 is predominantly expressed in the liver and has an intermediate binding affinity to both gAd and fAd. The two AdipoRs are highly structurally related and share 67% identity in protein sequence [145], T-cadherin is 41 the most recently discovered receptor for adiponectin which binds the hexameric and the HMW form in vitro. This receptor does not possess the transmembrane and cytoplasmic domain, thus its specific mechanism of intracellular signal transduction is currently unknown. Although its expression can be detected throughout the body, its highest expression is detected in the cardiovascular system [146]. APPLl (adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain and leucine zipper motif) was the first protein identified to bind to AdipoRl and AdipoR2 [147]. APPLl is composed of three domains: leucine-zipper (LZ), pleckstrin homology (PH), and phosphotyrosine binding (PTB) and binding of APPLl to adiponectin receptors is thought to be necessary for many adiponectin signaling pathways. For example, APPLl serves as an anchor protein and binds AdipoRl via the PTB domain and the upstream kinase of AMPK, LKB1, via the BAR domain and hence facilitates the phosphorylation of AMPK induced by adiponectin [148]. APPLl has also been shown to mediate the adiponectin- stimulated phosphorylation of p38 mitogen-activated protein kinase (MAPK) by acting as a scaffolding protein and facilitating the interaction of transforming growth factor- P-activated kinase 1 (TAK1) and mitogen-activated protein kinase kinase 3 (MKK3) [149]. 1.7.2 Adiponectin and Cardiac Remodeling Adiponectin is a well-known cardioprotective protein. Its cardioprotective action has been shown in animal models of volume overload induced by ischemia reperfusion (IR) and permanent ligation of the coronary artery [61,150-153], pressure overload induced by aortic banding [24, 25, 154], and viral cardiomyopathy [155]. 42 Many of adiponectin's cardioprotective effects have been shown to be mediated via AMPK-dependent pathway. For example, transfection of neonatal cardiomyocytes with a dominant negative mutant AMPKalpha2 subunit (AMPK-DN) attenuated the effects of adiponectin to inhibit apoptosis induced by simulated IR in vitro [61] and cardiomyocyte-specific AMPK-DN expression in mice prevented adiponectin's cardioprotective action and showed exaggeration of IR injury [27, 152]. AMPK is also required for adiponectin to reduce the activation of ERK or to enhance angiogenesis by increasing the release of VEGF in response to myocyte hypertrophy mediators such as pressure overload or alpha-adrenergic receptor stimulation [25, 154], Adiponectin has also been reported to mediate its cardioprotective action via AMPK-independent pathway to reduce the expression of TNF-alpha by upregulating the sphingosine kinase-cyclooxygenase-2-pathway [61, 156] and reduce the oxidative/nitrative stress by inhibiting the expression of gp91phox and inducible NOS stimulated by IR injury [27,152]. 1.8 Research Objectives CVD is a leading cause of death in the world [1] and one of its most common forms, heart failure [11], occurs as a result of progressive cardiac remodeling [13]. Diabetes is an independent risk factor for the development of heart failure. Diabetic patients suffer from higher incidence of morbidity and mortality after myocardial infarction [10]. However, in the presence of diabetes, the exact physiological and molecular changes associated with cardiac remodeling are still not clearly defined. Therefore this project was conducted to investigate the precise role of 43 diabetes in the regulation of cardiac remodeling events at both the molecular and the physiological levels. This project was comprised of work in three main parts: 1. in vivo temporal characterization of the effects of diabetes on cardiac function and various cardiac remodeling events after ischemia reperfusion injury. Although there are many literatures looking at the effects of diabetes on the development of heart failure, they neglect the extremely important progressive nature of cardiac remodeling events. This work was conduced to test the hypothesis that diabetes' effects on various cardiac remodeling events are time- dependent and are associated with adverse outcome after ischemia reperfusion injury. The heart samples were collected at an acute time point of 24hr and at a chronic time point of 6 weeks of reperfusion to delineate the time-dependent effects of diabetes on various cardiac remodeling events. 2. in vivo analysis of diabetes' role in cardiac remodeling post-myocardial infarction, with a focus on the ECM remodeling. This work was conducted under the hypothesis that aggravated cardiac remodeling in diabetic individuals after MI is due to enhanced ECM remodeling. 3. in vitro investigation of adiponectin's effect on hyperglycemia-induced apoptosis in H9c2 cells to test the hypothesis that adiponectin, an adipokine well known for its cardioprotective action, ameliorates hyperglycemia-induced apoptosis. 44 2 Chapter 2 Ischemia-Reperfusion Injury Leads to Distinct Temporal Cardiac Remodeling in Normal versus Diabetic Mice Megumi Eguchi, Young Hwa Kim, Keon Wook Kang, Chi Young Shim, Yangsoo Jang, Thierry Dorval, Kwang Joon Kim & Gary Sweeney Author Contributions: M.Eguchi contributed to planning, conducted all experiments except for the ones indicated below and wrote the initial manuscript draft. YH.Kim conducted the experiment found in Figure 2.2. CY. Shim assisted in the analysis of echocardiography data found in Figure 2.1,2.5 and Table 2.1. T.Dorval developed the software used to quantify TUNEL positive nuclei against total DAPI stained nuclei for the data used in Figure 2.3. G. Sweeney provided funding for project, designed experimental outline and edited manuscript drafts. 45 2.1 Abstract Diabetes is associated with a higher incidence of myocardial infarction (MI) and an increased propensity for subsequent events post-MI. I conducted a temporal analysis of the influence of diabetes on cardiac dysfunction and remodeling after ischemia reperfusion (IR) injury in mice. Diabetes was induced using streptozotocin and IR performed by ligation of the left anterior descending coronary artery for 30 min followed by reperfusion for up to 42 days. After 24 hours of reperfusion, echocardiography showed decreased systolic function indicated by a reduction in ejection fraction, fractional shortening and end systolic left ventricular volume (ESLW) in both control and diabetic mice. A smaller reduction of radial and longitudinal strain was seen in diabetic mice. After 24 hours of reperfusion, diabetic mice showed a reduced ability to uptake glucose which was determined by PET-CT using 2-deoxy-2-(18F)fluoro-D-glucose, reduced infarct size and less apoptosis indicated by TUNEL analysis in heart sections. This may be explained by increased levels of autophagy detected in diabetic mouse hearts. Over time, diabetic mice exhibited a decrease in both end diastolic left ventricular volume and ESLW with a decreased intraventricular space and thicker left ventricular wall, indicating concentric hypertrophy. This was associated with marked increases in fibrosis, indicated by Masson trichrome staining. In summary, we demonstrate that diabetes principally influences distinct IR-induced chronic changes in cardiac function and remodeling, while a smaller infarct size and elevated levels of autophagy with similar cardiac function are observed in acute phase. 46 2.2 Introduction Diabetes is associated with a higher incidence of heart failure and cardiac ischemic events [157], Patients with both type 1 and type 2 diabetes have a higher risk for encountering sudden death attributed to acute MI and these individuals also exhibit higher mortality rate and risk for developing left ventricular dysfunction after MI [158, 159]. Although the adverse influence of diabetes on left ventricular dysfunction is now a well established phenomenon, the precise temporal nature and mechanisms responsible are still incompletely understood. Due to inherent difficulties in the analysis of human myocardium, much of our knowledge on disease mechanisms comes from animal models showing structural, functional or mechanistic characteristics that are observed commonly in diabetic hearts [13, 160, 161]. Use of rodents to examine the influence of diabetes on the development and progression of cardiomyopathy has been studied in various transgenic and knockout models in vivo, in isolated perfused hearts and papillary muscles, or in vitro using isolated cardiomyocytes [160, 161]. However, many paradoxical observations exist and there is a need for the comparative analysis of remodeling events during the progression of heart failure. Cardiac remodeling is a progressive process involving changes in hypertrophy, cardiomyocyte apoptosis, inflammation, cardiac metabolism and fibrosis [13]. Each of these is altered in a time-dependent fashion subsequent to ischemia reperfusion injury (IR) that occurs during acute MI. Distinct remodeling events may initially be beneficial as they are initiated to compensate for failing cardiac function but remodeling will ultimately transition to heart failure [13], Changes in myocardial 47 metabolism with a reduced glucose uptake and oxidation and thus even higher reliance on fatty acids as a source of energy is one of the first observable remodeling events, often preceding functional indications of a failing heart [13, 160]. Although diabetes has been shown to be associated with increased incidence of acute MI and poorer clinical outcome after IR, the specific effects of diabetes on cardiac function and remodeling, as well as underlying mechanisms, after IR are unclear. Therefore, I conducted a time-dependent (up to 6 weeks) progressive analysis of IR-induced changes in cardiac performance in control and diabetic mice. We used the streptozotocin-induced diabetic mouse model and induced IR by ligating the left anterior descending coronary artery. I also investigated the remodeling events underlying the changes in cardiac function observed at various times during the course of the study as to investigate the extremely important progressive nature of cardiac remodeling events. 2.3 Materials and Methods 23.1 Generation of Diabetic Animals Diabetes was induced in male C57BL6 mice (aged 8-10 weeks) via single dose intraperitoneal injection of 150 mg/kg streptozotocin (Sigma, St. Louis, MO) in 0.1M citrate buffer, pH 4.1 given 7 days prior to surgery. Control animals received the citrate buffer for vehicle treatment. In every case we confirmed blood glucose levels of the mice at the time of surgery with the following data obtained: control 143.77 ± 5.89 mg/dL and STZ-treated 501.79 ± 14.06 mg/dL. Streptozotocin (STZ) is a glucose analogue with nitrosourea linked to its posterior C2 of D-glucose [162]. It is taken up 48 by the pancreatic P-cells through GLUT2 and causes DNA damage which subsequently induces apoptosis in these cells. It is a commonly used agent to induce experimental type 1 diabetes for its direct cytotoxic effect on pancreatic (3-cells [163]. Diabetic model induced by the injection of STZ is also recognized as a great model for studying the effects of hyperglycemia [164], 2.3.2 Induction of Cardiac Ischemia-Reperfusion Injury The cardiac ischemia reperfusion injury surgery was performed 1 week after the injection of STZ or vehicle solution. The mice were lightly anaesthetized with Zoletil 50 (lOmg/kg) and Rompun (2.5mg/kg) for intubation with a ventilation tube and they were seduced deeply with an additional 1.5% isofluorane. Left thoracotomy was performed between the fourth and fifth ribs and the pericardial tissue was removed before the ligation of LAD artery. LAD artery was ligated with 7-0 suture around a PF-10 tubing for 30 min and reperfused for 24 hours or 6 weeks. Sham- operated animals underwent the same surgical procedure except that the suture was not tied around the LAD artery. Infarct area was determined by staining the heart sections in 1% 2,3,5-triphenyltetrazolium chloride (TTC, Sigma) solution. Infarct area was calculated from the left ventricle cut into 5 slices as % non-TTC-stained volume/total ventricular volume. The infarct volume was calculated as the sum of non-TTC stained area x thickness of heart slice of each slice. All animals which died or did not show a decrease in fractional shortening within 24hr of surgery were excluded from the study. 49 2.3.3 Determination of Apoptotic Cells in Cardiac Tissue Apoptotic cells were visualized by TUNEL staining of frozen heart sections using In Situ Cell Death Detection Kit, Fluorescein (Roche) according to the manufacturer's protocol. The quantification of the number of apoptotic cells was performed using an image mining algorithm developed in-house. 2.3.4 Protein Expression Determination Using Western Blotting Hearts isolated from the animals were snap-frozen and homogenized in homogenization buffer (20mM Tris-HCl pH7.5, 150mM NaCl, ImM Na2EDTA, ImM EGTA, 1% Triton, 2.5mM sodium pyrophosphate, ImM P-glycerophosphate, ImM NajVO-i, lug/ml leupeptin, ImM PMSF supplemented with protease inhibitor cocktail (Sigma-Aldrich)). Protein samples (40ug) were run on SDS-PAGE and transferred onto a polyvinylidene fluoride membrane. The membranes were then probed with primary antibodies against LC3B (Cell Signaling), Cathepsin D (H-75), p62 (Sigma-Aldrich), and a-tubulin (P-16, Santa Cruz Biotechnology). 2.3.5 Echocardiography Images were obtained using Vevo2100 (Visual Sonics, Toronto, Canada) equipped with MS550D transducer. The mice were lightly anaesthetized using 1.5% isofluorane and restrained on a heated imaging table. The four limbs were attached to the ECG electrodes and hair on the chest was removed using Nair. Images were obtained from the B-mode parasternal long axis view, M-mode of the parasternal short-axis view and the pulse wave Doppler on the mitral valve from the apical four- chamber view; they were used to calculate cardiac, diastolic, and systolic functions. 50 The echocardiography analysis was performed on mice every week starting 24hr after the surgery up to 6 weeks. Echocardiograms were stored digitally and strain rate analysis was performed with Vevostrain software (Visual Sonics, Toronto, Canada). All parameters were averaged over at least three cardiac cycles. Peak systolic strain and systolic strain rate at 6 segments were obtained for quantitative analysis of global left ventricular systolic function, and the average values were calculated. Peak diastolic strain rate at 6 segments were obtained and their average values calculated for quantitative analysis of global diastolic function. 2.3.6 PET Imaging and Analysis Whole-body [18F]FDG PET/CT was performed using an animal PET/CT scanner (eXplore Vista DR PET/CT, GE Healthcare, Milwaukee, WI). The mice were maintained under fasting conditions for 12-14 hours. Glucose was orally administered (lOmg/g body weight) using zonde to enhance tracer uptake into the myocardium 30 min before the [18F]FDG injection. The PET/CT imaging acquisition was initiated 30 min after the introduction of 18.5 MBq/0.1 mL of [18F]FDG via the tail vein injection. Mice were maintained under isoflurane anesthesia (2% in 100% oxygen) on a heating pad during the injection of [18F]FDG, the uptake period, and during PET/CT scanning. Static whole-body PET scans were taken in 2 frames for 10 min. CT scan (40 kV, 250 pA) was taken in 2 frames for 11.5 min. The images were obtained by Fourier rebinning using ordered subsets expectation maximization (OSEM) reconstruction algorithm with decay-correction, attenuation-correction and random- correction from raw framed sinograms. The data were reconstructed over a 175 x 175 x 118 matrix with 0.77-mm slice thickness. Processed images were displayed in 51 coronal, transverse and sagital planes. For each PET scan, 3-dimensional regions of interest (ROI) were drawn over heart region on whole-body axial images. The standardized uptake value (SUV) for the calibrated and decay corrected series representing 3-D volume data was done as follows: SUV = ROI activity (MBq/g) / [injected dose (MBq) / body weight (g)]. 2.3.7 Histology The degree of fibrosis was examined by staining frozen heart sections for collagen using Masson Trichrome method. Quantification of the blue area representing collagen was performed as previously described [165] using Photoshop software. The number of macrophages infiltrating the heart was identified using antibody against CD68 (FA-11, Serotec) and quantified using an in-house image analysis software developed by Dr. Thierry Dorval (Institut Pasteur Korea). 2.3.8 Statistical Analysis All data are expressed as the means ± SEM. Statistical analysis was performed using t-test or one-way analysis of variance (ANOVA) and Tukey post-hoc test, and differences were determined to be statistically significant when p <0.05. 52 2.4 Results 2.4.1 Overall Cardiac Function 24h after IR Injury in Diabetic Mice Echocardiography analysis at 24 hours reperfusion revealed that IR injury significantly decreased the systolic function in both vehicle treated non-diabetic (VEH) mice and STZ treated diabetic (STZ) mice (table 1 and Figure 1). Both ejection fraction (EF) and fractional shortening were significantly reduced in both STZ and VEH animals after IR injury. End systolic left ventricular volume (ESLVV) was also increased without any increase in end diastolic left ventricular volume (EDLW), further indicating a decrease in the left ventricular (LV) contractility. In both groups, the decrease in EF comes from decreased contractility of the anterior wall (Figure 1A,B). VEH mice showed a decrease in both radial and longitudinal systolic strain and strain rate whereas STZ mice showed a decrease only in longitudinal systolic strain (Table 2.1, Figure 2.1C&E). VEH mice showed an increase in longitudinal diastolic strain but a decrease in both radial and longitudinal diastolic strain rate, all of which were not affected in STZ mice after IR (Figure 2.1D&E). 53 sham IR sham IR VEH ST2 D • Radial • Radial • Longitudinal IS • longitudnal sham ' IR 1 sham • IR sham IR sham IR VEH STZ VEH STZ • Radial • Radial • longitudinal • Longitudinal sham sham sham sham VEH STZ VEH STZ Figure 2.1: Echocardiography analysis performed to examine cardiac function after IR injury. IR injury caused a significant decrease in EF in both VEH and STZ animals but no difference was observed between the two IR groups (A). Representative M-mode images from each group where the numbers used for calculations were derived (B). The calculated systolic strain (C), diastolic strain (D), systolic strain rate (E), and diastolic strain rate (F) are derived from cardiac strain analysis. * indicates that p<0.05 compared to sham of the same treatment group. ** indicates that p<0.05 compared to VEH IR. All values are expressed as absolute values. n>10/group. 54 Table 2.1; Echocardiographic measurements 24 hours after IR VEH STZ sham IR sham IR BW (g) 25.0 ±0.7 26.1 ±0.9 23.44 ±0.7 22.6 ±0.5 HR (bmp) 446.1 ± 15.8 453.7 ±9.8 397.0 ± 17.8 395.6 ± 19.8 LVEDV (ul) 79.3 ± 3.4 79.8 ± 2.8 72.9 ± 2.6 73.8 ±2.8 LVESV (ul) 38.1 ±3.9 52.4 ±4.1* 35.5 ±2.4 46.3 ± 3.3* EF (%) 52.6 ±3.6 38.2 ±3.9* 51.3 ±2.8 37.4 ± 3.4* FS (%) 27.0 ±2.3 18.7 ±2.0* 26.0 ± 1.7 18.1 ± 1.8* Radial Systolic strain (%) 17.2 ±3.2 9.1 ± 1.3* 18.2 ±0.6 15.4 ± 1.2** Diastolic strain (%) -1.5 ±0.6 -1.4 ±0.5 -1.3 0.7 -1.7 ±0.5 Systolic strain rate (/s) 5.3 ±0.1 3.6 ±0.3* 4.7 ± 0.4 5.4 ±0.5** Diastolic strain rate (Is) -5.6 ±0.4 -4.3 ±0.5* -4.5 ±0.5 -4.8 ± 1.0 Longitudinal Systolic strain (%) -12.7 ± 1.2 -6.5 ± 0.7* -12.6 ± 1.0 -8.8 ± 0.8* Diastolic strain (%) 0.7 ± 0.2 1.8 ±0.4* 0.8 ± 0.2 1.2 ±0.3 Systolic strain rate (/s) -4.0 ± 0.2 -2.9 ± 0.3* -4.2 ± 0.2 -3.7 ± 0.3 Diastolic strain rate (/s) 4.3 ± 0.2 3.3 ±0.4* 4.3 ± 0.6 3.3 ± 0.3* BW: body weight, HR: heart rate, LVEDV: left ventricular end diastolic volume, LVESV: left ventricular end systolic volume, EF: ejection fraction, FS: fractional shortening. * p<0.05 when compared to its own sham. ** p<0.05 when compared to VEH IR. nalO/group. 2.4.2 Cardiac Glucose Uptake after IR is Reduced in Diabetic Mice [18F]-FDG was used to analyze the level of glucose uptake by the heart at 24 hr of reperfusion. [I8F]-FDG PET-CT method has been used widely in experimental and clinical settings to quantify cardiac glucose uptake [166]. When quantified, total [I8F]-FDG uptake by the heart was significantly reduced in STZ mice compared to VEH mice (Figure 2.2). 55 A VEH STZ B VEH STZ 5.0 2.5- Q Q • — ———VEH • t• STZ ^ Figure 2.2: Glucose uptake at 24hr reperfusion. 18FDG PET-CT was used to examine the level of cardiac glucose uptake in VEH and STZ mice. Representative scan images from transverse view (A) and other views (B) from each group are shown. The higher glucose uptake level is shown in the increase in the intensity of red as indicated on the color scale bar shown in each image. Quantification of the accumulated 18FDG in the heart shows that STZ heart has a significantly decreased glucose uptake level (C). SUV: standard uptake value, ^indicates that p<0.05 compared to VEH. n>4/group. 2.4.3 Infarct Size Induced by IR is Smaller in Diabetic Mice To examine the effects of STZ-diabetes on the extent of myocardial injury induced by IR, isolated hearts were stained with TTC to identify the infarct area size. Infarct area was significantly reduced in STZ animals compared to VEH animals at 24 hours of reperfusion time as shown visually and quantitatively in figure 2.3A and B, respectively. We also studied the extent of cell death using TUNEL staining of heart sections prepared after 24 hours of reperfusion. Consistent with infarct area data, the 56 number of apoptotic cells was significantly lower in the STZ mouse heart compared to VEH (Figure 2.3 C,D). 2.4.4 Autophagy is Upregulated in Diabetic Mice Hearts To assess the degree of autophagy in a mouse heart, whole tissue homogenate samples were run on Western blot to examine LC3I/II protein level, which is a commonly used marker to determine the extent of autophagy [167]. We observed that STZ-diabetic hearts showed an increased level of LC3II protein (Figure 2.3E), suggesting the upregulation of autophagosome formation. The upregulation of autophagy was further indicated by alterations in expression of cathepsin D and p62 (Figure 2.3E). 57 LC3 Cathepsin D Tubulin VEH STZ VEH STZ Figure 2.3: Effects of IR injury on cardiac cell death. A) Hearts were isolated at 24hr of reperfusion and stained with TTC for the measurement of infarct area. The viable part of the heart appears red and the infarct area appears white. B) Quantification of the infarct area shows that the infarct area is significantly smaller in STZ compared to VEH hearts. C) Apoptotic cells were identified using TUNEL method. TUNEL-positive nuclei are shown in green and total nuclei in DAPI, blue. D) Quantification of TUNEL positive nuclei reveals that the number of apoptotic cells is much lower in the hearts isolated from STZ animals. Analysis of autophagy-related proteins (E) indicates that autophagy is upregulated in STZ heart. The level of LC3II was significantly higher in the STZ heart compared to VEH and the increase in autophagy was confirmed by the increase in cathepsin D and decrease in p62. * indicates that p<0.05 compared to VEH. n>3/group. 58 2.4.5 IR-Induced Macrophage Infiltration is Not Affected by Diabetes Macrophage infiltration is part of the local inflammatory response following myocardial infarction and here I examined this process in VEH or STZ mice subjected to IR by staining for the macrophage marker CD68 (Figure 2.4). IR injury increased the number of macrophages recruited into the infarct area to a similar extent in VEH and STZ animals. 59 B I T -n 30- i 'i i i sham IR sham IR VEH STZ Figure 2.4: Macrophage infiltration into the heart. This was examined by immunofluorescent microscopy for macrophage marker CD68. Representative images taken from each group showing the presence of CD68 as red fluorescent (A). Quantification of the number of macrophages in defined area (B). No difference was observed between the VEH and STZ heart at 24hr of reperfusion. * indicates that p<0.05 compared to sham of the same treatment group. n>5/group. 60 2.4.6 Diabetes Leads to Distinct Progressive Changes in Cardiac Function Over Time Over the 6 week period that the study was carried out, VEH mice showed a small but significant increase in end diastolic left ventricular volume (EDLVV) (Figure 2.5A). These mice also exhibited a tendency to increase ESLVV (B) but this increase was not statistically significant. STZ animals on the other hand exhibited a decrease in both LVEDV and LVESV. Importantly, diabetic mice also showed an increase in ejection fraction after IR (C). STZ mice also developed at 42 days a decreased intraventricular space and thicker left ventricular wall suggesting the development of concentric hypertrophy (D,E). 61 LVEDV LVESV HKVEH Ml -VEHfc -A-STZ Ml -STZM Days post-IR Days post-IR VEH STZ -*-VEH Ml -A-STZ Ml | $ Days post-IR 3 £ 2.25i f 2.00 i 1.75 1.50 1.25 If 1.00 0.75 0.50 0.25 0.00 I I I 'I sham IR sham IR VEH STZ Figure 2.5: Echocardiography analysis followed up to 6 weeks of reperfusion. VEH mice developed a mild LV dilation as evidenced by a significant increase in LVEDV(A) and a tendency to increase LVESV(B). On the contrary, STZ mice developed concentric hypertrophy as shown by decreased LVEDV, LVESV and increased EF (A,B,C). M-mode image showing the increase in the LV wall and decrease in interstitial space in STZ heart (D). The development of hypertrophy in STZ heart after IR was confirmed by calculating the ratio of LV wall thickness/LV weight (E). * indicates that p<0.05 compared to DayO of the same treatment group, """indicates that p<0.05 compared to VEH on the same day post IR. n>3/group. 62 2.4.7 Diabetes Enhances Interstitial Collagen Deposition after IR Injury Fibrosis resulting from collagen deposition was detected using Masson trichrome staining in sections from hearts isolated at 6 weeks post-IR injury (Figure 2.6A,B). Our data revealed an increased level of interstitial collagen deposition in the remote area. STZ-diabetes in sham animals had no effect on the degree of fibrosis, yet the level of collagen deposition was significantly higher in STZ mouse heart after IR compared to VEH heart. Figure 2.6: Fibrosis is enhanced in STZ heart at 6 weeks of reperfusion. The degree of cardiac fibrosis was accessed by staining collagen using Masson Trichrome method. Quantification of the collagen area over the total area revealed that IR causes the development of fibrosis in both VEH and STZ heart but the degree of fibrosis is significantly greater in STZ animal (A). Representative image from each group (B). The blue area represents fibrillar collagen. * indicates that p<0.05 compared to sham of the same treatment group. ** indicates that p<0.05 compared to VEH IR. n>5/group. 63 30min ligation of LAD then reperfusion Acute 24hr Chronic up to 421 Changes in cardiac function Increased fibrosis Decreased systolic function STZ: greater interstitial fibrosis STZ: Better overall cardiac function Changes in cardiac structure Decrease In glucose uptake VEH: mild LV dilation .STZ: concentric hypertrophy Increase in cardiac cell death STZ: smaller infarct area and fewer apoptotic cells with upreguiation of autophagy No change in macrophage infiltration Figure 2.7: Summary schematic diagram. This describes the time span of the experiment with summary of observations made at each time point. Changes in cardiac remodeling events were observed either at acute time point (24hr reperfusion) or chronic time point (up to 6 weeks reperfusion) after 30min of ischemia. 2.5 Discussion Cardiac remodeling is a progressive and dynamic process composed of multiple components that show distinct temporal changes [13, 160, 161]. As mentioned previously, the current literature contains many apparently paradoxical observations on the contribution of diabetes to various remodeling events after the induction of IR. Various explanations are plausible, such as coexistence of obesity, lipotoxicity, the activation of renin-angiotensin system and cardiac insulin resistance [160]. Furthermore, I believe one principal factor is that studies were often conducted at a particular time point and although such studies are individually interesting, they 64 neglect the extremely important progressive nature of cardiac remodeling. Here I studied the temporal changes in cardiac structure and function after IR in the non- diabetic and STZ-induced diabetic mouse model and also examined the significance of distinct remodeling events in relation to these changes. I initially focused on myocardial structural and functional alterations which were determined using echocardiography. Speckle tracking strain analysis is a novel method that permits the assessment of myocardial deformation in 2 dimensions [168], In subjects with myocardial infarction, longitudinal strains are significantly reduced proportionately within the area of infarction, and correlate closely with peak infarct mass and ejection fraction. Hearts with smaller infarcts and preserved global left ventricular ejection fraction show sustained radial and longitudinal strain. Therefore, parameters from speckle tracking analysis are regarded as more sensitive than conventional echocardiographic parameters to detect myocardial dysfunction [168]. In my study, despite similar reduction of left ventricular ejection fraction after IR, the diabetic group revealed a smaller reduction of radial and longitudinal strain than the control group. These findings were consistent with a smaller infarct area in diabetic IR group. Changes in cardiac metabolism are one of the most important early events in the development of cardiomyopathy [13, 160]. Diabetic models consistently demonstrate increased fatty acid utilization and decreased glucose utilization, and the increased use of fatty acid oxidation as a source of ATP generation in the diabetic heart is associated with a higher oxygen consumption which leads to increased oxidative stress and decreased cardiac efficiency [169]. These changes in cardiac metabolism during the reperfusion period can have a significant effect on the function 65 of the heart after IR injury [170, 171]. Furthermore, the STZ-induced diabetes model is insulin deficient and likely to exhibit a reduced ability to efficiently use glucose as an energy source. I therefore speculated that the decrease in overall cardiac performance in diabetic animals after IR may be due to reduced ability of the heart to utilize glucose. I observed using [18F]-FDG and PET scanning that STZ mice show a significantly decreased level of cardiac glucose uptake compared to non-diabetic mice after IR. With regard to correcting this defect, it has been shown that stimulation of glucose metabolism using pharmacological interventions improves the degree of functional recovery after IR in both animals model and humans [170,172-175]. Whereas glucose uptake decreased, STZ animals exhibited a smaller infarct area and fewer TUNEL positive cells after 24 hours of reperfusion. In order to identify a possible mechanism of STZ-diabetes-induced protection against cardiomyocyte injury induced by IR, the level of cardiac autophagy was examined. Autophagy has been observed at elevated levels in the heart of patients or pigs with cardiomyopathy [98, 176, 177] and is thought to play an important role in the regulation and the development of heart failure [31, 37, 81, 102] and the upregulation of autophagy during ischemia has been shown to be beneficial to the heart [98, 100, 101]. During ischemia, autophagy reduces the level of apoptosis in the heart by degrading proteins and organelles that are damaged and harmful to the cell. The removal of damaged mitochondria is especially important as this will prevent the release of pro-apoptotic factors such as cytochrome c and ROS [102, 178]. Autophagy also enhances the recycling of amino and fatty acids to be used as energy sources in conditions of energy shortage such as ischemia [31, 102, 179]. In addition, insulin acting via mTOR is known to inhibit autophagy [180, 181] and in cardiac insulin 66 receptor knockout (CIRKO) mice, there is a constitutively activated level of autophagy [182]. I believe my data indicate that short-term activation of autophagy, likely at least in part due to the lack of insulin, in the STZ-diabetic mice may be beneficial in reducing the number of apoptotic cells and infarct size induced by IR. Indeed, Hill's group have shown previously that using aortic constriction to induce pressure overload also induced autophagy in the heart which peaks at 72 hours, is maintained for at least 3 weeks, and provides a protective influence on cardiomyocytes to promote functional recovery [98,104]. Macrophage infiltration is known to aggravate IR injury [183, 184], and local inflammation caused by macrophage infiltration is a potentially important contributor to changes in cardiac function after IR [185]. Macrophages are recruited from circulation to the site of injury and act to initiate the inflammatory response as well as clear debris and contribute to wound healing [185]. Macrophage infiltration into the heart may also be important in effective repair after IR injury as the recruited macrophages participate in producing cytokines and growth factors required for the proliferation of fibroblasts and vascularization [185]. However, long-term inflammation is known to induce excessive collagen deposition in the heart and ultimately cause heart failure [186, 187]. My data indicate that IR induced macrophage recruitment in heart tissue, but that the number of infiltrating macrophages was not different between the non-diabetic and diabetic groups after 24 hours of reperfiision. I performed further echocardiography study on VEH and STZ mice with and without IR over a period of 42 days to obtain a clear understanding of temporal changes in cardiac function. Through this time-course analysis, I observed that VEH 67 and STZ hearts responded differently to IR. IR injury induces a volume overload to the left ventricle and the heart normally compensates for this by mechanically increasing the left ventricle cavity by dilating it [15]. At 42 days post IR, VEH heart showed increase in EDLVY, indicating the development of a mild LV dilation. On the contrary, STZ animals developed concentric hypertrophy with a decrease in LV volume and intraventricular space and an increase in LV wall thickness. STZ heart compensated for IR injury by increasing the thickness of the LV wall. This is in contrast to VEH heart which compensated by dilating the LV. I hypothesized that the reason STZ heart responded differently is that its cavity dilation may have been limited by the excessive accumulation of collagen in the LV of STZ mice. Indeed, pre-existing fibrosis may be able to prevent dilation of the heart [188] and I observed that IR injury caused a significant increase in the amount of collagen deposition in the remote area, particularly in STZ-diabetic mice. This is also in agreement with established literature indicating that diabetes is associated with enhanced cardiac fibrosis [189-191]. My data also indicated that for up to 42 days after sham surgery, diabetes alone had no significant effect on the development of fibrosis. Thus, IR injury may have accelerated or enhanced the effects of diabetes on the accumulation of collagen in the heart. It is well known that the hemodynamic and neurohormonal changes in the period after MI stimulate events such as intense activation of both the circulating and the local renin-angiotensin-aldosterone system [192]. It has also been reported that diabetes is coupled with activation of the renin-angiotensin system in the heart [193]. Subsequent reactive and progressive interstitial fibrosis in the heart has been shown to be highly related to elevated angiotensin II and aldosterone. Pathologic changes such as this can induce LV hypertrophy and account for abnormal myocardial 68 stiffness and ventricular dysfunction [194]. Therefore, I suggest that the combination of impaired glucose metabolism and excessive neurohormonal activation after IR injury is a possible cause of more severe myocardial fibrosis in diabetic mice. The higher prevalence of heart failure after MI in diabetic patients [195] can be explained by different chronic cardiac remodeling and fibrosis confirmed in this study. Furthermore, the valsartan in acute myocardial infarction (VALIANT), a large scale clinical study of 15,000 patients, used echocardiography data to clearly demonstrate the existence of the two cardiac hypertrophic responses after IR injury [17, 196]. Concentric hypertrophy was associated with a worse prognosis, higher degree of morbidity and mortality than dilated (eccentric) hypertorophy. Our study also showed that STZ group developed concentric hypertrophy in response to IR injury over time whereas VEH mice responded differently and developed mild LV dilated hypertrophy. Although I did not examine heart failure and mortality at much later time points, the VALIANT findings correlate with the worse prognosis I observed in STZ diabetic mice. In conclusion, I have demonstrated here that STZ-diabetes confers differential effects on cardiac remodeling events after IR injury. Diabetic mice show a smaller infarct area post-MI, which may be the result of upregulated autophagy in the heart of these animals. However, in diabetic mice, glucose metabolism is compromised at this time. Long term follow up studies indicated that the diabetic mouse heart develops severe fibrosis and concentric hypertrophy leading to detrimental effects on functional performance. 69 2.6 Acknowledgements This work was supported by the Canadian Diabetes Association grant to G.S. and the Korea Research Foundation Grant funded by the Korean Government (MEST). M.E. was supported by the Heart & Stroke Foundation of Canada's Doctoral Research Award. 70 3 Chapter 3 Effects of Diabetes on Cardiac ECM Remodeling after Myocardial Infarction and the Subsequent Development of Cardiac Dysfunction Megumi Eguchi & Gary Sweeney Author Contributions: M.Eguchi planned and performed all experiments and wrote the initial manuscript draft. G.Sweeney provided funding for project, designed experimental outline and edited manuscript draft. 71 J 3.1 Abstract The cardiac activity of matrix metalloproteinases (MMPs) is increased in animal models and human patients with myocardial infarction (MI). This leads to degradation of the extracellular matrix (ECM) proteins such as collagen and is a key player in cardiac remodeling post-MI. High MMP activity is associated with an adverse left ventricular (LV) remodeling which will lead to the development of heart failure. Diabetes is classified as an independent risk factor for heart disease, and even though diabetic patients are known to have a worse prognosis of morbidity and mortality after MI, the precise way in which MMPs and associated LV remodeling events are regulated after MI in the presence of diabetes is still unclear. This study demonstrates that STZ-induced diabetes is associated with a higher mortality rate due to cardiac rupture and a greater degree of LV dilation. The STZ-diabetic animals exhibited a higher activity of MMP in the infarct area for an extended time period and excessive degradation of the normal collagen mesh structure. The STZ heart also showed an increased degree of cardiac dyssynchronic movement with development of fibrosis in remote areas. In vitro studies indicated that insulin treatment of cardiac fibroblasts inhibited the hypoxia-induced release of pro-MMP-9 and active MMP-2 while having no effect on collagen secretion. Extended, higher MMP activity in STZ animals, therefore, may have been caused by the absence of insulin in these animals. The greater degradation of ECM in the infarct area of the STZ mice may have been a cause for the increased incidence of cardiac wall rupture and the development of severe LV dilation. Development of mild fibrosis in the remote area may also have contributed to the establishment of severe cardiac dyssynchronicity in STZ animals. 72 This correlates with human studies where it has been proposed that the increased morbidity and mortality of diabetic individuals after MI is closely associated with enhanced activation of MMPs. 3.2 Introduction Coronary heart disease, including myocardial infarction (MI), is one of the most commonly identified causes of death in developed countries [197], MI is associated with cardiac remodeling events which involve complex left ventricular (LV) structural alterations. An ischemic insult is followed by an enlargement of the left ventricle and a progressive decrease in the cardiac function. It is often accompanied by diastolic stiffness and enhanced wall stress [198]. Mi-induced cardiac remodeling is associated with a robust activation of matrix metalloproteinases (MMPs). MMPs participate in the degradation of the cardiac ECM, a dynamic scaffold for the heart [105] which is constantly degraded and renewed. Its integrity and intact organization is critical for optimal cardiac performance [107, 113]. MMPs are thought to play a key role in ECM remodeling after MI or other cardiac pathological conditions [120] and various studies using both transgenic animals and pharmacological inhibitors have demonstrated that elevated activity of MMP in the heart is associated with adverse cardiac remodeling [110, 124-126, 134]. Importantly, a large number of MMP isoforms are expressed in the heart and are reported to be activated in different regions of the heart at different time points post-MI [132]. LV remodeling involves a change in the normal turnover of the ECM and the regulation of MMP activities; and a robust increase in the activation of MMPs seen post-MI causes the reduction in collagen cross-linking network due to an enhanced 73 degradation in the infarct area. This results in cardiac rupture, development of LV dilation and pump dysfunction [120,125,199]. Diabetes elevates mortality due to heart disease by 2-4 times [197] and is known as an independent risk factor for the development of heart failure after initial myocardial infarction [200, 201]. Various findings on the key roles of MMPs during cardiac remodeling after MI have been reported [120, 125, 126, 132, 134, 202], However, the precise way in which MMPs and LV remodeling events are regulated after MI in the presence of diabetes is yet to be clearly identified. Here, I investigated the effects of diabetes on Ml-induced development of left ventricular dysfunction from the perspective of MMP activation, ECM remodeling and the consequent development of cardiac dysfunction. 3 J Materials and Methods 33.1 Generation of Diabetic Mice and Induction of MI Diabetes was induced in male C57BL6 mice (aged 8-10 weeks) via a single dose intraperitoneal injection of 150 mg/kg streptozotocin (Sigma, St. Louis, MO) in 0.1M citrate buffer, pH 4.1. Animals were confirmed diabetic when the blood glucose level reached >300mg/dL four days after the STZ administration (STZ animals) (Table 3.1). Control (VEH) animals received the citrate buffer for vehicle treatment. These animals were randomly separated into MI and sham groups and MI was induced in these animals as previously described [203]. For this experiment, the left anterior descending coronary artery was ligated permanently and the heart samples were collected post-mortem after spontaneous death or sacrifice on Day 1, 3, 7, 14 74 and 28 post-MI. Detailed information on how the surgery was performed can be found in Chapter 2.3.2. After the surgery, all dead animals were subjected to autopsy. Cardiac rupture was confirmed by the presence of blood pool in the chest cavity and a rupture of the cardiac wall in the infarct area of the heart. All animals which died or did not show a decrease in fractional shortening within 24hr of surgery were excluded from the study. Tab e 3.1: Blood glucose leve prior to STZ injection VEH STZ Blood glucose (mg/dL) 142.14 ± 5.88 514.06 ± 14.84 3J.2 Masson Trichrome Staining an Infarct Area Calculation Infarct area was examined by staining heart sections with standard Masson trichrome method as previously described [198, 204]. Seven 5um sections of frozen heart isolated at Day 3, 7 and 14 post-MI were prepared from top to the apex of the heart. Each stained section was scanned and quantified using ImageJ software. The infarct area was measured as the ratio (%) of the infarct area divided by the entire LV area. 3 J J Echocardiography Analysis Images were obtained using Vevo2100 (Visual Sonics, Toronto, Canada) equipped with MS550D transducer. The mice were lightly anaesthetized using 1.5% isofluorane mixed with 100% O2 during imaging. The images were obtained from the B-mode long axis view and the M-mode of the parasternal short-axis view. All parameters were averaged over at least three cardiac cycles for analysis. Speckle- tracking cardiac strain analysis was performed using Vevostrain software incorporated 75 into Vevo2100 from the movies acquired from the B-mode long-axis view. The tracking quality was visually inspected and the tracing was confirmed acceptable when the traced line moved along with the moving heart image for at least 3 cardiac cycles. These cardiac cycles were used for the analysis. 3.3.4 MMP Activity Analysis Using Fluorescent Molecular Tomography The degree and site of MMP activation in the heart was analyzed ex vivo with a fluorescent molecular tomography (FMT) system, Visen FMT2500, using MMPSense680 probe (Perkin Elmer, MA). MMPSense680 is a near-infrared fluorescence agent activated by key matrix MMPs including MMP-2, -3, -9 and -13. The animals were anesthetized with 3% isofluorane and injected with O.lnmol/g MMPSence680 via retro-orbital injection 24hr prior to excision of the heart and imaging. This technique allows rapid visualization and quantification of MMP activity. The heart was excited at 680nm and the emission detected at 700nm. 3.3.5 Scanning Electron Microscopy Analysis of Collagen Structure Heart samples for scanning electron microscopy (SEM) were fixed in 2% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.3 for at least 2hr, rinsed in the buffer and dehydrated in a graded ethanol series. The samples were critical point dried in a Bal-tec CPD030 critical point dryer, mounted on aluminum stubs, gold coated in a Denton Desk II sputter coater, and the collagen structure in the infarct area was examined in an FEI XL30 SEM. SEM analysis was then performed as fee for service by Mr. Douglas Holmyard from Mount Sinai Hospital, Pathology and Laboratory Medicine. 76 3.3.6 Primary Cardiac Fibroblasts Isolation Primary mouse cardiac fibroblasts were isolated from the heart of 8-12 weeks old C57BL6 mice as previously described [205] with a few modifications. Briefly, 2-3 hearts were excised from mice and rinsed in ice-cold Krebs-Henseleit (KH) buffer. The hearts were then minced into tiny pieces and digested in 0.6mg/ml collagenase type II (Calbiochem) dissolved in KH buffer at 37°C. Supernatant was collected and replaced with fresh collagenase solution every 15minutes until all tissue was digested. Collected supernatant was pelleted by centrifusion at l,200rpm for 2 min then resuspended in F12:DMEM with 10% FBS and plated on four 10cm dishes. The cells were passaged once to increase the purity of the myofibroblast culture before they were used for experiments. Cells were used for experiments only until they reached passage number 4. 3.3.7 Gelatin Zymography Cultured isolated primary adult mouse cardiac fibroblasts were incubated in a hypoxic chamber for 48hr with an Anaeropack for Cell Culture (Mitsubishi Gas Chemical) as was previously described [206] in serum free DMEM/F12 media. Cells were treated with different concentrations of insulin (Humulin R, Eli Lily) for the entire treatment period. Control oxygenated cells were incubated in a regular incubator with 5% CO2, balanced with air. Conditioned media collected from these cells were concentrated using the Centricon-10 system (Millipore). Protein concentration of media was quantified using BCA protein assay kit (Pierce, Rockford, Illinois), and concentrated media containing 20 |ig of protein was run on a 10% SDS polyacrylamide gel containing 0.1% gelatin. The gel was rinsed for 30 min with a 77 gentle agitation in a 2.5% Triton X-100 solution of to remove SDS from the gel and renature the proteins, followed by a rinse in distilled water 3 times for 15 min. The gel was then equilibrated in developing buffer (1 M Tris-HCl, pH 7.6 with 100 mM CaCh) at 37°C for 24-48hrs to allow the degradation of gelatin by gelatinases present on the gel. The activity of MMPs was terminated by soaking the gel in 100 mM EDTA for 15 min. The gel was stained for 30 min in 0.25% Coomassie blue R-250 to identify the proteolytic activity of MMPs which was quantified using Image J software. MMP-2 activity assay was performed using SensoLyte490 MMP-2 assay kit, fluorometric (AnaSpec, CA) according to the manufacturer's protocol. Briefly, concentrated media containing 20 |xg of protein was incubated with EDANS/DABCYL FRET MMP-2 substrate peptide for lhr at room temperature. The amount MMP-2 activity is converted into fluorescence intensity which was measured at Ex/Em=340 ± 30 nm/490 ±30 nm. The fluorescence reading from the substrate control was taken for the background fluorescence reading and subtracted from the readings of sample readings to get relative fluorescence unit (RFU). The FRET substrate in this kit can also be cleaved by MMP-8,12,13, and-14. 3-3.8 Collagen Content Assay The same conditioned media samples used for zymography were used for determining the release of fibrillar collagen by cardiac fibroblasts. Media containing 20ug protein was first dried at 37°C for 24hrs in a humidified chamber followed by another incubation at 37°C in a dry condition for 24hrs. Picrosirius red solution (0.1% picrosirius red in saturated aqueous picric acid solution) was added to the dried media 78 sample and incubated at room temperature for lhr. The tube containing the sample was centrifuged at 12,000rpm for lOmin using a tabletop centrifuge to pellet the insoluble collagen bound with picrosirius red. The pellet was washed 5 times with ice- cold lOmM HCl. lOOul of 0.5M NaOH solution was added to the tube to elute the stain and the absorbance was read at 555nm. 3.3.9 Statistical Analysis Comparison of survival rates between the VEH and STZ groups was performed by Kaplan-Meier analysis. Whether they were sacrificed for experimental purposes or died of natural cause, all animals were included in the analysis. All other data are expressed as the means ± SEM and analyzed using t-test or one-way ANOVA and Tukey post-hoc test. Differences were determined to be statistically significant when p <0.05. 3.4 Results 3.4.1 Survival and Infarct Area Size of VEH and STZ Mice after MI Kaplan-Meier survival analysis indicated that the mortality rate of STZ mice after MI is significantly higher than VEH mice. Whereas the decline in the survival of VEH mice occurred within the first week post-MI and stabilized afterwards, the survival rate of STZ mice continued to decline until Day 14 post-MI (Figure 3.1). In both treatment groups, most deaths were due to cardiac wall puncture (data not shown). Neither VEH nor STZ heart exhibited an expansion of the infarct area and no significant difference in the infarct area size was observed between the two MI groups (Figure 3.2). Whereas collagen accumulation is evident in the infarct area of VEH mice as is indicated by the blue color, this cannot be observed in STZ heart (figure 2A). Survival of VEH and STZ-diabetic mice after Ml 100 'VEH •STZ 8 12 16 20 24 28 Days Post Ml Figure 3.1: Survival rate of VEH and STZ mice up to Day 28 post-MI. The decline in the survival rate of VEH and STZ mice is similar up to Day7 post-MI. The mortality rate stops declining starting Day7 post-MI in VEH mice but the STZ mice continue to show a decline in survival rate up to Day 14 post-MI. The survival rate of the STZ animals is significantly lower compared to that of VEH mice. n>30/group. 80 Day 3 Day 7 Day 14 VEH ©0© STZ CCC7 70 60 £50 10 a 40 re • VEH S 30 £ • STZ £ 20 10 0 ri3 fi7 r*14 Days post-MI Figure 3.2: Change in infarct area over time after MI. Masson trichrome staining of frozen heart sections indicates the myocytes in red, collagen in blue, and area with dead myocytes without collagen deposition in white. (B) Quantification of the infarct area (blue or white area in the heart sections) expressed as a percentage of the total LV area. n>3. 3.4.2 MMP Activation and Collagen Degradation is Greater in STZ Mice after MI MI is known to induce the activation of MMPs [110]. Here the extent and location of MMP activation in the heart was analyzed ex vivo using FMT (Figure 3.3A). Starting Day 3 post-MI, an upregulation of MMP activity was observed in the infarct area of both VEH and STZ mice. Whereas the highest MMP activity was observed on Day 3 post-MI in VEH heart and started to decrease from that point 81 onward, the peak MMP activity occurred on Day 7 in the STZ heart. On Day 3, MMP activity of VEH and STZ hearts were comparable and peak MMP activity observed on Day7 in STZ heart was significantly higher than that of VEH heart. On Day 14, the VEH MMP activity level declined to a level similar to the sham controls. Although the MMP activity level declined following its peak activation in both VEH and STZ hearts, MMP activity of the STZ-diabetic heart was still significantly higher than that of shams at this time (Figure 3.3B). In terms of localization, MMP activation was exclusively limited to the infarct area in both VEH and STZ hearts (Figure 3.3A). SEM analysis is often used to visually examine the composition and the organization of collagen network on the cardiac surface [111, 112, 122], and this technique was utilized to study heart samples collected on Day 7 post-MI. Both VEH and STZ sham healthy hearts showed fine mesh-like collagen weave structures on the surface as previously reported [111, 112, 122] (Figure 3.3B). There was no obvious difference observed between the two sham groups. MI resulted in the disturbance of this fine collagen network in the infarct area in both VEH and STZ. On Day 7 post- MI, the VEH heart exhibited an increase in the density of the collagen mesh composed of thin collagen filaments whereas the STZ heart showed more disorganized network of collagen scaffold composed of numerous larger collagen fiber. 82 I High VEH £ > • • t • C STZ • • •• Low 1 3 7 14 Days Post-MI I lVEH«h»m 1STZ sham |=jVEHMI HHI STZ Ml Days Post-MI E 2« Figure 3.3: Changes in cardiac MMP activity and collagen structure post-MI. Fluorescent molecular tomography (FMT) was used to assess the location and the degree of MMP activation in the heart post-MI at different time points (A). Images of 83 the heart are representing FMT images for each MI treatment group at indicated time point. Quantification of MMP activity peaks on Day3 in VEH heart whereas the peak is seen on Day 7 in STZ heart (B). Scanning electron microscope (SEM) analysis demonstrates that disturbance in the fine collagen mesh network by MI on Day7 post- injury (C). The disturbance is much greater in STZ animals. White bars: 5um. * indicates p<0.05 vs its own sham group. ** indicates that p<0.05 vs VEH MI. n>3/group. 3.4.3 STZ Mice Develop Severe LV Dilation and Dyssynchronicity Echocardiography analysis demonstrated the development of LV dilation after MI in both VEH and STZ animals as indicated by an increase in end diastolic (Figure 3.4A) and systolic (B) LV volume. Compared to its own sham, the degree of dilation was significantly larger in STZ animals compared to VEH animals. The effects of MI on cardiac function was measured in fractional shortening (FS) (C) and cardiac output (CO) (D). FS was significantly decreased by MI in both VEH and STZ heart but STZ animals exhibited a further decrease in FS on Day 1 post-MI. MI also lead to a significant decrease in CO in both VEH and STZ animals and STZ animals showed a further decrease in CO compared to VEH. However this initial decrease in CO was brought back to the level similar to that of sham controls by Day 14 post-MI in both MI groups. A summary of echocardiography measurements is found in Table 3.2. Neither VEH nor STZ demonstrated a change in diastolic function on Day 1 post-MI as was confirmed by the mitral valve early filling/atrial filling (MV E/A). Novel speckle-tracking analysis of cardiac strain on Day 1-post MI revealed that diastolic strain rate was not decreased at all in the STZ heart whereas the radial strain rate was significantly decreased in the VEH heart (Figure 3.4E). VEH heart also showed a decline in longitudinal strain rate during systole was affected in VEH. 84 However, both radial and longitudinal rates were affected during systole in STZ heart (F). Table 3.2: Echocardiography measurements on Day 1 post-MI VEH STZ sham MI sham MI BW (g) 23.1 ± 0.6 22.8 ± 0.5 20.4 ± 0.8 19.8 ±0.8 HR (bmp) 482 ±8 528 ± 9* 396 ±10** 406 ± 28** SV (ul) 21.9 ± 1.4 12.8 ± 1.3* 26.6 ± 1.5** 14.3 ± 1.3* CO (ml/min) 10.6 ±0.7 6.7 ±0.7* 10.6 ±0.6 5.8 ±0.7* EDLW (ul) 69.5 ± 2.2 82.3 ± 2.9* 57.0 ±2.3** 76.7 ±3.1* ESLW (ul) 47.6 ±1.6 69.5 ± 3.3* 30.3 ± 2.4** 62.4 ±3.2* EF (%) 38.2 ±1.8 23.3 ± 1.9* 50.9 ±2.9** 26.1 ±2.1* FS (%) 18.4 ±1.0 10.1 ±0.9* 25.8 ± 1.9** 12.1 ±1.0* MV E/A 1.6 ±0.1 1.7 ±0.1 1.5 ±0.1 1.5 ±0.1 HR: heart rate, SV: stroke volume, CO: cardiac output, EDLW: end diastolic left ventricular volume, ESLW: end systolic left ventricular volume, EF: ejection fraction, FS: fractional shortening, MV E/A: mitral valve early filling/atrial filling, ""indicates p<0.05 vs sham of its own treatment group. **indicates p<0.05 vs VEH of its own surgery group. n>15/group. 85 Change In EDLW Change in ESLW 80 70 •'*# —VEH ,T 60 —VEH — STZ 50 -STZ 340 30 20 10 0 1 7 14 1 7 14 Days post-MI Days post-MI Change In FS Chang* In CO Days post-MI Days post-MI 1 7 14 VEH — STZ I" Diastolic strain rate Systolic strain rate •RadU •Radial •Longitudinal •longitudinal > 4 sham sham sham sham Figure 3.4: Changes in cardiac structure and functions post-MI compared to sham control. Both VEH and STZ animals develop LV dilation as indicated by an increase in end diastolic LV volume (EDLW) (A) and end systolic LV volume (ESLW) (B) post-MI. The degree of LV dilation is more severe in STZ animals. MI also leads to a decrease in fractional shortening over time (C). Cardiac output is initially decreased but brought back to the normal level by Day 14 post-MI (D). Graphs (A-D) are shown as a change compared to the sham control of each treatment group. Diastolic (E) and systolic (F) strain rate analysis indicated that STZ animals did not exhibit any decrease in diastolic strain rate whereas its systolic strain rate was significantly reduce in both radial and longitudinal movements. * indicates p<0.05 vs its own sham group. n>3/group/time point. 86 Speckle-tracking analysis was also performed on Day 14-post MI. Both diastolic (Figure 3.5A) and systolic (B) strain rates were reduced in both VEH and STZ animals. No significant differences were observed in any of the strain rate analysis between the two MI groups. LV synchronicity was examined by a common method of calculating the wall delay [207-209]. This analysis divides the heart into 6 separate segments: basal, mid, and apex from the posterior and anterior wall and calculates the difference in time it takes for each of 6 segments of the heart to reach its peak strain. Wall delay measurement revealed that VEH mice developed wall dyssynchrony in both radial and longitudinal movement during diastole but only in longitudinal movement during systole. STZ animals demonstrated LV dyssynchrony in radial and longitudinal movement during both diastole and systole and the degree of diastolic longitudinal dyssynchrony was significantly greater than that of VEH animals (Figure 3.5C&D). Overall, the degree of cardiac dyssynchronicity was greater in STZ heart (E, F). 87 diastolic Strain Rate Systolic Strain Rata •Radial •Radial •Longitudinal •longitudinal aRadial 0 •Radial Diastolic Wad May •Longitudinal Systolic Wall May •longitudinal Figure 3.5: Cardiac function measured on Day 14-post-MI. Both diastolic (A) and systolic (B) strain rates are reduced in VEH and STZ heart. MI causes the heart to develop radial and longitudinal dyssynchrony during diastole (C) and systole (D). (E) The color-coded images depicting the LV divided into six sector indicates that the 88 degree of cardiac dyssynchrony is greater in STZ heart. The numbers indicated on the images in each LV sector indicate the difference in time between the peak strain rate of each sector to the average peak strain rate. This information is also displayed in % from -50 to +50. The dyssynchronic movement of the STZ heart is also demonstrated in (F) where the movement of each sector is not aligned. The colors on graphs correspond to the colors of the sectors indicated on the pericardial portion in (E). * indicates p<0.05 vs its own sham group. ** indicates that p<0.05 vs VEH MI. n>3/group/time point. 3.4.4 Collagen Deposition in the Remote Area Post-MI Injury The amount of collagen accumulation in the area remote from the infarct area was examined by staining heart sections with Masson trichrome (Figure 3.6) as MI is known to induce fibrosis in the remote area of the heart over longer time periods [126]. There was an insignificant but slight increase in collagen deposition in VEH remote area after MI compared to the sham control (sham data not shown). STZ animals demonstrated a significant increase in collagen deposition on Day 7 and Day 14 compared to sham animals at the same time intervals. 89 A Day 3 Day 7 Day 14 3 7 14 Day* post-MI Figure 3.6: Collagen deposition in the remote area after MI. (A) Representative images from each treatment group are shown. (B) Quantification of Masson trichrome images indicated that STZ heart exhibit a significant increase in the deposition of collagen in the remote area post-MI. n>4/group. '•'indicates p<0.05 vs sham of its own group. 90 3.4.5 Regulation of MMP and Collagen Secretion by Insulin STZ animals become hyperglycemic primarily due to the lack of insulin in circulation from the loss of insulin secreting pancreatic beta-cells. In order to investigate the effect of insulin on MMP and collagen secretion from cardiac fibroblasts under a stressed condition, primary cardiac fibroblasts isolated from adult mouse hearts were treated with or without insulin and incubated under hypoxic condition, an in vitro approach to mimic the state of MI. Cells were treated with different concentrations of insulin (0 to 50nM) and media collected from each sample was analyzed by gelatin zymography to determine the activity of MMP-2 and -9 and the abundance of these MMPs released into the media. Hypoxia treatment significantly increased release of active MMP-2 as was indicated by an increased gelatinolytic activity at the active MMP-2 molecular weight of 67kDa. This increase in activity, however, was significantly inhibited by the treatment of insulin in a dose dependent manner (Figure 3.7A&B). The ability of insulin to reduce the release of active MMP-2 induced by hypoxia was also confirmed by MMP-2 activity assay kit (C). Insulin treatment also reduced the release of pro-MMP-9 into media under hypoxic condition in a dose dependent manner (A). Insulin increased the release of fibrillar collagen at a concentration of 50nM under oxygenated conditions (D). Hypoxic condition for 48hrs slightly but significantly decreased the release of fibrillar collagen and also inhibited the effect of insulin on collagen release. 91 Pro-MMP9 band Intensity 2.5 pro-MMP9-> 2 I ei.5 glycosylated MMP2->- 8 pro-MMP2~> activated MMP2-^ I*u. Insulin 0.1 10 0.01 0.1 10 50 OS 0 0 0> N \0 Activated-MMP2 band intensity MMP2 activity • 02 2.5- • 02 20000 • H • H 2.0- 16000 12000 | 1fr 8000 1ii. IO- 0.5- 4000 0- 0.0" o % •\0 \ t \ oI lil N \® <#> hsuin Treatment (nM) Insulin Treatment (nM) Fibrillar collagen secretion 2.5- • 03 2.0- • H 1.5- 1.0- 0.5- O.O 0 0.1 \ \0«p llllllO ^ \ \0 Insulin Treatment (nM) Figure 3.7: Effects of insulin on MMP activation and fibrillar collagen secretion in primary cardiac fibroblasts. 48hr hypoxia treatment increases the release of pro- MMP-9 and active MMP-2 from adult cardiac fibroblasts and this increase is inhibited by insulin treatment in a dose dependent manner(A, B). The quantification of the active MMP-2 band is shown in graph (C). The changes in MMP activity induced by hypoxia and insulin treatments were also tested with MMP-2 activity assay kit (D). Insulin induces the secretion of fibrillar collagen at 50nM concentration only under oxygenated condition. Hypoxia treatment results in a small but significant decrease in the secretion of collagen and blocks the effect of insulin on the release of collagen into to extracellular space (D). O2: oxygenated condition, H: hypoxic condition. * indicates p<0.05 vs OnM insulin treatment under oxygenated condition. ** indicates p<0.05 vs OnM insulin treatment under hypoxic condition. n=3 except for 1 and 50nM Insulin treatment in (B) and the MMP-2 activity assay (C) where n=l. 92 In vivo In vitro VEH STZ Insulin t MMP activation f T f MMP activaty duration 11 MMP-2 activation MMP-9 release 11 Mature scar formation f t LV dilation ft t Mortality f t t Fibrosis of remote area t f Cardiacdyssynchrony ft Figure 3.8: Summary of results. The findings of this study both in vivo and in vitro are summarized in this figure, f indicates an increase compared to the sham control. 3.5 Discussion Diabetic patients are known to suffer from severe cardiac dysfunction and a significantly higher mortality rate after the incident of MI [200, 201]. Although diabetes is recognized as an independent risk factor for the development of heart disease, the mechanism of how diabetes impacts cardiac remodeling events post-MI is not entirely clear. This current study indicated that STZ animals suffer from enhanced and accelerated LV remodeling post-MI. I observed that the first and most apparent evidence for this was a higher mortality rate, which was found to be mostly due to an increased incidence of cardiac wall rupture and an intensive dilation of the left ventricle. An important component of LV remodeling involves the reorganization of the cardiac ECM [107, 120] and increased expression and activity of MMPs is well established to occur in infarcted heart [122, 125-127, 132, 202]. The use of the 93 pharmacological inhibitors of MMP and genetically modified animals have proven the effectiveness of MMP inhibition in improving survival rate and cardiac remodeling after MI [125, 202, 210] and there is a clear evidence that regulation of MMPs during the cardiac ECM remodeling plays a key role in the progression of LV remodeling and the survival of MI patients. Previously, it has been reported that increased MMP activation is associated with the incidence of cardiac wall rupture most likely due to the disruption of the ECM structures in the infarct area [125, 211, 212], Prolonged degradation of the ECM contributes to the slippage of viable cells and wall thinning and with a very little ECM support, the heart is not able to resist the stress generated during pumping and the cardiac wall ruptures [122]. Cardiac wall rupture often occurs within the first week of the onset of MI [125], and this reported finding is consistent with the finding of the currently study. Most deaths observed in VEH animals occurred within the first 7 days post-MI. VEH mice exhibited a rapid increase in the MMP activity which was reduced by Day 7 post-MI and higher accumulation of collagen was evident in the infarct area of VEH animal. The VEH heart also demonstrated a better organization of the reconstructed collagen scaffold and a formation of a mature scar in the infarct area by this time. A further increase in the mortality rate of these animals may have been abrogated by this rapid formation of scar in the infarct area. However, STZ heart demonstrated an increase in MMP activity higher than that of VEH mice for up to day 7 and exhibited a high degree of MMP activity until day 14. This must have contributed to the poor formation of a mature scar within the infarct area with smaller collagen deposition and disorganized collagen mesh network and be responsible for the higher mortality rate of these animals. 94 It has been reported that the loss of collagen fibroskeleton is associated not only with cardiac rupture but also with myocyte slippage leading to the dilation of the LV [213-215]. MI induced LV dilation in both VEH and STZ hearts, but in this study, the degree of LV dilation was much more severe in STZ heart. A greater degree of decomposition in the collagen network, and the lack of thick, mature scar formation in the STZ-diabetic heart may be in part responsible for greater degree of LV dilation. Another reason leading to an intensive dilation of the LV in STZ heart is that these animals also exhibited a greater decrease in FS and cardiac output on Day 1 post-MI. Initially, LV dilation is known to be a compensating mechanism of the heart to make up for the volume overload caused by MI to sustain a normal CO [216]. Indeed, the initial significant decrease in CO observed on Day 1 post-MI was brought back to the normal levels by Day 14 in both VEH and STZ animals. LV dilation most likely contributed to the compensated increase in CO and STZ heart may have compensated for the greater decrease in cardiac output by a greater dilation of the LV. A loss of integrate collagen network in the ECM is also associated with infarct area expansion [213-215]. In this study, although the increase of MMP activity was much greater in STZ animals, no evidence of infarct area expansion was observed in the heart of these animals post-MI. The speckle-tracking cardiac strain analysis is a novel and powerful tool used to quantitatively assess the deformation of the LV wall and changes in overall LV contraction and relaxation in an intact mouse [208]. As previously reported, this study also demonstrated that MI induces the reduction in the ability of the heart to deform as the heart contracts and dilates [208, 217]. I first speculated that STZ diabetes may have a negative effect on the cardiac strain but the existence of diabetes had no 95 significant effect on the changes in the strain rate. MI also caused the development of dyssynchronic cardiac wall motion in both VEH and STZ animals, however STZ animals exhibited a greater disturbance in the synchronic motion of the cardiac wall. In the heart, ECM not only serves as the structural framework of the organ but also gives structural integrity to convert contraction of each individual myocytes into an overall heart contraction [107]. The greater disturbance in the synchronic movement of the heart may therefore be caused by a greater remodeling of the ECM in the heart of the STZ-diabetic animals. Although there are no data currently available on mice, it has been reported previously that human patients exhibiting a higher degree of cardiac fibrosis are more likely to develop cardiac dyssynchrony [218]. STZ animals demonstrated a further disturbance in cardiac synchronicity and I speculated that this may be due to an increased fibrosis in the remote area of these animals. Although the STZ animals showed a slightly higher accumulation of collagen in the remote area compared to the VEH mice, there was no statistically significant difference between the two MI groups. However it may be important to analyze the type of collagen depositing in the heart between the two groups as the heart is comprised mainly of collagen type I and III, each of which has different characteristics (refer to Chapter 1.5.2 for detailed information), a so a further experiment to investigate this may be beneficial in answering the question. The presence of dyssynchronic heart movement is also regarded as an indicator of increased mortality, mitral regurgitation, and the progression of LV remodeling. Indeed, cardiac resynchronization therapy has been recently demonstrated to improve the function of the heart and survival in heart failure patients [209] and a higher mortality rate of STZ mice post-MI may have also been caused by an existing cardiac dyssynchrony. 96 STZ animals are diabetic primarily due to lack of insulin in the circulation. Therefore, I hypothesized that the differences in the MMP activity observed between VEH and STZ may be at least in part caused by the presence or absence of insulin. Fibroblasts are the most predominant cells in the heart and play an essential role in ECM remodeling events. In response to a stimulus such as MI, cardiac fibroblasts become highly activated and increase the secretion of MMPs and collagen [219] to initiate the repair of the infarct area. Hypoxia treatment of cardiac fibroblasts increased the secretion of both pro and active forms of MMPs, as was previously reported in various cancer cells and human macrophages [220-222]. In my study, insulin inhibited hypoxia-induced release of active MMP-2 and pro-MMP-9. Although it was previously reported by our lab that insulin treatment of rat glomerular mesangial cells resulted in an increased activity of MMP-2 [223], this could be due to a difference in cell type. Recently, Schuyler et al. reported that insulin treatment of diabetic apoE-/- mice resulted in a decreased MMP-9 expression in aortic atherosclerotic plaque [224]. It has also been shown in human MI patients that the infusion of insulin inhibits the release of MMP-1 into serum presumably from the infarcted cardiac tissue [225] and insulin infusion into obese non-diabetic human subjects decreases plasma MMP-9 level by 1/3 [226]. Although extrapolation of data should be done with caution, the ability of insulin to suppress the activation of MMPs correlates well with the enhanced MMP activity seen in STZ animals. Collagen secretion, however, was decreased under hypoxia condition. Several reports have demonstrated that hypoxia increases the production of collagen in cardiac fibroblast and in other cell types [227-230]. Since none of them employed the same experimental conditions as this study, it is possible that slight differences in the 97 experimental condition lead to this discrepancy. However, it is worthwhile to investigate the effect of insulin on collagen secretion under both normoxic and hypoxic conditions using alternate methods to validate the finding of this work. In this study, the activity and the localization of MMP in the infarcted heart were analyzed using FMT with a probe that emits near-infrared fluorescence when cleaved by MMPs. The use of near-infrared fluorescence imaging of MMP activity has been used previously in various studies including cancer [231], atherosclerosis [232], and pulmonary inflammation [233]; and this method provides a direct quick and reliable quantification of MMP activity in intact tissue in vivo and ex vivo. Here I developed the use of this probe with FMT for analysis of cardiac MMP activity for the first time. The advantage of this method in examining the cardiac MMP activity post-MI is that it not only offers sensitive quantification of MMP activity but also the location of the activation. If possible, it will also allow the researcher to track the same animal and monitor the changes in the location and the activity of MMPs over time. This will also allow the reduction in the number of experimental animals needed to complete a project. Nonetheless, the protocol for the use of this method to study infarcted heart in vivo is not yet established and it is currently difficult to obtain fluorescent readings in beating hearts. Analysis of MT-1 MMP promoter activity ligated to luciferase gene using a bioluminescent scan has been previously performed in the infarcted heart of a transgenic mouse[126] and this analysis was also done ex vivo. Despite this minor disadvantage, it is still a very powerful tool to study cardiac MMP activity in animal models as shown here for the first time. Since MMPSense680 probe used for this experiment can be activated by several MMP isoforms, MMP-2, - 3, -9 and -13, so it would be necessary to identify the MMP that is the most activated 98 in the heart after MI in the future. In summary, STZ mice show a significantly higher activity of MMP for an extended time period in the infarct area after MI and this was associated with poor scar formation in the infarct area and a higher mortality rate. These animals exhibited a greater LV dilation with a greater disturbance in the synchronic movement of the heart. Insulin treatment of cardiac fibroblasts demonstrated the ability of insulin to inhibit the expression of MMP-9 and the activation of MMP-2. The lack of insulin in STZ-diabetic mice may be playing a key role in the exacerbated ECM remodeling after MI. This study did not investigate the direct role of insulin on MMP activity in vivo and does not provide direct evidence of the involvement of MMPs in the subsequent development of the LV dysfunction. Future experiments should be performed to investigate these to further understand the regulation of ECM remodeling post-MI in diabetes. 3.6 Acknowledgement This work was supported by the Canadian Diabetes Association grant to G.S. and the Korea Research Foundation Grant funded by the Korean Government (MEST). M.E. was supported by the Heart & Stroke Foundation of Canada's Doctoral Research Award. 99 4 Chapter 4 Adiponectin Protects H9c2 Cells from Hyperglycemia-induced Apoptosis Through the Activation of p38 MAPK Megumi Eguchi, Tien Cung, Ying Liu, Riya Ganguly & Gary Sweeney Author Contributions: M.Eguchi contributed to planning, conducted all experiments and wrote the initial manuscript draft. T.Cung assisted in all the experiments except for Figure 4.6 & 4.7 as part of her undergraduate thesis project under the direct supervision of M.Eguchi. R. Ganguly ran Western blot for LC3 used in Figure 4.6C. Y. Liu ran Western blot for LC3 used in Figure 4.7C. G. Sweeney provided funding for project, designed experimental outline and edited manuscript drafts. 100 4.1 Abstract Diabetes has been associated with a higher incidence of heart failure. It has been proposed that one contributing factor to heart failure is the elevation of cardiomyocyte apoptosis. This may be due to hyperglycemia, which has previously been shown to induce apoptosis in cardiomyocytes. Adiponectin is a cardioprotective adipokine and its effects include attenuation of ischemia/reperfusion-induced apoptosis in the heart. However, whether adiponectin mediates direct cardioprotective effects against hyperglycemia-induced apoptosis is still unknown. Therefore, in this study I investigated the role of globular (gAd) and full-length (fAd) adiponectin in cardiomyocyte apoptosis induced by hyperglycemia. Incubation of H9c2 cells in hyperglycemic condition (25mM glucose) decreased cell viability and induced apoptosis as observed via an increase in annexin V binding, caspase 3 activity, and TUNEL staining. All of these were reversed by treatment with gAd or fAd. Western blot analysis showed that acute treatment with either gAd or fAd resulted in an increase in the phosphorylation of p38 MAPK at T180/Y182. By demonstrating that adiponectin loses its ability to protect cells from apoptosis in the presence of the p38 inhibitor SB203580,1 confirmed that adiponectin's anti-apoptotic effect was mediated through the activation of this kinase. I also used LC3I/II Western blot to demonstrate that adiponectin attenuates hyperglycemia-induced inhibition of autophagy. In summary, adiponectin exerts a protective effect on H9c2 cardiomyocytes against hyperglycemia-induced apoptosis through the activation of p38 MAPK, and possibly through the maintenance of cell autophagy. This work adds further to evidence suggesting adiponectin is a viable therapeutic target to treat heart failure in diabetic 101 patients. 4.2 Introduction Diabetes is associated with a higher incidence of cardiovascular diseases; the Framingham study indicates that diabetes is an independent risk factor for developing heart failure in both men and women [200, 201]. Furthermore, the mortality rate from cardiovascular complications is 2-4 times higher in patients with diabetes compared to those without it [234]. Diabetic individuals may exhibit diabetic cardiomyopathy which is the development of cardiac dysfunction in the absence of hypertension or coronary artery diseases [235]. The role of diabetic factors such as hyperglycemia's direct action on the myocardium has therefore generated great academic interest. Recent major clinical trials such as ACCORD (Action to Control Cardiovascular Risk in Diabetes) [236]; ADVANCE (Action in Diabetes and Vascular Disease: Preterax and Diamicron Modified Release Controlled Evaluation) [237]; and VADT (Veterans Affairs Diabetes Trial) [238] however have shown that intensive glycaemic control in type 2 diabetic patients has no significant reduction in the outcome of macrovascular complications. This suggests that the role of hyperglycemia on overall cardiovascular function may be minor. Nonetheless, the DCCT (Diabetes Control and Complications Trial) follow-up study indicates the importance of proper glycemic control in reducing the development of cardiovascular diseases (CVD) in type 2 diabetic individuals [239]. Differential outcomes between these studies may be explained by factors such as the duration of diabetes, and the 102 preexistence of CVD at the start of the study. Still, the importance of proper glycemic management on the incidence of cardiovascular events remains uncertain. An increased apoptotic rate of cardiomyocytes is observed in heart failure, and this is known to play an important role in the progression of the disease [240]. Accordingly, apoptosis has emerged as a potential therapeutic target for treating heart failure, especially as cardiomyocytes have a very limited ability to proliferate, and, even a small increase in the rate of apoptosis is detrimental to cardiac function [28], Furthermore, hyperglycemia is already well understood to be at least partly responsible for beta-cell apoptosis in diabetic individuals [241-243]; and it has been shown that high concentrations of glucose are directly associated with cardiomyocyte apoptosis [244,245] and the development of diabetic cardiomyopathy [246]. Adiponectin is a 30kDa adipokine [144] normally found in circulation at a high concentration (~3-30(ig/ml) [247]. Full-length adiponectin (fAd) in circulation exists in several oligomeric forms; high-molecular weight (HMW), mid-molecular weight, and trimeric forms [140] and can produce globular C-terminus fragment (gAd) through proteolytic cleavage by leukocyte elastase [139]. It has been demonstrated that both forms of adiponectin, fAd and gAd, have anti-inflammatory, anti-diabetic, and cardioprotective roles [248]. Heretofore, the cardioprotective action of adiponectin has been demonstrated by its ability to: 1) reduce infarct area [61, 249, 250]; 2) induce revascularization [151, 251] after IR; 3) prevent concentric hypertrophy of the heart caused by pressure overload [154]; and 4) minimize the progression of systolic dysfunction after MI [150]. (Refer to Chapter 1.6.2 for more detailed information.) Circulating HMW adiponectin level is significantly reduced in obese individuals[247] and type 2 diabetic patients who exhibit a higher incidence of 103 heart failure and cardiovascular complications [252, 253]. Although adiponectin's cardioprotective role against severe acute insults to the heart such as ischemia reperfusion injury and pressure overload hypertrophy have been established [61, 154, 249, 250], the direct effects of adiponectin on cardiomyocyte apoptosis induced by hyperglycemia is yet to be determined. Lower circulating adiponectin concentration in diabetic individuals may be related to the incidence of heart failure seen in diabetic individuals and since hyperglycemia is a characteristic of diabetes that is directly associated with diabetic cardiomyopathy and cardiomyocyte apoptosis, I investigated the role of adiponectin on cardiomyocyte apoptosis induced by hyperglycemia. Since there is a close relationship between apoptosis and autophagy (Refer to Chapter 1.4.2.2), I also examined the role of adiponectin on cardiomyocyte apoptosis in terms of its ability to regulate autophagy. 43 Materials and Methods 4.3.1 Cell Culture and Induction of Apoptosis H9c2 cells were cultured as described previously [254]. In brief, cells were maintained in DMEM with 5.5mM glucose supplemented with 10% (v/v) fetal bovine serum and 1% penicillin/streptomycin (v/v). Cells were routinely grown to 80% confluence in 75 cm2 flasks at 37 °C with an atmosphere of 5% CO2 prior to passage and seeding for experiments. For the study of adiponectin's effect on hyperglycemia- induced apoptosis, cells incubated in hyperglycemic media (HG, 25mM glucose) or normoglycemic control media (NG, 5.5mM glucose + 19.5mM mannitol to account 104 for osmolarity effects) were treated with gAd (lug/ml) or fAd (5ug/ml) for various time points indicated in figure legends. 4.3.2 Production of fAd and gAd Recombinant gAd and post-translationally modified fAd was produced in our own lab as previously described [255]. gAd was produced by subcloning murine gAd cDNA (a kind gift from Dr. Philipp Scherer, Albert Einstein College of Medicine, New York city) into the pTriEx4 expression vector (Novagen, Canada) and fAd was produced in a mammalian expression system. 4.3.3 Cell Viability Assay Cell viability was assessed using MTT (3-(4,5- Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) and trypan blue exclusion assay as previously described [254, 256]. For inhibition of p38 MAPK, cells were treated with SB203580 (Calbiochem, Darmstadt, Germany). Control cells were treated with vehicle, DMSO. 4.3.4 Detection of Apoptosis Apoptosis was analyzed using Annexin V binding assay, caspase 3 activity assay and TUNEL as described previously [254]. 4.3.5 Western Blot Western blot analysis was performed as previously described [255] using antibodies specific to phospho-p38 at T180/Y182, phospho-AMPK at T172 and LC3B (all at 1:1000, CellSignaling, MA). HRP-linked secondary anti-rabbit (1:10000, CellSignaling, MA) was used as secondary antibody and proteins were detected by 105 enhanced chemiluminescence reagent. The membranes were stripped each time and re-probed with antibodies against beta-actin (1:1000, CellSignaling, MA) for loading controls. Band intensity was quantified using Scion Image software. 4.3.6 Statistical Analysis All data are expressed as mean values ± SEM. Statistical analysis was performed using Student's Mest or one-way ANOVA and Tukey post-hoc test. Differences between groups were considered statistically significant when p<0.05 and are indicated by symbols as described in figure legends. 4.4 Results 4.4.1 Hyperglycemia Induces Apoptosis in H9c2 Cells Tiypan blue exclusion assay was conducted to study the effect of hyperglycemic condition on the viability of H9c2 cardiomyocytes. Cells were incubated with NG and HG media for 48hrs and 72hrs and it was found that HG treatment resulted in an increase in the number of dead cells compared to NG treatment (Figure 4.1 A). Then I tested the characteristics unique to apoptosis such as phosphatidylserine (PS) externalization to the outer membrane of the phospholipid bilayer and activation of caspase 3 in order to assess the form of cell death induced by hyperglycemia. Cells began to expose PS to the external membrane after 24hr of incubation with HG media as was shown by FITC-annexin V binding (Figure 4.IB). Cells were counterstained with propidium iodide (PI) to help distinguish between apoptosis and necrosis. A significant increase in caspase 3 activity, which was 106 measured as the amount of cleaved DEVD-pNA, was detected upon 48hr incubation with HG media (C). These results indicate that the cell death induced by HG was mediated through apoptosis rather than necrosis. A DNG |25 i ^T -HG L20 • 1 Url 48 72 24hr Incubation time (h) C inAO L • ; QNG«HG !i i| «ao LIrB I^H— rmHL 48 72 Incubation time (h) Figure 4.1: Hyperglycemia induces apoptosis in H9c2 cells. (A) Cell viability was assessed by trypan blue exclusion assay after H9c2 cardiomyocytes were incubated with NG or HG media for 48hr and 72hr. The number of dead cells under NG incubation was normalized to 1. HG treatment increased the number of dead cells by ~3 times at 48hr and ~5 times at 72hr. (B) Annexin V binding assay shows that HG treatment of 24hr and 48hr increases PS exposure to the outer membrane. Annexin V is shown in green and PI is shown in red fluorescence. (C) Activity of caspase 3 was significantly increased after incubation with hyperglycemic media for 48hr and 72hr. 72hr incubation under NG increased caspase 3 activity by ~2.5 times compared to 48hr in NG. n>4/treatment. * indicates p<0.05 vs NG. Graph changed to % trypan blue instead of fold over control. 107 4.4.2 Adiponectin Protects Cells from Hyperglycemia-Induced Apoptosis Cells were treated with two forms of adiponectin, fAd (5p.g/ml) or gAd (lug/ml), to study the effects of adiponectin on hyperglycemia-induced apoptosis. The concentrations used are based on my preliminary data as well as those used in a previous publication [257]. As shown in Figure 4.2, trypan blue exclusion assay showed that treatment with either form of adiponectin significantly abrogated the effects of hyperglycemia on H9c2 cell viability. When assessed for its effects on apoptosis, the treatment of cells with either form of adiponectin resulted in the following decreases: 1) annexin-V binding; 2) caspase 3 activation; and 3) DNA strand breaks measured by TUNEL. These results indicate that adiponectin protected cells from hyperglycemia-induced apoptosis (Figure 4.3A-D). Adiponectin treatment alone had no significant effect on cell viability or apoptosis under NG. CON fAd gAd Figure 4.2: Adiponectin treatment attenuates the effect of hyperglycemia. Trypan blue exclusion assay shows that both gAd (lug/ml) and fAd (5ug/ml) protect H9c2 cells from hyperglycemia-induced cell death. n=3. * indicates p<0.05 vs Con NG and ** indicates p<0.05 vs Con HG. Graph changed to % trypan blue instead of fold over control. 108 NG HG con fAd 9M con fM 9Ad NG HG Figure 4.3: Adiponectin protects H9c2 from HG-induced apoptosis. Adiponectin's ability to prevent hyperglycemia-induced apoptosis was shown in its ability to suppress PS exposure (A), caspase 3 activation (B), and DNA fragmentation measured by TUNEL (C). TUNEL staining of DNA fragmentation is shown in green and total nuclei in blue stained with DAPI. Adiponectin treatment alone had no effect on cell viability or apoptosis. n>4/treatment. * indicates p<0.05 vs Con NG and ** indicates p<0.05 vs Con HG. 4.4.3 Adiponectin's Anti-Apoptotic Action is Mediated Through the Activation of p38 Next, I investigated intracellular signaling mechanisms by which adiponectin acts to exert its anti-apoptotic effect. Western blot analysis showed treatment by either fAd or gAd increased the phosphorylation level of p38 MAPK at T180/Y182 in H9c2 cells within 5 min after treatment (Figure 4.4). In order to examine whether activation of this kinase is necessary for adiponectin's anti-apoptotic action against hyperglycemia, cells were treated with the inhibitor for p38a and P isoforms, SB203580 (SB, lOuM) before treatment with adiponectin and the level of caspase 3 109 activity was examined. I found that adiponectin was no longer able to inhibit the increase in caspase 3 activity caused by HG treatment when p38 activity was inhibited (Figure 4.5A). Similarly, cell viability was assessed using MTT assay to examine whether this inability of adiponectin to suppress the increase in caspase 3 activity resulted in the loss of adiponectin's protective action against hyperglycemia-induced apoptosis. Indeed, treatment by fAd or gAd failed to significantly abrogate the effect of HG on cell viability when p38 activation was suppressed by SB (Figure 4.5B). A B fAd 0 5 10 20 30 min Time (mm) Figure 4.4: Adiponectin stimulates phosphorylation of p38 MAPK at T180/Y182. (A) Western blot analysis shows that acute treatment of H9c2 cells with gAd (lug/ml) and fAd (5ug/ml) induces an increase in the phosphorylation of p38 MAPK at T180/Y182. Blots were quantified and average values shown in graph (B). n=5. * indicates p<0.01 vs 0 min treatment of its own treatment. 110 DMSO DMSO 1.4 • NG 1.2 • HG ?1.0 £ c 0.8 5 «| 0.6 i°I°'| 4 0.2 0.0 fAd • - gAd - + DMSO SB DMSO Figure 4.5: Activation p38 is essential for adiponectin's ability to protect H9c2 cells from hyperglycemia-induced apoptosis. (A) Caspase 3 activity was measured from cells pretreated with p38 inhibitor (SB, lOuM) followed by fAd (5fig/ml or gAd lug/ml) treatment in HG media for 48hr. Both forms of adiponectin completely lost their ability to inhibit caspase 3 activation in the presence of SB. (B) Cell viability was measured by MTT assay after incubation with HG media for 72hr. After which, neither form of adiponectin was able to attenuate cell death induced by HG when cells were pre-treated with SB prior to treatment with gAd (1 jig/ml) and fAd (5[ig/ml). n=3. * indicates p<0.05 vs NG of its treatment group. ** indicates p<0.05 vs HG of its treatment group without adiponectin. Ill 4.4.4 Hyperglycemia Inhibits Autophagy Since autophagy and apoptosis are closely related and the upregulation of autophagy is implicated in the protection against apoptosis [18, 32,Yan, 2005 #454], I investigated the role of autophagy in HG-induced apoptosis. First, I investigated to see if HG treatment affects autophagy at all by performing Western blot for LC3II, a commonly used marker for autophagy [93, 167]. (Refer to Chapter 1.4.2.4 for more detailed information.) Dose-dependent treatment of H9c2 cells with BafAl indicated that lOnM concentration is sufficient to block the fusion of autophagosome with lysosomes; therefore, this concentration of BafAl was used for further experiments (Figure 4.6A). HG treatment either for 24hr or 48hr resulted in a smaller accumulation of LC3II both at steady and accumulation states (Figure 4.6B). This indicates the reduction of autophagosome formation and inhibition of autophagy. DMSO BafAl OnM SnM 10nM 2SnM 50nM 100nM 5 10 25 50 100 Concentration (nM) X I1! Bffi LC3I LC31I NG HG BaUl 24hf 48hr DmDMSO1L BrfAl JDMSO B«fA1 24hr 4thr Figure 4.6: HG treatment of H9c2 cells induces autopbagy. The autophagy flux was measured under NG and HG conditions with or without BafAl treatment. (A) Appropriate concentration of BafAl to treat H9c2 cells to inhibit autophagosomal degradation by lysosome was determined using Western blot. (B) Quantification of the blot confirms that autophagosome degradation was blocked starting at 5nM of BafAl (B). n=l for (A) and (B). (C) Cells were incubated in NG or HG media with or without treatment of lOnM BafAl. At 24hr incubation, cells incubated in HG media show a reduced level of autophagy as is shown by a smaller accumulation of LC3II in the presence of BafAl. Cells not treated with BafAl were treated with DMSO and represent the steady state of autophagy. Cells treated with BafAl represent the accumulation state of autophagy and is an indication of autophagosome flux. (D) Quantification of Western blot of n=3. Image for (C) shown here are representative blot from n=3. * indicates p<0.05 vs NG of its own group. 113 4.4.5 Adiponectin Prevents the Suppression of Autophagy Induced by Hyperglycemia Next, I examined the role of adiponectin on the regulation of autophagy. Adiponectin is well known for its ability to induce AMPK activation and I confirmed that treatment with fAd or gAd increases the phosphorylation of AMPK in H9c2 cells (Figure 4.7A&B). Since AMPK is known to be able to control autophagy through the mTOR pathway, I investigated to see whether or not adiponectin is able to regulate autophagy. With a 48hr treatment of either fAd or gAd, HG-induced inhibition of autophagosome formation was restored to the level similar to that of control LG treated cells (Figure 4.7C). Treatment with adiponectin resulted in more accumulation of LC3II under HG incubation with BafAl. This clearly indicated that treatment with either form of adiponectin enhanced both the production and the degradation of autophagosome. 114 B (Ad 0 5 10 20 30 min P-AMPKT172 10 • (Ad f^adn —— 2J5 U *-5 gAd 0 5 10 15 20 30 K 11.0 P-AMPKT172 , 05 00 AMPK alphas- 5 10 20 Tlme(mtn) DMSO Ba(A1 7.5 n jjjjjjNGHG >5.0- . 2.5" m1DB31DEw con lAd gAd con fAd gAd DMSO BafA1 Figure 4.7: Adiponectin induces phosphorylation of AMPK and reverses the effect of HG on autophagy. (A) Both fAd and gAd treatment results in a rapid increase in the phosphorylation level of AMPK at T172. Blots were quantified and average values are shown in graph (B). * indicates p<0.01 vs 0 min treatment of its own treatment. (C) Adiponectin treatment blocks the decrease in autophagy induced by HG. Accumulation of LC3II with BafAl treatment is increased in the presence of fAd or gAd. Quantification of LC3II is shown in graph (D). * indicates p<0.05 vs NG of its own group. ** indicates p<0.05 vs HG of its own group without adiponectin treatment. n=3. 115 4.5 Discussion Circulating HMW adiponectin concentration is significantly decreased in type 2 diabetic individuals. Studies have shown that this decrease is positively correlated with the development of cardiovascular disease, including heart failure, in these individuals [247, 252, 253]. However, the mechanisms by which adiponectin may be directly associated with heart failure are not entirely clear. Apoptosis is an important component of cardiac remodeling leading to heart failure but the current literature has primarily shown direct effects of adiponectin against cardiomyocyte apoptosis in acute ischemia/reperfusion injury [61, 152, 250]. Hyperglycemia has been identified as a direct cause of diabetic cardiomyopathy and cardiomyocyte apoptosis [244, 245, 258-260] thus hyperglycemia in diabetic patients may be playing an important role in the development of heart failure in these individuals. Diabetes is known to have a negative effect on the development of heart failure, while adiponectin's effect on heart failure is known to be positive. Since diabetes and adiponectin are associated with the development of the disease where cardiomyocyte apoptosis plays an important role, my objective was to study the effect of adiponectin on apoptosis induced by hyperglycemia. I first confirmed that incubation of H9c2 cardiomyocytes in HG media significantly increases the number of apoptotic cells as was previously shown [244, 245, 258-260]. Treatment of these cells with two forms of adiponectin, fAd or gAd, demonstrated that both forms of adiponectin are directly able to act on cardiomyocytes to counteract the effect of hyperglycemia and prevent the initiation of apoptosis. PS exposure to the outer membrane is one of the earliest markers of 116 apoptosis that can be detected [261]. Adiponectin's ability to inhibit this event shows that adiponectin's action to inhibit apoptosis occurs at an early stage. Adiponectin's anti-apoptotic capacity was also confirmed by its ability to prevent the activation of caspase 3 and DNA fragmentation. The increase in the activity of caspase 3 caused by HG was completely reversed back to the basal level with both adiponectin treatments. DNA fragmentation measured by TUNEL method also showed that treatment with adiponectin dramatically decreased the number of TUNEL positive cells under HG condition. Since treatment of H9c2 cells with either form of adiponectin resulted in a rapid increase in the phosphorylation level of p38, I investigated whether this activation of p38 also contributed to adiponectin's protective action on cardiomyocyte against hyperglycemia-induced apoptosis. Inhibition of p38 by SB203580 resulted in the complete loss of adiponectin's ability to both inhibit the activation of caspase 3 and cell death indicating that p38 activation is involved in mediating adiponectin's cardioprotective action against hyperglycemia. Although my results indicate the anti- apoptotic role of p38, the function of p38 in deciding cell fate in cardiomyocytes is debatable as there are many publications that support both pro-and anti-apoptotic effects of p38. For example, p38 has been shown to induce apoptosis by phosphorylating Bcl-2 and targeting it for degradation [262] or stimulating Bax translocation to mitochondria in cardiac myocytes [263]. Inhibition of p38 has also been shown to be cardioprotective by reducing the injury after ischemia/reperfusion [264, 265]. However, the activation of p38 is implicated in the upregulation of Pim3 to prevent apoptosis in cardiomyocytes [266]. It has also been suggested that p38 activates heat shock protein 27 (Hsp27) which acts as an anti-apoptotic protein by 117 reducing ROS and Bax:Bcl2 ratio to prevent cardiomyocyte apoptosis induced by doxorubicin [267]. Increased activation of p38 is important for its ability to precondition the heart for ischemic injury [268-271], and a decreased expression of p38a may be involved in the development of heart failure [272]. The role of p38 in regulating apoptosis and the development of heart failure is therefore perplexing. Whether or not p38 plays a pro-apoptotic or anti-apoptotic role in the development of heart failure may depend on other coexisting factors; it may also depend on the localization of this kinase within the cellular matrix. Nonetheless, my results indicate that activation of p38 by adiponectin plays an anti-apoptotic role in response to hyperglycemia-induced apoptosis and the activation of this kinase plays an important role in mediating adiponectin's anti-apoptotic effect. Indeed, adiponectin has been shown to promote cell survival in human primary hepatocytes by inducing the production of survival factor CXCL8 through the activation of p38 [273]. However, this study is the first to demonstrate the anti-apoptotic action of adiponectin on cardiomyocytes through the activation of p38. Apoptosis is closely associated with autophagy, and the upregulation of autophagy is implicated in the reduction of apoptosis in cardiomyocytes [98, 100]. Autophagy can be regulated by AMPK signaling pathway in the heart [101] and since adiponectin is a potent activator of AMPK, I speculated that adiponectin may be able to regulate autophagy activation. HG treatment inhibited autophagy in H9c2 cells and indeed, either fAd or gAd treatment of H9c2 cells reversed the effect of HG on autophagy. HG treatment resulted in the inhibition of autophagosome formation, thus the initiation of autophagy, as was evidenced by a smaller accumulation of LC3II under HG treatment in the presence of BafAl. Although I was not able to confirm the 118 exact mechanism by which adiponectin regulates autophagy, I consider that adiponectin may be able to do this at least in part through AMPK activation. Indeed, it has previously been shown that AMPK activation results in a decrease in phosphorylation of mTOR at Ser2481 residue and in Thr389 phosphorylation of downstream target of mTOR, p70S6K and this was associated with an increase in starvation-induced autophagy in neonatal cardiomyocytes [101]. Recently, Xie et al. [274] also reported that type 1 diabetic OVE26 mice exhibit decreased cardiac autophagy with reduction in AMPK activation. Overexpression of dominant negative AMPK in cardiomyocytes resulted in further inhibition of cardiac autophagy in these animals, which was associated with exacerbated cardiac dysfunction and increased mortality. On the other hand, the chronic activation of AMPK with metformin in these diabetic animals significantly stimulated the activation of autophagy, demonstrating the important role of AMPK in regulation of autophagy in the heart. Although there is little known about the role of p38 in the regulation of autophagy, p38 has also been implicated in the activation of autophagy induced by lipopolysaccharide and TNF-alpha in HL-lcardiomyocyte [275]. p38 activation is also reported to be involved in the upregulation of autophagy in cancer cells. For example, p38 activation is necessary in the autophagic cell death of breast cancer cells induced by pineapple extract, bromelain, by increasing the expression of Beclin-1 [276]; and of liver cancer cells induced by berberine, a Chinese medicine extract [277]. Therefore p38 may also be able to regulate autophagy in cardiomyocytes, and although I was not able to link the activation of p38 by adiponectin to the activation of autophagy in this study, it is worthwhile to conduct this study in the future. 119 p38 has been proposed to be a downstream target of AMPK in skeletal muscle cells [278] and this finding may also apply to our system with H9c2. Yoon et al. [278] reported that treatment of C2C12 myotubes with adiponectin increases the phosphorylation level of both AMPK and p38. However, when these cells were treated with araA, an AMPK inhibitor, phosphorylation of p38 MAPK by adiponectin was inhibited, whereas p38 MAPK inhibition with SB treatment did not affect AMPK phosphorylation level. When H9c2 cells are treated with adiponectin, I also see an increase in the phosphorylation level of both AMPK and p38. Therefore it is possible that the phosphorylation of AMPK by adiponectin treatment could be an upstream signaling leading to an elevation in p38 phosphorylation in H9c2 cardiomyocytes . However, it has also been reported by Ho et al. [279] that p38 is not a downstream component of the AMPK pathway, as the treatment of L6 skeletal myoblasts and myotubes with AICAR, an AMPK activator, did not result in the increase in p38 phosphorylation. Moreover, transgenic mice expressing the inactive form of AMPKalpha2 showed no abnormality in contraction-mediated p38 phosphorylation. Although they presented clear evidences that p38 is not a downstream target of AMPK, they did not examine whether AMPK stimulation by adiponectin can affect the phosphorylation off p38. Since p38 phosphorylation mediated by AMPK may be specific to adiponectin signaling, this needs to be tested and merits further study in cardiomyocytes as well. It was previously reported by our lab that incubation of L6 skeletal muscle cells in HG media leads to a decrease in AdipoRl expression and the development of adiponectin resistance [257]. However it is not clear whether or not cardiomyocytes will respond to chronic incubation with HG in the same way, and it will be important 120 to investigate this to understand the adiponectin's action under chronic incubation with HG. In summary, incubation of H9c2 cardiomyocytes under hyperglycemic condition results in decreased cell viability due to increased level of apoptosis. Treatment with fAd or gAd forms of adiponectin protects cells from hyperglycemia- induced apoptosis as early as PS externalisation to the outer membrane, caspase 3 activation and ultimately DNA fragmentation. This is the first study to demonstrate adiponectin's potential cardioprotective effects against hyperglycemia-induced apoptosis. It is also the first study to demonstrate that such anti-apoptotic action occurs through a p38MAPK-dependent signaling mechanism. HG treatment also resulted in the inhibition of autophagy which could be reversed with fAd or gAd treatment. Thus, adiponectin's anti-apoptotic effect may also involve its ability to regulate autophagy. Since both apoptosis and autophagy are potential therapeutic targets to treat heart failure, adiponectin's anti-apoptotic effect under hyperglycemic condition may be of significant physiological and therapeutic relevance. 121 Adiponectin (fAd, gAd) / \ ? AMPK > p38 HG Autophagy -J Apoptosis Figure 4.8: Adiponectin's action against HG-induced apoptosis: summary schematic diagram. Solid lines indicate the pathways that have been confirmed in the present study. Dotted lines indicate the pathways which have been verified or suggested by other publications but were not confirmed in this study and merit examination in future experiments. 4.6 Acknowledgement This work was supported by the Doctoral Research Award from the Heart and Stroke Foundation of Canada which was granted to ME. It was also supported by the Heart and Stroke Foundation of Canada operating grant and Canadian Institutes of Health Research (CIHR) New Investigators Award to GS. 122 5 Chapter 5 Conclusion 123 This project was conducted to investigate the role of diabetes in the regulation of cardiac remodeling events at both molecular and physiological levels. The summary of the finding of this work are as follows: 1. Diabetes is associated with a higher incidence of heart failure and cardiac ischemic events [157]. Patients with both type 1 and type 2 diabetes have a higher risk for encountering sudden death after acute MI and these individuals also exhibit higher mortality rate and risk for developing left ventricular dysfunction after MI [158,159]. Although the adverse influence of diabetes on left ventricular dysfunction is now a well established phenomenon, the temporal nature of cardiac remodeling events is often neglected and require further investigation. This work provided finding that the presence of diabetes exerted a differential effect on the cardiac remodelling pattern after IR. In the acute stage after IR, diabetes may confer some beneficial effect on the heart by upregulating autophagy and reducing the infarct size. Upregulation of autophagy during ischemia has been shown to be beneficial [100, 101]. At the same time, however, uptake was compromised in diabetic mice. In a long-term post-IR, diabetic mice developed severe accumulation of interstitial collagen and concentric hypertrophy whereas VEH mice showed the development of mild LV dilation. Concentric hypertrophy and fibrosis are associated with a higher degree of morbidity and mortality [17], leading to detrimental effects on cardiac functional performance in STZ heart. 2. MMP activity and ECM remodeling are known to be important components of cardiac remodeling after MI [120, 125, 126, 132, 134, 202], In this work, I 124 have also demonstrated that MI induced cardiac ECM remodelling as indicated by an activation of MMPs and a reorganization of the cardiac collagen scaffold. This remodelling process was aggravated in the presence of diabetes which caused a further activation of MMPs in the infarct area that was likely associated with excessive ECM degradation, dilation of the left ventricle and elevated mortality rate after MI. Hearts from diabetic animals exhibited a greater degree of cardiac dyssynchrony, which is known to be an indicator of an increased mortality, mitral regurgitation, and the progression of LV remodeling [209]. I have demonstrated insulin as a regulator of MMP activation and the hypoinsulinemia seen in diabetic individuals may be one factor responsible for the aggravated response in ECM remodeling. Insulin was able to inhibit the secretion of pro-MMP-9 and active MMP-2; therefore, the absence of insulin in the STZ-diabetic animals may be one mechanism leading to the excessive activation of MMPs. MI is associated with a high mortality rate due to cardiac wall rupture [125, 211, 212] and the higher incidence of death in the diabetics may be due to excessive ECM remodeling leading to the weakening of the LV wall. This work also established and used a near-infrared MMP substrate probe with FMT for analysis of cardiac MMP activity for the first time. The exact isoform of MMP that is mainly activated in the heart after MI needs to be identified in the future, along with the type of collagen (type I vs type III) depositing in the remote area of the heart post-MI between VEH and STZ heart. 125 3. A significant decrease in the circulating HMW adiponectin level in type 2 diabetic individuals is positively correlated with the development of cardiovascular disease, including heart failure, in these individuals [247, 252, 253] and my work has provided evidence that adiponectin is able to counteract the cytotoxic effect of hyperglycemia on H9c2 cells. Hyperglycemia induced apoptosis in H9c2 cells and this was inhibited by adiponectin. The anti- apoptotic effect of adiponectin was mediated through the activation of p38, and possibly through upregulating the level of autophagy, which was downregulated in hyperglycemic condition. Whether upregulation of autophagy by adiponectin under hyperglycaemic condition is regulated by the activation of p38 is a question that needs to be investigated in the future. The involvement of AMPK in the activation of p38, as well as the effect of chronic incubation of H9c2 cardiomyocytes under hyperglycemic condition on the development of adiponectin resistance require further examination. In conclusion, through these projects, I have demonstrated that diabetes is a critical regulator of cardiac remodeling events and confers complex time dependent differential effects both at molecular and physiological levels on many aspects of cardiac remodeling events. I have demonstrated in this PhD project using both in vivo and in vitro models, how hyperglycemia, hypoinsulinemia, and changes in adiponectin level contribute to the acceleration of the cardiac remodeling events via negatively acting on cardiomyocyte apoptosis, ECM remodeling, cardiac metabolism and contractility. 126 Although this work investigated only a few of the diabetic characteristics on some of the cardiac remodeling events, each of the findings arising from this work further developed our understanding of both the physiological and the molecular consequences of diabetes in the progression of cardiac remodeling, ultimately leading to heart failure. Accordingly, these findings will help in defining possible therapeutic targets to treat or prevent the development of heart failure in diabetic patients. 127 6 References 1. Joshi, R., et al., Global inequalities in access to cardiovascular health care: our greatest challenge. J Am Coll Cardiol, 2008. 52(23): p. 1817-25. 2. Statistics, [cited 2011 June 15]; Available from: http://www.heartandstroke.eom/site/c.ikIOLcMWJtE/b.3483991/k.34A8/ Statistics.htm. 3. Association, C.D. Diabetes facts. 2011 [cited 2011 September 15]. 4. Nussey, S. and S. Whitehead, Endocrinology: An Integrated Approach. 2001: Oxford: BIOS Scientific Publishers. 5. Fox, C.S., et al., Lifetime risk of cardiovascular disease among individuals with and without diabetes stratified by obesity status in the Framingham heart study. Diabetes Care, 2008.31(8): p. 1582-4. 6. Franco, O.H., et al., Associations of diabetes mellitus with total life expectancy and life expectancy with and without cardiovascular disease. Arch Intern Med, 2007.167(11): p. 1145-51. 7. Gustafsson, I., et al., Unrecognized glycometabolic disturbance as measured by hemoglobin Ale is associated with a poor outcome after acute myocardial infarction. Am Heart J, 2007.154(3): p. 470-6. 8. Stratton, I.M., et al., Association of glycaemia with macrovascular and microvascular complications of type 2 diabetes (UKPDS 35): prospective observational study. BMJ, 2000.321(7258): p. 405-12. 9. Velagaleti, R.S., et al., Relations of lipid concentrations to heart failure incidence: the Framingham Heart Study. Circulation, 2009.120(23): p. 2345-51. 10. Prevention, C.f.D.C.a. (2011) National diabetes fact sheet: national estimates and general information on diabetes and prediabetes in the United States, 2011. 11. ter Keurs, H.E., Heart failure and Starling's Law of the heart Can J Cardiol, 1996.12(10): p. 1047-57. 12. Konstam, M.A., et al., Left ventricular remodeling in heart failure: current concepts in clinical significance and assessment JACC Cardiovasc Imaging, 2011.4(1): p. 98-108. 13. Abel, E.D., S.E. Litwin, and G. Sweeney, Cardiac remodeling in obesity. Physiol Rev, 2008.88(2): p. 389-419. 14. Kai, H., [Cardiac remodeling in chronic heart failure]. Nippon Rinsho, 2006.64(5): p. 855-60. 15. Pfeffer, M.A., Left ventricular remodeling after acute myocardial infarction. Annu Rev Med, 1995.46: p. 455-66. 16. Cohn, J.N., R. Ferrari, and N. Sharpe, Cardiac remodeling—concepts and clinical implications: a consensus paper from an international forum on cardiac remodeling. Behalf of an International Forum on Cardiac Remodeling. J Am Coll Cardiol, 2000.35(3): p. 569-82. 17. V erma, A., et al., Prognostic implications of left ventricular mass and geometry following myocardial infarction: the VALIANT (VALsartan In 128 Acute myocardial iNfarcTioit) Echocardiographic Study. JACC Cardiovasc Imaging, 2008.1(5): p. 582-91. 18. Hamacher-Brady, A., N.R. Brady, and R.A. Gottlieb, The interplay between pro-death and pro-survival signaling pathways in myocardial ischemia/reperfusion injury; apoptosis meets autophagy. Cardiovasc Drugs Ther, 2006.20(6): p. 445-62. 19. Centurion, O.A., The open artery hypothesis: beneficial effects and long- term prognostic importance ofpatency of the infarct-related coronary artery. Angiology, 2007.58(1): p. 34-44. 20. McMurray, J.J. and M.A. Pfeffer, Heart failure. Lancet, 2005.365(9474): p. 1877-89. 21. Mookadam, F. and S.E. Moustafa, Prevention of late postmyocardial infarction left ventricular remodeling: an update. Curr Heart Fail Rep, 2009.6(4): p. 245-53. 22. Rumberger, J.A., et al., Nonparallel changes in global left ventricular chamber volume and muscle mass during the first year after transmural myocardial infarction in humans. J Am Coll Cardiol, 1993.21(3): p. 673- 82. 23. Clarke, M., M. Bennett, and T. Littlewood, Cell death in the cardiovascular system. Heart, 2007.93(6): p. 659-64. 24. Denzel, M.S., et al., T-cadherin is critical for adiponectin-mediated cardioprotection in mice. J Clin Invest. 120(12): p. 4342-52. 25. Shimano, M., et al., Adiponectin deficiency exacerbates cardiac dysfunction following pressure overload through disruption of an AMPK-dependent angiogenic response. J Mol Cell Cardiol, 2010.49(2): p. 210-20. 26. Crow, M.T., et al., The mitochondrial death pathway and cardiac myocyte apoptosis. Circ Res, 2004.95(10): p. 957-70. 27. Wang, Y., et al., Cardioprotective effect of adiponectin is partially mediated by its AMPK-independent antinitrative action. Am J Physiol Endocrinol Metab, 2009.297(2): p. E384-91. 28. Dalton, S., Cardiac stem cells: at the heart of cell therapy. Regen Med, 2008.3(2): p. 181-8. 29. Kunapuli, S., S. Rosanio, and E.R. Schwarz, "How do cardiomyocytes die?" apoptosis and autophagic cell death in cardiac myocytes. J Card Fail, 2006.12(5): p. 381-91. 30. Reeve, J.L., et al., Don't lose heart—therapeutic value of apoptosis prevention in the treatment of cardiovascular disease. J Cell Mol Med, 2005.9(3): p. 609-22. 31. Gustafsson, A.B. and R.A. Gottlieb, Eat your heart out: Role of autophagy in myocardial ischemia/reperfusion. Autophagy, 2008.4(4): p. 416-21. 32. Nishida, K., O. Yamaguchi, and K. Otsu, Crosstalk between autophagy and apoptosis in heart disease. Circ Res, 2008.103(4): p. 343-51. 33. Clerk, A., et al., Regulation of cardiac myocyte cell death. Pharmacol Ther, 2003.97(3): p. 223-61. 34. Pop, C. and G.S. Salvesen, Human caspases: activation, specificity, and regulation. J Biol Chem, 2009.284(33): p. 21777-81. 129 35. Stennicke, H.R., et al., Pro-caspase-3 is a major physiologic target of caspase-8. J Biol Chem, 1998. 273(42): p. 27084-90. 36. Tyas, L., et al., Rapid caspase-3 activation during apoptosis revealed using fluorescence-resonance energy transfer. EMBO Rep, 2000.1(3): p. 266-70. 37. Whelan, R.S., V. Kaplinskiy, and R.N. Kitsis, Cell death in the pathogenesis of heart disease: mechanisms and significance. Annu Rev Physiol, 2010. 72: p. 19-44. 38. Ashkenazi, A. and V.M. Dixit, Death receptors: signaling and modulation. Science, 1998.281(5381): p. 1305-8. 39. Zha, J., et al., Posttranslational N-myristoylation of BID as a molecular switch for targeting mitochondria and apoptosis. Science, 2000.290(5497): p. 1761-5. 40. Acehan, D., et al., Three-dimensional structure of the apoptosome: implications for assembly, procaspase-9 binding, and activation. Mol Cell, 2002.9(2): p. 423-32. 41. Qin, H., et al., Structural basis of procaspase-9 recruitment by the apoptotic protease-activating factor 1. Nature, 1999.399(6736): p. 549-57. 42. Bratton, S.B., et al., Recruitment, activation and retention of caspases-9 and -3 by Apaf-1 apoptosome and associated XIAP complexes. EMBO J, 2001. 20(5): p. 998-1009. 43. Suzuki, Y., Y. Nakabayashi, and R. Takahashi, Ubiquitin-protein ligase activity ofX-linked inhibitor of apoptosis protein promotes proteasomal degradation of caspase-3 and enhances its anti-apoptotic effect in Fas- induced cell death. Proc Natl Acad Sci USA, 2001.98(15): p. 8662-7. 44. Janicke, R.U., et al., Caspase-3 is required for DNA fragmentation and morphological changes associated with apoptosis. J Biol Chem, 1998. 273(16): p. 9357-60. 45. Leers, M.P., et al., Immunocytochemical detection and mapping of a cytokeratin 18 neo-epitope exposed during early apoptosis. J Pathol, 1999. 187(5): p. 567-72. 46. Yang, F., et al., Antibody to caspase-cleaved actin detects apoptosis in differentiated neuroblastoma and plaque-associated neurons and microglia in Alzheimer's disease. Am J Pathol, 1998.152(2): p. 379-89. 47. Sallmann, F.R., et al., Characterization of antibodies specific for the caspase cleavage site on poly(ADP-ribose) polymerase: specific detection of apoptotic fragments and mapping of the necrotic fragments ofpoly(ADP- ribose) polymerase. Biochem Cell Biol, 1997. 75(4): p. 451-6. 48. Antonsson, B., Box and other pro-apoptotic Bcl-2 family "killer-proteins" and their victim the mitochondrion. Cell Tissue Res, 2001.306(3): p. 347- 61. 49. Wei, M.C., et al., Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death. Science, 2001.292(5517): p. 727-30. 50. Antonsson, B., et al., Box oligomerization is required for channel-forming activity in liposomes and to trigger cytochrome c release from mitochondria. Biochem J, 2000.345 Pt 2: p. 271-8. 130 51. Lassus, P., X. Opitz-Araya, and Y. Lazebnik, Requirement for caspase-2 in stress-induced apoptosis before mitochondrialpermeabilization. Science, 2002.297(5585): p. 1352-4. 52. Miyashita, T. and J.C. Reed, Tumor suppressor p53 is a direct transcriptional activator of the human box gene. Cell, 1995. 80(2): p. 293-9. 53. Wood, D.E. and E.W. Newcomb, Cleavage of Box enhances its cell death function. Exp Cell Res, 2000. 256(2): p. 375-82. 54. Chen, Z., et al., Overexpression of Bcl-2 attenuates apoptosis and protects against myocardial I/R injury in transgenic mice. Am J Physiol Heart Circ Physiol, 2001.280(5): p. H2313-20. 55. Cheng, E.H., et al., BCL-2, BCL-X(L) sequester BH3 domain-only molecules preventing BAX- and BAK-mediated mitochondrial apoptosis. Mol Cell, 2001.8(3): p. 705-11. 56. Wang, K., et al., BID: a novel BH3 domain-only death agonist Genes Dev, 1996.10(22): p. 2859-69. 57. van Heerde, W.L., et al., Markers of apoptosis in cardiovascular tissues: focus on Annexin V. Cardiovasc Res, 2000.45(3): p. 549-59. 58. Watanabe, M., et al., The pros and cons of apoptosis assays for use in the study of cells, tissues, and organs. Microsc Microanal, 2002. 8(5): p. 375- 91. 59. Olivetti, G., et al., Apoptosis in the failing human heart N Engl J Med, 1997.336(16): p. 1131-41. 60. Matsui, T., et al., Akt activation preserves cardiac function and prevents injury after transient cardiac ischemia in vivo. Circulation, 2001.104(3): p. 330-5. 61. Shibata, R., et al., Adiponectin protects against myocardial ischemia- reperfusion injury through AMPK- and COX-2-dependent mechanisms. Nat Med, 2005.11(10): p. 1096-103. 62. Kim, A.S., et al., A small molecule AMPK activator protects the heart against ischemia-reperfusion injury. J Mol Cell Cardiol, 2011.51(1): p. 24- 32. 63. Bishopric, N.H., et al., Molecular mechanisms of apoptosis in the cardiac myocyte. Curr Opin Pharmacol, 2001.1(2): p. 141-50. 64. Curtis, L.H., et al., Incidence and prevalence of heart failure in elderly persons, 1994-2003. Arch Intern Med, 2008.168(4): p. 418-24. 65. Liu, M., et al., Aging might increase myocardial ischemia / reperfusion- induced apoptosis in humans and rats. Age (Dordr), 2011. 66. Shih, H., et al., The aging heart and post-infarction left ventricular remodeling. J Am Coll Cardiol, 2011.57(1): p. 9-17. 67. Xu, X.H., et al., VEGF attenuates development from cardiac hypertrophy to heart failure after aortic stenosis through mitochondrial mediated apoptosis and cardiomyocyte proliferation. J Cardiothorac Surg, 2011.6: p. 54. 68. Chen, M.S., et al., Statins initiated after hypertrophy inhibit oxidative stress and prevent heart failure in rats with aortic stenosis. J Mol Cell Cardiol, 2004.37(4): p. 889-96. 131 69. Yang, Z. and D.J. Klionsky, Mammalian autophagy: core molecular machinery and signaling regulation. Curr Opin Cell Biol, 2010.22(2): p. 124-31. 70. Cuervo, A.M., Chaperone-mediated autophagy: selectivity pays off. Trends Endocrinol Metab, 2010.21(3): p. 142-50. 71. Klionsky, D.J., A.M. Cuervo, and P.O. Seglen, Methods for monitoring autophagy from yeast to human. Autophagy, 2007.3(3): p. 181-206. 72. Mehrpour, M., et al., Autophagy in health and disease. 1. Regulation and significance of autophagy: an overview. Am J Physiol Cell Physiol, 2010. 298(4): p. C776-85. 73. Elmore, S.P., et al., The mitochondrial permeability transition initiates autophagy in rat hepatocytes. FASEB J, 2001.15(12): p. 2286-7. 74. Mizushima, N., The role of the Atgl/ULKl complex in autophagy regulation. Curr Opin Cell Biol, 2010. 22(2): p. 132-9. 75. Young, A.R., et al., Starvation and ULKl-dependent cycling of mammalian Atg9 between the TGN and endosomes. J Cell Sci, 2006.119(Pt 18): p. 3888-900. 76. Nowak, J., et al., The TP53INP2 protein is required for autophagy in mammalian cells. Mol Biol Cell, 2009.20(3): p. 870-81. 77. Ropolo, A., et al., The pancreatitis-induced vacuole membrane protein 1 triggers autophagy in mammalian cells. J Biol Chem, 2007. 282(51): p. 37124-33. 78. Takahashi, Y., C.L. Meyerkord, and H.G. Wang, Bif-l/endophilin Bl: a candidate for crescent driving force in autophagy. Cell Death Differ, 2009. 16(7): p. 947-55. 79. Liang, C., et al., Beclinl-binding UVRAG targets the class C Vps complex to coordinate autophagosome maturation and endocytic trafficking. Nat Cell Biol, 2008.10(7): p. 776-87. 80. Klionsky, D.J. and S.D. Emr, Autophagy as a regulated pathway of cellular degradation. Science, 2000.290(5497): p. 1717-21. 81. Gottlieb, R.A. and R.M. Mentzer, Autophagy during cardiac stress: joys and frustrations of autophagy. Annu Rev Physiol, 2010. 72: p. 45-59. 82. Gustafsson, A.B. and R.A. Gottlieb, Autophagy in ischemic heart disease. Circ Res, 2009.104(2): p. 150-8. 83. Levine, B., S. Sinha, and G. Kroemer, Bcl-2 family members: dual regulators of apoptosis and autophagy. Autophagy, 2008.4(5): p. 600-6. 84. Wei, Y., et al., JNKl-mediated phosphorylation of Bcl-2 regulates starvation-induced autophagy. Mol Cell, 2008.30(6): p. 678-88. 85. Zalckvar, E., et al., DAP-kinase-mediatedphosphorylation on the BH3 domain ofbeclin 1 promotes dissociation ofbeclin 1 from Bcl-XL and induction of autophagy. EMBO Rep, 2009.10(3): p. 285-92. 86. Sinha, S. and B. Levine, The autophagy effector Beclin 1: a novel BH3-only protein. Oncogene, 2008.27 Suppl 1: p. SI37-48. 87. Luo, S. and D.C. Rubinsztein, Apoptosis blocks Beclin 1-dependent autophagosome synthesis: an effect rescued by Bcl-xL. Cell Death Differ, 2010.17(2): p. 268-77. 132 88. Lee, J.S., et al., FLIP-mediated autophagy regulation in cell death control Nat Cell Biol, 2009.11(11): p. 1355-62. 89. Wu, Y. and B.P. Zhou, Kinases meet at TSC. Cell Res, 2007.17(12): p. 971-3. 90. Hosokawa, N., et al., Nutrient-dependent mTORCl association with the ULKl-Atgl3-FIP200 complex required for autophagy. Mol Biol Cell, 2009. 20(7): p. 1981-91. 91. Gwinn, D.M., et al., AMPK phosphorylation of raptor mediates a metabolic checkpoint Mol Cell, 2008.30(2): p. 214-26. 92. Liang, J., et al., The energy sensing LKB1-AMPKpathway regulates p27(kipl) phosphorylation mediating the decision to enter autophagy or apoptosis. Nat Cell Biol, 2007.9(2): p. 218-24. 93. Klionsky, D.J., et al., Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy, 2008.4(2): p. 151- 75. 94. Bjorkoy, G., et al., p62/SQSTMl forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death. J Cell Biol, 2005.171(4): p. 603-14. 95. Ivanova, S., et al., Lysosomes in apoptosis. Methods Enzymol, 2008.442: p. 183-99. 96. Takemura, G., et al., A utophagy maintains cardiac function in the starved adult. Autophagy, 2009.5(7): p. 1034-6. 97. Cao, D J., T.G. Gillette, and J.A. Hill, Cardiomyocyte autophagy: remodeling, repairing, and reconstructing the heart Curr Hypertens Rep, 2009.11(6): p. 406-11. 98. Yan, L., et al., Autophagy in chronically ischemic myocardium. Proc Natl Acad Sci USA, 2005.102(39): p. 13807-12. 99. Valentim, L., et al., Urocortin inhibits Beclinl-mediated autophagic cell death in cardiac myocytes exposed to ischaemia/reperfusion injury. J Mol Cell Cardiol, 2006.40(6): p. 846-52. 100. Hamacher-Brady, A., N.R. Brady, and R.A. Gottlieb, Enhancing macroautophagy protects against ischemia/reperfusion injury in cardiac myocytes. J Biol Chem, 2006.281(40): p. 29776-87. 101. Matsui, Y., et al., Distinct roles of autophagy in the heart during ischemia and reperfusion: roles of AMP-acHvatedprotein kinase and Beclin 1 in mediating autophagy. Circ Res, 2007.100(6): p. 914-22. 102. Nishida, K., et al., The role of autophagy in the heart Cell Death Differ, 2009.16(1): p. 31-8. 103. Matsui, Y., et al., Molecular mechanisms and physiological significance of autophagy during myocardial ischemia and reperfusion. Autophagy, 2008. 4(4): p. 409-15. 104. Zhu, H., et al., Cardiac autophagy is a maladaptive response to hemodynamic stress. J Clin Invest, 2007.117(7): p. 1782-93. 105. Libby, P. and R.T. Lee, Matrix matters. Circulation, 2000.102(16): p. 1874-6. 106. Alberts, B., Molecular biology of the cell. 4th ed. 2002, New York: Garland Science, xxxiv, [1548] p. 133 107. Fedak, P.W., et al., Cardiac remodeling and failure From molecules to man (Part II). Cardiovasc Pathol, 2005.14(2): p. 49-60. 108. Spotnitz, H.M., Macro design, structure, and mechanics of the left ventricle. J Thorac Cardiovasc Surg, 2000.119(5): p. 1053-77. 109. Factor, S.M. and T.F. Robinson, Comparative connective tissue structure- function relationships in biologic pumps. Lab Invest, 1988.58(2): p. 150-6. 110. Spinale, F.G., Matrix metalloproteinases: regulation and dysregulation in the failing heart Circ Res, 2002.90(5): p. 520-30. 111. Spinale, F.G., et al., Time-dependent changes in matrix metalloproteinase activity and expression during the progression of congestive heart failure: relation to ventricular and myocyte function. Circ Res, 1998.82(4): p. 482- 95. 112. Trueblood, N.A., et al., Exaggerated left ventricular dilation and reduced collagen deposition after myocardial infarction in mice lacking osteopontin. Circ Res, 2001.88(10): p. 1080-7. 113. Fomovsky, G.M., S. Thomopoulos, and J.W. Holmes, Contribution of extracellular matrix to the mechanical properties of the heart. J Mol Cell Cardiol, 2010.48(3): p. 490-6. 114. Weber, K.T., Cardiac interstitium in health and disease: the fibrillar collagen network. J Am Coll Cardiol, 1989.13(7): p. 1637-52. 115. Tyagi, S.C., Proteinases and myocardial extracellular matrix turnover. Mol Cell Biochem, 1997.168(1-2): p. 1-12. 116. Tyagi, S.C., A. Ratajska, and K.T. Weber, Myocardial matrix metalloproteinase(s): localization and activation. Mol Cell Biochem, 1993. 126(1): p. 49-59. 117. Gross, J. and C.M. Lapiere, Collagenolytic activity in amphibian tissues: a tissue culture assay. Proc Natl Acad Sci USA, 1962.48: p. 1014-22. 118. Brew, K. and H. Nagase, The tissue inhibitors of metalloproteinases (TIMPs): an ancient family with structural and functional diversity. Biochim Biophys Acta. 1803(1): p. 55-71. 119. Visse, R. and H. Nagase, Matrix metalloproteinases and tissue inhibitors of metalloproteinases: structure, function, and biochemistry. Circ Res, 2003. 92(8): p. 827-39. 120. Creemers, E.E., et al., Matrix metalloproteinase inhibition after myocardial infarction: a new approach to prevent heart failure? Circ Res, 2001.89(3): p. 201-10. 121. Ra, H J. and W.C. Parks, Control of matrix metalloproteinase catalytic activity. Matrix Biol, 2007.26(8): p. 587-96. 122. Spinale, F.G., Myocardial matrix remodeling and the matrix metalloproteinases: influence on cardiac form and function. Physiol Rev, 2007. 87(4): p. 1285-342. 123. Nuttall, R.K., et al., Expression analysis of the entire MMP and TIMP gene families during mouse tissue development. FEBS Lett, 2004.563(1-3): p. 129-34. 124. Koskivirta, I., et al., Mice with tissue inhibitor of metalloproteinases 4 (Timp4) deletion succumb to induced myocardial infarction but not to cardiac pressure overload. J Biol Chem, 2010. 285(32): p. 24487-93. 125. Matsumura, S., et al., Targeted deletion or pharmacological inhibition of MMP-2 prevents cardiac rupture after myocardial infarction in mice. J Clin Invest, 2005.115(3): p. 599-609. 126. Spinale, F.G., et al., Cardiac restricted overexpression of membrane type-1 matrix metalloproteinase causes adverse myocardial remodeling following myocardial infarction. J Biol Chem, 2010. 285(39): p. 30316-27. 127. Tyagi, S.C., et al., Matrix metalloproteinase activity expression in infarcted, noninfarcted and dilated cardiomyopathy human hearts. Mol Cell Biochem, 1996.155(1): p. 13-21. 128. Spinale, F.G., et al., A matrix metalloproteinase induction/activation system exists in the human left ventricular myocardium and is upregulated in heart failure. Circulation, 2000.102(16): p. 1944-9. 129. Thomas, C.V., et al., Increased matrix metalloproteinase activity and selective upregulation in LV myocardium from patients with end-stage dilated cardiomyopathy. Circulation, 1998.97(17): p. 1708-15. 130. Gunja-Smith, Z., et al., Remodeling of human myocardial collagen in idiopathic dilated cardiomyopathy. Role of metalloproteinases and pyridinoline cross-links. Am J Pathol, 1996.148(5): p. 1639-48. 131. van den Borne, S.W., et al., Increased matrix metalloproteinase-8 and -9 activity in patients with infarct rupture after myocardial infarction. Cardiovasc Pathol, 2009.18(1): p. 37-43. 132. Peterson, J.T., et al., Evolution of matrix metalloprotease and tissue inhibitor expression during heart failure progression in the infarcted rat Cardiovasc Res, 2000.46(2): p. 307-15. 133. Mukherjee, R., et al., Selective spatiotemporal induction of matrix metalloproteinase-2 and matrix metalloproteinase-9 transcription after myocardial infarction. Am J Physiol Heart Circ Physiol, 2006.291(5): p. H2216-28. 134. Kandalam, V., et al., TIMP2 deficiency accelerates adverse post-myocardial infarction remodeling because of enhanced MT1-MMP activity despite lack ofMMP2 activation. Circ Res, 2010.106(4): p. 796-808. 135. Liu, Y., et al., Functional significance of skeletal muscle adiponectin production, changes in animal models of obesity and diabetes, and regulation by rosiglitazone treatment Am J Physiol Endocrinol Metab, 2009.297(3): p. E657-64. 136. Pineiro, R., et al., Adiponectin is synthesized and secreted by human and murine cardiomyocytes. FEBS Lett, 2005.579(23): p. 5163-9. 137. Maddineni, S., et al., Adiponectin gene is expressed in multiple tissues in the chicken: food deprivation influences adiponectin messenger ribonucleic acid expression. Endocrinology, 2005.146(10): p. 4250-6. 138. Arita, Y., et al., Paradoxical decrease of an adipose-specific protein, adiponectin, in obesity. Biochem Biophys Res Commun, 1999.257(1): p. 79-83. 139. Waki, H., et al., Generation ofglobular fragment of adiponectin by leukocyte elastase secreted by monocytic cell line THP-1. Endocrinology, 2005.146(2): p. 790-6. 135 140. Wang, Y., et al., Post-translational modifications of the four conserved lysine residues within the collagenous domain of adiponectin are required for the formation of its high molecular weight oligomeric complex. J Biol Chem, 2006. 281(24): p. 16391-400. 141. Matsuzawa, Y., Adiponectin: Identification, physiology and clinical relevance in metabolic and vascular disease. Atheroscler Suppl, 2005.6(2): p. 7-14. 142. Vu, V., et al., Coculture with primary visceral rat adipocytes from control but not streptozotocin-induced diabetic animals increases glucose uptake in rat skeletal muscle cells: role of adiponectin. Endocrinology, 2007.148(9): p. 4411-9. 143. Guo, Z., et al., Cardiac expression of adiponectin and its receptors in streptozotocin-induced diabetic rats. Metabolism, 2007. 56(10): p. 1363-71. 144. Fang, X. and G. Sweeney, Mechanisms regulating energy metabolism by adiponectin in obesity and diabetes. Biochem Soc Trans, 2006.34(Pt 5): p. 798-801. 145. Yamauchi, T., et al., Cloning of adiponectin receptors that mediate antidiabetic metabolic effects. Nature, 2003.423(6941): p. 762-9. 146. Hug, C., et al., T-cadherin is a receptor for hexameric and high-molecular- weight forms ofAcrp30/adiponectin. Proc Natl Acad Sci USA, 2004. 101(28): p. 10308-13. 147. Mao, X., et al., APPL1 binds to adiponectin receptors and mediates adiponectin signalling and function. Nat Cell Biol, 2006.8(5): p. 516-23. 148. Zhou, L., et al., Adiponectin activates AMP-activatedprotein kinase in muscle cells via APPLl/LKBl-dependent andphospholipase C/Ca2+/Ca2+/calmodulin-dependent protein kinase kinase-dependent pathways. J Biol Chem, 2009.284(33): p. 22426-35. 149. Xin, X., et al., APPL1 mediates adiponectin-stimulated p38 MAPK activation by scaffolding the TAKl-MKK3-p38 MAPK pathway. Am J Physiol Endocrinol Metab, 2011.300(1): p. E103-10. 150. Shibata, R., et al., Adiponectin protects against the development of systolic dysfunction following myocardial infarction. J Mol Cell Cardiol, 2007. 42(6): p. 1065-74. 151. Shibata, R., et al., Adiponectin stimulates angiogenesis in response to tissue ischemia through stimulation of amp-activated protein kinase signaling. J Biol Chem, 2004.279(27): p. 28670-4. 152. Wang, Y., et al., AMP-activated protein kinase deficiency enhances myocardial ischemia/reperfusion injury but has minimal effect on the antioxidant/antinitrative protection of adiponectin. Circulation, 2009. 119(6): p. 835-44. 153. Wang, Y., et al., Cardiomyocyte-derived adiponectin is biologically active in protecting against myocardial ischemia-reperfusion injury. Am J Physiol Endocrinol Metab. 298(3): p. E663-70. 154. Shibata, R., et al., Adiponectin-mediated modulation of hypertrophic signals in the heart Nat Med, 2004.10(12): p. 1384-9. 136 155. Takahashi, T., et al., Adiponectin replacement therapy attenuates myocardial damage in leptin-deficient mice with viral myocarditis. J Int Med Res, 2005.33(2): p. 207-14. 156. Ikeda, Y., et al., Cyclooxygenase-2 induction by adiponectin is regulated by a sphingosine kinase-1 dependent mechanism in cardiac myocytes. FEBS Lett, 2008. 582(7): p. 1147-50. 157. Flaherty, J.D. and C J. Davidson, Diabetes and coronary revascularization. Jama, 2005. 293(12): p. 1501-8. 158. Fisher, B.M., Heart abnormalities in IDDM. Diabetologia, 1997.40 Suppl 2: p. S127-9. 159. Katayama, T., et al., Clinical outcomes and left ventricular function in diabetic patients with acute myocardial infarction treated by primary coronary angioplasty. Int Heart J, 2005.46(4): p. 607-18. 160. Bugger, H. and E.D. Abel, Rodent models of diabetic cardiomyopathy. Dis Model Mech, 2009. 2(9-10): p. 454-66. 161. Breckenridge, R., Heart failure and mouse models. Dis Model Mech. 3(3- 4): p. 138-43. 162. Mossman, B.T., G.L. Wilson, and J.E. Craighead, Chlorozocin. A diabetogenic analogue of streptozocin with dissimilar mechanisms of action on pancreatic beta cells. Diabetes, 1985.34(6): p. 602-10. 163. Ray at, G.R., et al., Immunization with streptozotocin-treated NOD mouse islets inhibits the onset of autoimmune diabetes in NOD mice. J Autoimmun, 2003.21(1): p. 11-5. 164. Wu, K.K. and Y. Huan, Streptozotocin-Induced Diabetic Models in Mice and Rats. Current Protocols in Pharmacology, 2008.40(5): p. 47.1-47.14. 165. Dahab, G.M., et al., Digital quantification of fibrosis in liver biopsy sections: description of a new method by Photoshop software. J Gastroenterol Hepatol, 2004.19(1): p. 78-85. 166. Kofoed, K.F., et al., Glucose metabolism in reperfused myocardium measured by [2-18F] 2-fluorodeoxyglucose and PET. Cardiovasc Res, 2000. 45(2): p. 321-9. 167. Kabeya, Y., et al., LC3, a mammalian homologue of yeast ApgSp, is localized in autophagosome membranes after processing. EMBO J, 2000. 19(21): p. 5720-8. 168. Geyer, H., et al., Assessment of myocardial mechanics using speckle tracking echocardiography: fundamentals and clinical applications. J Am Soc Echocardiogr. 23(4): p. 351-69; quiz 453-5. 169. Taha, M. and G.D. Lopaschuk, Alterations in energy metabolism in cardiomyopathies. Ann Med, 2007.39(8): p. 594-607. 170. Kantor, P.F., J.R. Dyck, and G.D. Lopaschuk, Fatty acid oxidation in the reperfused ischemic heart Am J Med Sci, 1999.318(1): p. 3-14. 171. Finegan, B.A., et al., Adenosine alters glucose use during ischemia and reperfusion in isolated rat hearts. Circulation, 1993.87(3): p. 900-8. 172. Broderick, T X., et al., Beneficial effect of carnitine on mechanical recovery of rat hearts reperfused after a transient period of global ischemia is accompanied by a stimulation of glucose oxidation. Circulation, 1993. 87(3): p. 972-81. 173. Lopaschuk, G.D., et al., Etomoxir, a carnitine palmitoyltransferase I inhibitor, protects hearts from fatty acid-induced ischemic injury independent of changes in long chain acylcarnitine. Circ Res, 1988.63(6): p. 1036-43. 174. Lopaschuk, G.D., et al., Glucose andpalmitate oxidation in isolated working rat hearts reperfused after a period of transient global ischemia. Circ Res, 1990.66(2): p. 546-53. 175. Carvalho, G., et al., Cardioprotective Effects of Glucose and Insulin Administration While Maintaining Normoglycemia (GIN Therapy) in Patients Undergoing Coronary Artery Bypass Grafting. J Clin Endocrinol Metab. 176. Sugimoto, S., A novel vacuolar myopathy with dilated cardiomyopathy. Autophagy, 2007.3(6): p. 638-9. 177. Kostin, S., et al., Myocytes die by multiple mechanisms in failing human hearts. Circ Res, 2003.92(7): p. 715-24. 178. Gustafsson, A.B. and R.A. Gottlieb, Mechanisms of apoptosis in the heart. J Clin Immunol, 2003.23(6): p. 447-59. 179. Rabinowitz, J.D. and E. White, Autophagy and metabolism. Science. 330(6009): p. 1344-8. 180. Levine, B. and G. Kroemer, Autophagy in the pathogenesis of disease. Cell, 2008.132(1): p. 27-42. 181. Salih, D.A. and A. Brunet, FoxO transcription factors in the maintenance of cellular homeostasis during aging. Curr Opin Cell Biol, 2008.20(2): p. 126-36. 182. Boudina, S., et al., Contribution of impaired myocardial insulin signaling to mitochondrial dysfunction and oxidative stress in the heart. Circulation, 2009.119(9): p. 1272-83. 183. Lutz, J., K. Thurmel, and U. Heemann, Anti-inflammatory treatment strategies for ischemia/reperfusion injury in transplantation. J Inflamm (Lond), 2010. 7: p. 27. 184. Sisley, A.C., et al., Neutrophil depletion attenuates human intestinal reperfusion injury. J Surg Res, 1994. 57(1): p. 192-6. 185. Frangogiannis, N.G., C.W. Smith, and M.L. Entman, The inflammatory response in myocardial infarction. Cardiovasc Res, 2002.53(1): p. 31-47. 186. Haudek, S.B., et al., Monocytic fibroblast precursors mediate fibrosis in angiotensin-II-induced cardiac hypertrophy. J Mol Cell Cardiol, 2010. 49(3): p. 499-507. 187. Ren, J., et al., Proinflammatory Protein CARD9 Is Essential for Infiltration of Monocytic Fibroblast Precursors and Cardiac Fibrosis Caused by Angiotensin II Infusion. Am J Hypertens, 2010. 188. Cleland, J.G., A. Torabi, and N.K. Khan, Epidemiology and management of heart failure and left ventricular systolic dysfunction in the aftermath of a myocardial infarction. Heart, 2005.91 Suppl 2: p. ii7-13; discussion ii31, ii43-8. 189. Aragno, M., et al., Oxidative stress triggers cardiac fibrosis in the heart of diabetic rats. Endocrinology, 2008.149(1): p. 380-8. 138 190. Ban, C.R. and S.M. Twigg, Fibrosis in diabetes complications: pathogenic mechanisms and circulating and urinary markers. Vase Health Risk Manag, 2008.4(3): p. 575-96. 191. Li, Q., et al., Effects of benazepril on cardiac fibrosis in STZ-induced diabetic rats. Acta Cardiol, 2010.65(4): p. 431-9. 192. Foy, S.G., et al., Neurohormonal changes after acute myocardial infarction. Relationships with haemodynamic indices and effects of ACE inhibition. Eur Heart J, 1995.16(6): p. 770-8. 193. Sechi, L.A., C.A. Griffin, and M. Schambelan, The cardiac renin- angiotensin system in STZ-induced diabetes. Diabetes, 1994.43(10): p. 1180-4. 194. Weber, K.T. and C.G. Brilla, Pathological hypertrophy and cardiac interstitium. Fibrosis and renin-angiotensin-aldosterone system. Circulation, 1991.83(6): p. 1849-65. 195. Aneja, A., et al., Diabetic cardiomyopathy: insights into pathogenesis, diagnostic challenges, and therapeutic options. Am J Med, 2008.121(9): p. 748-57. 196. Verma, A., et al., The relationship between renalfunction and cardiac structure, function, and prognosis after myocardial infarction: the VALIANT Echo Study. J Am Coll Cardiol, 2007.50(13): p. 1238-45. 197. Roger, V.L., et al., Heart disease and stroke statistics—2011 update: a report from the American Heart Association. Circulation, 2011.123(4): p. el8- e209. 198. Gao, L., et al., Role of kallistatin in prevention of cardiac remodeling after chronic myocardial infarction. Lab Invest, 2008.88(11): p. 1157-66. 199. Spinale, F.G., et al., Matrix metaUoproteinase inhibition during the development of congestive heart failure: effects on left ventricular dimensions and function. Circ Res, 1999.85(4): p. 364-76. 200. Marma, A.K. and D.M. Lloyd-Jones, Systematic examination of the updated Framingham heart study general cardiovascular risk profile. Circulation, 2009.120(5): p. 384-90. 201. Preis, S.R., et al., Trends in all-cause and cardiovascular disease mortality among women and men with and without diabetes mellitus in the Framingham Heart Study, 1950 to 2005. Circulation, 2009.119(13): p. 1728-35. 202. Rohde, L.E., et al., Matrix metaUoproteinase inhibition attenuates early left ventricular enlargement after experimental myocardial infarction in mice. Circulation, 1999.99(23): p. 3063-70. 203. Tarnavski, O., et al., Mouse cardiac surgery: comprehensive techniques for the generation of mouse models of human diseases and their application for genomic studies. Physiol Genomics, 2004.16(3): p. 349-60. 204. Jin, J., et al., Transplantation of mesenchymal stem cells within a poly(lactide-co-epsilon-caprolactone) scaffold improves cardiac function in a rat myocardial infarction model Eur J Heart Fail, 2009.11(2): p. 147-53. 205. Haudek, S.B., et al., Bone marrow-derived fibroblast precursors mediate ischemic cardiomyopathy in mice. Proc Natl Acad Sci USA, 2006.103(48): p. 18284-9. 206. Kamiya, T., et al., A simplified model of hypoxic injury in primary cultured rat hepatocytes. In Vitro Cell Dev Biol Anim, 1998.34(2): p. 131-7. 207. Ng, A.C., et al., Left ventricular longitudinal and radial synchrony and their determinants in healthy subjects. J Am Soc Echocardiogr, 2008.21(9): p. 1042-8. 208. Gnyawali, S.C., et al., High-frequency high-resolution echocardiography: first evidence on non-invasive repeated measure of myocardial strain, contractility, and mitral regurgitation in the ischemia-reperfused murine heart J Vis Exp, 2010(41). 209. Suffoletto, M.S., et al., Novel speckle-tracking radial strain from routine black-and-white echocardiographic images to quantify dyssynchrony and predict response to cardiac resynchronization therapy. Circulation, 2006. 113(7): p. 960-8. 210. Mukherjee, R., et al., Myocardial infarct expansion and matrix metalloproteinase inhibition. Circulation, 2003.107(4): p. 618-25. 211. Fang, L., et al., Differences in inflammation, MMP activation and collagen damage account for gender difference in murine cardiac rupture following myocardial infarction. J Mol Cell Cardiol, 2007.43(5): p. 535-44. 212. Arikawa, M., et al., Donepezil, Anti-Alzheimer's Disease Drug, Prevents Cardiac Rupture during Acute Phase of Myocardial Infarction in Mice. PLoS One, 2011.6(7): p. e20629. 213. D'Armiento, J., Matrix metalloproteinase disruption of the extracellular matrix and cardiac dysfunction. Trends Cardiovasc Med, 2002.12(3): p. 97-101. 214. Gao, X.M., et al., Lower risk of postinfarct rupture in mouse heart overexpressing beta 2-adrenergic receptors: importance of collagen content. J Cardiovasc Pharmacol, 2002.40(4): p. 632-40. 215. Ichihara, S., et al., Targeted deletion of angiotensin II type 2 receptor caused cardiac rupture after acute myocardial infarction. Circulation, 2002. 106(17): p. 2244-9. 216. Hutchinson, K.R., J.A. Stewart, Jr., and P.A. Lucchesi, Extracellular matrix remodeling during the progression of volume overload-induced heart failure. J Mol Cell Cardiol, 2010.48(3): p. 564-9. 217. Oishi, Y., et al., Myocardial Strain Imaging is a Superior Method for Evaluating Papillary Muscle Contractility in Patients With Myocardial Infarction. J Echocardiogr, 2005.3(4): p. 148-152. 218. Tigen, K., et al., Diffuse late gadolinium enhancement by cardiovascular magnetic resonance predicts significant intraventricular systolic dyssynchrony in patients with non-ischemic dilated cardiomyopathy. J Am Soc Echocardiogr, 2010.23(4): p. 416-22. 219. Porter, K.E. and N.A. Turner, Cardiac fibroblasts: at the heart of myocardial remodeling. Pharmacol Ther, 2009.123(2): p. 255-78. 220. Burke, B., et al., Hypoxia-inducedgene expression in human macrophages: implications for ischemic tissues and hypoxia-regulated gene therapy. Am J Pathol, 2003.163(4): p. 1233-43. 140 221. Canning, M.T., et al., Oxygen-mediated regulation of gelatinase and tissue inhibitor of metalloproteinases-1 expression by invasive cells. Exp Cell Res, 2001. 267(1): p. 88-94. 222. Munoz-Najar, U.M., et al., Hypoxia stimulates breast carcinoma cell invasion through MT1-MMP and MMP-2 activation. Oncogene, 2006. 25(16): p. 2379-92. 223. Lee, M.P. and G. Sweeney, Insulin increases gelatinase activity in rat glomerular mesangial cells via ERK- and PI-3 kinase-dependent signalling. Diabetes Obes Metab, 2006.8(3): p. 281-8. 224. Schuyler, C.A., et al., Insulin treatment attenuates diabetes-increased atherosclerotic intimal lesions and matrix metalloproteinase 9 expression in apolipoprotein E-deficient mice. J Endocrinol, 2011.210(1): p. 37-46. 225. Chaudhuri, A., et al., Effect of modified glucose-insulin-potassium on free fatty acids, matrix metalloproteinase, and myoglobin in ST-elevation myocardial infarction. Am J Cardiol, 2007.100(11): p. 1614-8. 226. Dandona, P., et al., Insulin suppresses plasma concentration of vascular endothelial growth factor and matrix metalloproteinase-9. Diabetes Care, 2003. 26(12): p. 3310-4. 227. Agocha, A., H.W. Lee, and M. Eghbali-Webb, Hypoxia regulates basal and induced DNA synthesis and collagen type I production in human cardiac fibroblasts: effects of transforming growth factor-betal, thyroid hormone, angiotensin Hand basic fibroblast growth factor. J Mol Cell Cardiol, 1997. 29(8): p. 2233-44. 228. Hu, C.P., et al., Blockade of hypoxia-reoxygenation-mediated collagen type I expression and MMP activity by overexpression ofTGF-betal delivered by AA V in mouse cardiomyocytes. Am J Physiol Heart Circ Physiol, 2007. 293(3): p. H1833-8. 229. Sodhi, C.P., et al., Hypoxia and high glucose cause exaggerated mesangial cell growth and collagen synthesis: role of osteopontin. Am J Physiol Renal Physiol, 2001.280(4): p. F667-74. 230. Tamamori, M., et al., Stimulation of collagen synthesis in rat cardiac fibroblasts by exposure to hypoxic culture conditions and suppression of the effect by natriuretic peptides. Cell Biol Int, 1997. 21(3): p. 175-80. 231. Zhang, Q., et al., The role of the intravascular microenvironment in spontaneous metastasis development Int J Cancer, 2010.126(11): p. 2534- 41. 232. Deguchi, J.O., et al., Inflammation in atherosclerosis: visualizing matrix metalloproteinase action in macrophages in vivo. Circulation, 2006.114(1): p. 55-62. 233. Cortez-Retamozo, V., et al., Real-time assessment of inflammation and treatment response in a mouse model of allergic airway inflammation. J Clin Invest, 2008.118(12): p. 4058-66. 234. Devereux, R.B., et al., Impact of diabetes on cardiac structure and function: the strong heart study. Circulation, 2000.101(19): p. 2271-6. 235. Hayat, S.A., et al., Diabetic cardiomyopathy: mechanisms, diagnosis and treatment. Clin Sci (Lond), 2004.107(6): p. 539-57. 141 236. Gerstein, H.C., et al., Effects of intensive glucose lowering in type 2 diabetes. N Engl J Med, 2008.358(24): p. 2545-59. 237. Patel, A., et al., Intensive blood glucose control and vascular outcomes in patients with type 2 diabetes. N Engl J Med, 2008.358(24): p. 2560-72. 238. Duckworth, W., et al., Glucose control and vascular complications in veterans with type 2 diabetes. N Engl J Med, 2009.360(2): p. 129-39. 239. Skyler, J.S., et al., Intensive glycemic control and the prevention of cardiovascular events: implications of the ACCORD, ADVANCE, and VA Diabetes Trials: a position statement of the American Diabetes Association and a Scientific Statement of the American College of Cardiology Foundation and the American Heart Association. J Am Coll Cardiol, 2009. 53(3): p. 298-304. 240. Lee, Y. and A.B. Gustafsson, Role of apoptosis in cardiovascular disease. Apoptosis, 2009.14(4): p. 536-48. 241. Hou, Z.Q., et al., Involvement of chronic stresses in rat islet and INS-1 cell glucotoxicity induced by intermittent high glucose. Mol Cell Endocrinol, 2008.291(1-2): p. 71-8. 242. McCarty, M.F., J. Barroso-Aranda, and F. Contreras, NAPDH oxidase mediates glucolipotoxicity-induced beta cell dysfunction - Clinical implications. Med Hypotheses, 2009. 243. Park, K.G., et al., Glucotoxicity in the INS-1 rat insulinoma cell line is mediated by the orphan nuclear receptor small heterodimer partner. Diabetes, 2007. 56(2): p. 431-7. 244. Cai, L., et al., Hyperglycemia-induced apoptosis in mouse myocardium: mitochondrial cytochrome C-mediated caspase-3 activation pathway. Diabetes, 2002.51(6): p. 1938-48. 245. Kumar, S. and S.L. Sitasawad, N-acetylcysteine prevents glucose/glucose oxidase-induced oxidative stress, mitochondrial damage and apoptosis in H9c2 cells. Life Sci, 2009.84(11-12): p. 328-36. 246. Cai, L. and YJ. Kang, Oxidative stress and diabetic cardiomyopathy: a brief review. Cardiovasc Toxicol, 2001.1(3): p. 181-93. 247. Liu, Y., et al., Total and high molecular weight but not trimeric or hexameric forms of adiponectin correlate with markers of the metabolic syndrome and liver injury in Thai subjects. J Clin Endocrinol Metab, 2007. 92(11): p. 4313-8. 248. Shetty, S., C.M. Kusminski, and P.E. Scherer, Adiponectin in health and disease: evaluation of adiponectin-targeted drug development strategies. Trends Pharmacol Sci, 2009. 249. Gonon, A.T., et al., Adiponectin protects against myocardial ischaemia- reperfusion injury via AMP-activatedprotein kinase, Akt, and nitric oxide. Cardiovasc Res, 2008.78(1): p. 116-22. 250. Tao, L., et al., Adiponectin cardioprotection after myocardial ischemia/reperfusion involves the reduction of oxidative/nitrative stress. Circulation, 2007.115(11): p. 1408-16. 251. Ohashi, K., et al., Adiponectin promotes revascularization of ischemic muscle through a cyclooxygenase 2-dependent mechanism. Mol Cell Biol, 2009.29(13): p. 3487-99. 252. Seino, Y., et al., High molecular weight multimer form of adiponectin as a useful marker to evaluate insulin resistance and metabolic syndrome in Japanese men. Metabolism, 2007.56(11): p. 1493-9. 253. Tabara, Y., et aL, Reduced high-molecular-weight adiponectin and elevated high-sensitivity C-reactive protein are synergistic risk factors for metabolic syndrome in a large-scale middle-aged to elderly population: the Shimanami Health Promoting Program Study. J Clin Endocrinol Metab, 2008. 93(3): p. 715-22. 254. Eguchi, M., et al., Leptin protects H9c2 rat cardiomyocytes from H202- induced apoptosis. FEBS J, 2008.275(12): p. 3136-44. 255. Palanivel, R., et al., Globular and full-length forms of adiponectin mediate specific changes in glucose and fatty acid uptake and metabolism in cardiomyocytes. Cardiovasc Res, 2007. 256. L'Ecuyer, T., et al., Glutathione S-transferase overexpression protects against anthracycline-induced H9C2 cell death. Am J Physiol Heart Circ Physiol, 2004.286(6): p. H2057-64. 257. Fang, X., et al., Hyperglycemia- and hyperinsulinemia-induced alteration of adiponectin receptor expression and adiponectin effects in L6 myoblasts. J Mol Endocrinol, 2005.35(3): p. 465-76. 258. Li, Y., et al., Calpain activation contributes to hyperglycaemia-induced apoptosis in cardiomyocytes. Cardiovasc Res, 2009.84(1): p. 100-10. 259. Malhotra, A., et al., Inhibition of p66ShcA redox activity in cardiac muscle cells attenuates hyperglycemia-induced oxidative stress and apoptosis. Am J Physiol Heart Circ Physiol, 2009.296(2): p. H380-8. 260. Shen, E., et al., Racl is requiredfor cardiomyocyte apoptosis during hyperglycemia. Diabetes, 2009.58(10): p. 2386-95. 261. Brumatti, G., C. Sheridan, and S.J. Martin, Expression and purification of recombinant annexin V for the detection of membrane alterations on apoptotic cells. Methods, 2008.44(3): p. 235-40. 262. Markou, T., et al., Regulation of Bcl-2phosphorylation in response to oxidative stress in cardiac myocytes. Free Radic Res, 2009.43(9): p. 809-16. 263. Capano, M. and M. Crompton, Box translocates to mitochondria of heart cells during simulated ischaemia: involvement of AMP-activated and p38 mitogen-activatedprotein kinases. Biochem J, 2006.395(1): p. 57-64. 264. Martin, J.L., et al., Antiischemic effects of SB203S80 are mediated through the inhibition ofp38alpha mitogen-activated protein kinase: Evidence from ectopic expression of an inhibition-resistant kinase. Circ Res, 2001.89(9): p. 750-2. 265. Saurin, A.T., et al., The role of differential activation ofp38-mitogen- activatedprotein kinase in preconditioned ventricular myocytes. FASEB J, 2000.14(14): p. 2237-46. 266. Liu, D., et al., Pim-3 protects against cardiomyocyte apoptosis in anoxia/reoxygenation injury via p38-mediated signal pathway. Int J Biochem Cell Biol, 2009.41(11): p. 2315-22. 267. Venkatakrishnan, C.D., et al., Heat shock protects cardiac cells from doxorubicin-induced toxicity by activating p38 MAPK and phosphorylation 143 of small heat shock protein 27. Am J Physiol Heart Circ Physiol, 2006. 291(6): p. H2680-91. 268. Maulik, N., et al., Ischemic preconditioning triggers the activation of MAP kinases and MAPKAP kinase 2 in rat hearts. FEBS Lett, 1996.396(2-3): p. 233-7. 269. Maulik, N., et al., Ischemic preconditioning triggers tyrosine kinase signaling: a potential role for MAPKAP kinase 2. Am J Physiol, 1998. 275(5 Pt 2): p. H1857-64. 270. Nagy, N., et al., Ischemic preconditioning involves dual cardio-protective axes with p38MAPK as upstream target J Mol Cell Cardiol, 2007.42(5): p. 981-90. 271. Sato, M., et al., SAPKs regulation of ischemic preconditioning. Am J Physiol Heart Circ Physiol, 2000.279(3): p. H901-7. 272. Lemke, L.E., et al., Decreased p38 MAPK activity in end-stage failing human myocardium: p38 MAPK alpha is the predominant isoform expressed in human heart J Mol Cell Cardiol, 2001.33(8): p. 1527-40. 273. Wanninger, J., et al., Adiponectin-stimulated CXCL8 release in primary human hepatocytes is regulated by ERK1/ERK2, p38 MAPK, NF-kappaB, and STAT3 signaling pathways. Am J Physiol Gastrointest Liver Physiol, 2009.297(3): p. G611-8. 274. Xie, Z., C. He, and M.H. Zou, AMP-activatedprotein kinase modulates cardiac autophagy in diabetic cardiomyopathy. Autophagy, 2011. 7(10). 275. Yuan, H., et al., LPS-induced autophagy is mediated by oxidative signaling in cardiomyocytes and is associated with cytoprotection. Am J Physiol Heart Circ Physiol, 2009.296(2): p. H470-9. 276. Bhui, K., et al., Pineapple bromelain induces autophagy, facilitating apoptotic response in mammary carcinoma cells. Biofactors, 2010.36(6): p. 474-82. 277. Wang, N., et al., Berberine induces autophagic cell death and mitochondrial apoptosis in liver cancer cells: the cellular mechanism. J Cell Biochem, 2010.111(6): p. 1426-36. 278. Yoon, M.J., et al., Adiponectin increases fatty acid oxidation in skeletal muscle cells by sequential activation ofAMP-activated protein kinase, p38 mitogen-activated protein kinase, and peroxisome proliferator-activated receptor alpha. Diabetes, 2006. 55(9): p. 2562-70. 279. Ho, R.C., et al., Dissociation ofAMP-activated protein kinase and p38 mitogen-activated protein kinase signaling in skeletal muscle. Biochem Biophys Res Commun, 2007.362(2): p. 354-9. 144