Accepted Manuscript

Molecular mechanisms of cardiac pathology in diabetes –

Experimental insights

U. Varma, P. Koutsifeli, V.L. Benson, K.M. Mellor, L.M.D.

Delbridge

PII: S0925-4439(17)30412-X
DOI: doi:10.1016/j.bbadis.2017.10.035
Reference: BBADIS 64947
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Received date: 28 July 2017
Revised date: 9 October 2017
Accepted date: 27 October 2017

Please cite this article as: U. Varma, P. Koutsifeli, V.L. Benson, K.M. Mellor, L.M.D.
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 ACCEPTED MANUSCRIPT

Molecular mechanisms of cardiac pathology in diabetes – experimental insights

Varma U,2 Koutsifeli P,1,2 Benson VL,1 Mellor KM,*1,2,3 Delbridge LMD*2
1. Department of Physiology, University of Auckland, New Zealand
2. Department of Physiology, University of Melbourne, Melbourne, Victoria, Australia
3. Auckland Bioengineering Institute, University of Auckland, New Zealand

*Authors contributed equally.

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Word count: 5112
Figures: 2

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Tables: 1
References: 200 NU
Keywords: diabetes, heart, metabolism, autophagy, oxidative stress
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Corresponding Author:
Prof LMD Delbridge
Department of Physiology
University of Melbourne
Parkville, Victoria
Australia 3010
lmd@unimelb.edu.au
Ph.: +61 3 83445853

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ABSTRACT

Diabetic cardiomyopathy is a distinct pathology independent of co-morbidities such as coronary

artery disease and hypertension. Diminished glucose uptake due to impaired insulin signaling and

decreased expression of glucose transporters is associated with a shift towards increased reliance on

fatty acid oxidation and reduced cardiac efficiency in diabetic hearts. The cardiac metabolic profile

in diabetes is influenced by disturbances in circulating glucose, insulin and fatty acids, and

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alterations in cardiomyocyte signaling. In this review, we focus on recent preclinical advances in

understanding the molecular mechanisms of diabetic cardiomyopathy. Genetic manipulation of

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cardiomyocyte insulin signaling intermediates has demonstrated that partial cardiac functional

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rescue can be achieved by upregulation of the insulin signaling pathway in diabetic hearts.
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Inconsistent findings have been reported relating to the role of cardiac AMPK and β-adrenergic

signaling in diabetes, and systemic administration of agents targeting these pathways appear to elicit
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some cardiac benefit, but whether these effects are related to direct cardiac actions is uncertain.

Overload of cardiomyocyte fuel storage is evident in the diabetic heart, with accumulation of
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glycogen and lipid droplets. Cardiac metabolic dysregulation in diabetes has been linked with
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oxidative stress and autophagy disturbance, which may lead to cell death induction, fibrotic
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‘backfill’ and cardiac dysfunction. This review examines the weight of evidence relating to the
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molecular mechanisms of diabetic cardiomyopathy, with a particular focus on metabolic and

signaling pathways. Areas of uncertainty in the field are highlighted and important knowledge gaps
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for further investigation are identified.

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1. Introduction

Diabetic patients have a high risk of cardiovascular disease and mortality. A distinct

cardiomyopathy has been identified in patients with type 1 and type 2 diabetes (T1D, T2D),

characterized by cardiac dysfunction, fibrosis, oxidative stress and metabolic disturbance. Although

extensive co-morbidities are common in diabetic patients, the occurrence of diabetic

cardiomyopathy is independent of vascular abnormalities such as coronary artery disease, and

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hypertension [1-4]. Cardiac outcomes are influenced by diabetic systemic insult including

hyperglycemia, dyslipidemia and hyperinsulinemia (T2D)/hypoinsulinemia (T1D), resulting in

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altered cardiomyocyte molecular signaling and metabolism [5]. Additionally, volume-loading from

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obesity can elicit significant effects on cardiac structural remodeling, with subsequent functional
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disturbance.
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Diastolic dysfunction is an early manifestation of diabetic cardiomyopathy, prevalent in more than

50% of asymptomatic diabetic patients [6-8], and linked to an increased risk of heart failure and

mortality, independent of systolic functional decline [9]. Impaired heart relaxation involves
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increased ventricular wall stiffness and abnormal cardiac filling [10, 11]. The early occurrence of
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diastolic dysfunction in asymptomatic diabetic patients is evident even in patients with normal

blood pressure, no vascular complications and normal contractility during systole [7, 12, 13].
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Although hyperglycemia certainly elicits marked effects on cardiomyocyte function, evidence

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suggests that glycemic status is not necessarily predictive of early cardiac functional decline [14].

Treatment options targeting diastolic dysfunction are limited and understanding the molecular basis

for dysfunction in the diabetic heart is an important priority.

Clinically, diabetic patients exhibit worse outcomes post-ischemia, with a higher incidence of

post-ischemic heart failure despite smaller infarct size, and are less responsive to the protective

effects of ischemic preconditioning [15-17]. Experimental studies with diabetic rodent models have

reported inconsistent findings relating to post-ischemic outcomes and pre-conditioning (reviewed in

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[16]), with some reports suggesting that the diabetic heart is resistant to ischemic injury and

progression to heart failure [18-21]. Further work is required to elucidate the interaction between

diabetes and ischemic events, with particular focus on aligning the animal models with clinical

phenotypes for mechanistic interrogation.

At the molecular level, the energy stress associated with diabetes has been shown to induce

alterations in myocardial substrate and energy metabolism [22]. Increased free fatty acids, oxidative

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stress and impaired protein clearance have been implicated in activating the endoplasmic reticulum

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stress response, linked with activation of autophagy and apoptotic cell death in the diabetic heart

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(reviewed in [23]). High extracellular glucose has been linked to formation of cross-linking

advanced glycation end-products (AGEs) on proteins such as collagens, contributing to ventricular

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stiffness, and more recently AGE modification of intracellular proteins has been identified which

may provide a novel route of cellular damage in diabetic cardiomyopathy [24-26]. Glucose-
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mediated post-translational modifications have been well-described in diabetes, in the heart and

other tissues. O-GlcNAcylation of key Ca2+ handling proteins and signaling kinases has been shown
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to play an important role in mediating cardiac dysfunction in diabetes (reviewed in [24, 27]). Ca2+
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handling disturbance appears to be a key feature of diabetic cardiomyocytes, involving prolonged

SERCA2-mediated Ca2+ removal from the cytosol during diastole, changes in myofilament Ca2+
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sensitivity, and heightened ryanodine receptor-mediated Ca2+ leak from the sarcoplasmic reticulum

contributing to arrhythmia susceptibility [28, 29]. From an epigenetics perspective, dysregulation of

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miRNAs has recently received attention for contributing to changes in expression of genes involved

in diabetic cardiomyopathy (reviewed in [30]). An understanding of the key miRNAs and their

targets is emerging and may open up new therapeutic opportunities in the field.

The focus of this review is on the molecular mechanisms of diabetic cardiomyopathy, with

comprehensive analysis of the literature relating to cardiomyocyte metabolic and signaling

pathways, accumulation of fuel stores (glycogen and lipid droplets), oxidative stress, and autophagy

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disturbance. We highlight areas of uncertainty in the field and identify important knowledge gaps

for further investigation.

2. Metabolic disturbances in the diabetic heart

Adult cardiomyocytes preferentially use fatty acid oxidation for ATP production, with a smaller

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contribution from glucose metabolism (up to 40%) [31]. Sarcolemmal glucose uptake is primarily

mediated by insulin-independent glucose transporter (GLUT) 1 and insulin-dependent GLUT4

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transporters. Decreased expression of GLUT1 and GLUT4, and reduced insulin-stimulated GLUT4

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translocation to the sarcolemma, are associated with decreased glucose uptake in both T1D and

T2D rodent hearts [32, 33]. Sodium-glucose co-transporters (SGLTs) are upregulated in the kidney
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and intestinal tissue of human diabetic patients and rodent diabetic models, facilitating glucose
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absorption [34]. Specifically, SGLT1 is increased in hearts of T2D patients and obese insulin

resistant mice (ob/ob), but decreased in T1D mice [35]. Cardiac SGLT1 expression is increased

with exposure to leptin [35], and upregulation of SGLT1 in T2D may provide an alternate route for
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glucose uptake when GLUT1 and GLUT4 are downregulated. Systemic inhibition of SGLT1 and/or
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SGLT2 has been advanced as an effective anti-hyperglycemic therapy (reviewed in [34]) and

multiple clinical trials are underway investigating the cardiac therapeutic potential of SGLT1
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inhibition. In T1D mice, pharmacological inhibition of histone deacetylases (HDACs) increases

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cardiac expression of both GLUT1 and GLUT4 and improves cardiac function [36], suggesting that

GLUT downregulation may be linked to HDAC regulation and constitutes a central component of

diabetic cardiomyopathy.

Transporters for galactose (GLUT1,3,8,11) and fructose (GLUT5,8,11) are also expressed in

cardiomyocytes and may provide alternative fuel sources in energy stress settings [37, 38]. In

glucose-deprived isolated adult cardiomyocytes, exposure to fructose facilitates contractile function,

indicating that cardiomyocytes have the capacity to transport and utilize fructose [39]. Cardiac
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fructose content is increased in diabetes [40, 41] and may influence protein glycation damage and

metabolic disturbance [42]. Cardiomyocyte fructose accumulation may be due to over-activation of

the sorbitol pathway and/or endogenous fructose exposure from dietary sources. Understanding the

nuances of alternative fuel sources in the diabetic heart may provide new opportunities for

intervention.

Coincident with impaired glucose uptake, reduced glucose oxidation is a common observation in

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diabetic cardiomyocytes [43], associated with a shift towards increased reliance on fatty acid

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oxidation [22] (Figure 2). Additionally, increased fatty acid oxidation (driven by increased fatty

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acid uptake) might further suppress glucose oxidation via the Randle cycle [44]. In genetic mouse

models of obesity (ob/ob) and T2D (db/db), decreased glucose oxidation is observed by 4 weeks of
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age, associated with an increase in fatty acid oxidation and reduced cardiac efficiency, preceding

hyperglycemia [43]. Interventions to upregulate glucose oxidation, eg. via dichloroacetic acid-
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induced increased pyruvate dehydrogenase activity, have been shown to improve cardiac function

in both T2D (high fat diet) and T1D (STZ) rats [45, 46]. Similarly, rosiglitazone-induced PPAR-γ
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activation in db/db mice improved cardiac efficiency associated with increased glucose oxidation
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and decreased fatty acid oxidation [47]. Together, these findings suggest that normalization of the

metabolic profile in the diabetic heart may be an important target for ameliorating cardiac function
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in diabetes.
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Given that de novo synthesis of fatty acids in the heart is limited, intracellular fatty acid availability

is reliant on circulating levels of fatty acids, uptake kinetics, and intracellular lipid depots [48]. An

increase in circulating fatty acids has been observed clinically and in T2D rodent models, associated

with impaired glucose tolerance and insulin sensitivity in muscle tissue [49]. Fatty acyl-CoA-

mediated inhibition of insulin receptor substrate signaling pathways, hexokinase inhibition and

peroxisome proliferator-activated receptor α (PPAR-α) activation may be involved [49]. PPAR-α

activation stimulates fatty acid transport while suppressing glucose utilization, further promoting

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the shift towards fatty acid oxidation [50]. High fat-fed mice with global PPAR-α gene deletion

exhibit improved insulin sensitivity compared to their wildtype counterparts. Cardiac outcomes

were not investigated in this study [51]. In contrast, db/db mice with long term exposure to a PPAR-

α agonist exhibit enhanced cardiac glucose uptake and utilization associated with a normalization of

blood glucose and insulin, but stabilization of the cardiac metabolic profile in this setting does not

lead to functional improvement [52, 53]. HDACs may also play an important role via PPARα-

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mediated regulation of glucose and lipid homeostasis. HDAC inhibitors have gained attention as a

promising therapeutic intervention to improve systemic insulin resistance and glucose handling

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[54], and an understanding of their role in the diabetic heart is emerging. Streptozotocin (STZ)-

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induced T1D rats treated with HDAC inhibitor, MPT0E104, exhibit improved in vivo cardiac

function and electrocardiogram profile linked with restored cardiac expression of PPARα [55].
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Whether these effects are secondary to the observed improvement in systemic glucose and lipid
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profile, or mediated by intrinsic cardiomyocyte mechanisms is not yet clear, and investigation into

cardiac-specific modulation of HDACs is required.

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Collectively, the metabolic adaptations in the diabetic heart appear to have a detrimental impact on
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cardiac efficiency and function and may be attributed to changes in circulating glucose, insulin and

fatty acids. Advanced knowledge of the complex signaling networks connecting glucose utilization
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and fatty acid oxidation might yield valuable therapeutic targets to stabilize the metabolic profile in

the diabetic heart.

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3. Cardiac metabolic signaling dysregulation in diabetes

3.1 Insulin signaling

Cardiac insulin signaling mediates cellular homeostasis via control of substrate utilization, protein

synthesis, autophagy and cell survival. Suppression of this pathway is associated with increased

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cardiac dysfunction and susceptibility to stress-induced heart failure [56]. Physiologically, binding

of the insulin ligand to the sarcolemmal insulin receptor (IR) activates insulin receptor substrates

(IRS1 and 2) and downstream phosphoinositide-3-kinase class I (PI3K(I))-protein kinase B (Akt)

pathways. Akt activation stimulates the translocation of GLUT4 to the cell membrane and

subsequent uptake of glucose [32]. The mechanisms mediating impaired insulin signaling in T2D

are not well understood. Recently, an E3 ubiquitin ligase, MG53, has been implicated as an

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important regulator of the insulin signaling pathway [57]. Multiple rodent diabetic models (db/db

mice and high fat diet (HFD) mice) exhibit elevated cardiac MG53 protein content, linked with

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increased proteosomal degradation of IR and IRS1, and activation of PPARα [57, 58]. Cardiac-

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specific overexpression of MG53 induces a phenotype which mimics many of the features of

diabetic cardiomyopathy including downregulation of the insulin signaling pathway, increased

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fibrosis and cardiac dysfunction [58]. However, low MG53 expression in the human myocardium
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makes it an unlikely cardiac specific therapeutic target in diabetes [59].

Some insights into the role of insulin signaling in diabetic cardiac pathology have been gained via
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genetic manipulation of signaling intermediates. Cardiac insulin receptor knockout (α-myosin heavy
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chain (αMHC) promotor) decreases cardiomyocyte glucose uptake, increases cardiac oxidative

stress, and reduces mitochondrial function and cardiac efficiency [60-62]. Similarly, cardiac double
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knockout of IRS1 and IRS2 (α-MHC promotor) reduces cardiomyocyte ATP content, increases

fibrosis, impairs cardiac metabolism and function, and increases apoptosis leading to eventual
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cardiac failure [63]. STZ-induced T1D cardiac pathology is exacerbated in IR-KO mice, with a

greater extent of mitochondrial dysfunction and reduced cardiac efficiency relative to WT mice

[62]. These gene deletion studies suggest that the upstream components of the insulin signaling

pathway (IR, IRS1, IRS2) play an important role in diabetes-associated cardiac pathology, and

investigation of the efficacy of overexpression of these proteins in providing a metabolic rescue

strategy would be informative.

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Impaired glucose uptake in the diabetic heart is often linked with reduced expression or activity of

the downstream intermediates in the insulin signaling pathway (Figure 2). Decreased cardiac basal

and insulin-stimulated phosphorylation of Akt is evident in diabetic rodent models [64-66] and

studies using genetic manipulation of the PI3K-Akt nexus in diabetic mice have demonstrated that

these signaling intermediates play a central role in diabetic cardiac pathology. STZ-induced T1D

cardiac pathology is exacerbated in mice with downregulated cardiomyocyte PI3K activity

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(dominant-negative PI3K(I) mice, αMHC promotor), and prevented in mice with upregulated

cardiomyocyte PI3K activity (constitutively active PI3K(I) mice, αMHC promotor) [67]. Similarly,

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upregulation of PI3K via administration of constitutively active PI3K(I) viral constructs in mice

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with established diabetic cardiac pathology (STZ-induced T1D), partially rescued cardiac

dysfunction [68]. Surprisingly, insulin-stimulated glucose uptake has been observed to be decreased
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by upregulation of cardiomyocyte PI3K-Akt signaling (via inducible constitutively active PI3K and
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myristoylated (active) Akt, αMHC promotor) [69], suggesting that cardiac functional rescue

induced by increased PI3K activity may not be related to restoration of glucose uptake. Given that
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PI3K is an important signaling focal point for regulation of multiple pathways, it is likely that
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favorable cardiac outcomes are related to altered metabolism, protein synthesis and/or cell survival.
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Interestingly, not all animal studies suggest that diabetes is detrimental in the context of a secondary
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cardiac insult. In response to pressure overload surgery, diabetic mice (induced by STZ, db/db, high

fat diet) exhibit less severe (or even abrogated) systolic dysfunction, attributed to the absence of
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insulin-dependent Akt activation of downstream hypertrophic signaling pathways [21, 70-72].

IR-KO mice and Akt1-deficient mice also exhibit preserved systolic function following pressure

overload, suggesting that the insulin pathway plays an important role in mediating the progression

to heart failure induced by pressure overload [21]. These findings have important implications for

the possible adverse effects of insulin supplementation in settings of high pressure-mediated cardiac

remodeling such as in hypertensive diabetic patients.

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3.2 AMPK signaling

AMP-activated kinase (AMPK) is composed of a catalytic α subunit and regulatory β and γ

subunits, and is activated in conditions of stress and starvation via an increase in cellular AMP

concentration [73]. The binding of AMP to AMPK promotes the auto-phosphorylation of threonine

172 and prevents dephosphorylation of AMPK by phosphatases [73]. AMPK is involved in

numerous cellular processes including regulation of glycolysis and fatty acid oxidation, autophagy

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initiation, and lipid, glycogen and protein synthesis [74] (Figure 2). AMPK mediates tight

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regulation of glucose handling via promoting GLUT4 translocation [75], GLUT4-independent

glucose uptake [76], and glycogen synthase phosphorylation [77, 78]. AMPK also binds to

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glycogen via its β subunit carbohydrate domain [79, 80], although the role of AMPK in regulating
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glycogen content in the heart is not well understood.
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Clinically, AMPK activators such as metformin have been used extensively to control

hyperglycemia in T2D patients for over 60 years [81]. Trials are currently underway to evaluate the
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therapeutic cardiac effects of metformin in T1D patients [82] and numerous studies have
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demonstrated that metformin treatment improves cardiac outcomes in T1D and T2D rodent models
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[83, 84]. Cardiac AMPK downregulation has been reported in animal models of T1D and T2D and

may be an important intervention target [84, 85]. However not all studies have reported decreased
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AMPK activity with diabetes. Obese insulin resistant mice (ob/ob) exhibit increased cardiac AMPK
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phosphorylation (Thr172) which can be restored by captopril treatment, suggesting involvement

from the renin-angiotensin system [86]. Interestingly, cardiac AMPK modulation in T1D appears

dependent on diabetes duration - AMPK phosphorylation is increased in STZ rats at 4 days post-

injection, yet not different from control at 6 weeks post-STZ injection [87]. Activation of AMPK

via metformin treatment in cultured cardiomyocytes exposed to high glucose improves cell survival

relative to high glucose alone, an effect associated with a pro-autophagic/anti-apoptotic dissociation

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of Beclin-Bcl2 [88]. Whether cardiac benefit observed with AMPK activation is secondary to

systemic improvement, or due to intrinsic cardiomyocyte mechanisms is yet to be elucidated.

3.3 β-adrenergic signaling

Diabetic patients exhibit impaired exercise capacity, sympathetic activity and blunted inotropic

response to β‐adrenergic stimulation [89, 90]. Experimentally, β-adrenergic involvement in diabetic

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cardiomyopathy appears to be different in T1D and T2D settings. In STZ-induced T1D rodents,

cardiac β-adrenergic receptors are observed to be desensitized and downregulated [91-95]. In obese

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T2D rats, heightened sensitivity to β1- but not β2-adrenergic receptor-mediated chronotropy is

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evident [96]. Interestingly, global deletion of the β2 receptor prevented both diastolic and systolic
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dysfunction in high fat-fed mice via inhibition of insulin-mediated activation of phosphodiesterase

4D [97]. Given that β2-adrenergic receptors are present in only ~5% of ventricular cardiomyocytes
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[98], β2-receptor knockout-induced cardiac outcomes may be related to systemic or atrial changes.

The relationship between β-adrenergic signaling and insulin resistance is emerging as an important
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focal point in diabetic cardiac pathology (Figure 2). Short term stimulation of the β-adrenergic
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pathway in cultured cardiomyocytes increases insulin-dependent glucose uptake through

phosphorylation of Akt via protein kinase A [99]. In contrast, long term β-adrenergic stimulation
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inhibits insulin-dependent glucose uptake in cultured cardiomyocytes [99]. In vivo, sustained

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systemic activation of the β-adrenergic receptor (daily injections of isoproterenol) induces insulin

resistance, and reduces glucose uptake and GLUT4 expression in cardiac tissue; effects which could

be ameliorated with β-blocker treatment [100]. Hyperinsulinemia may play a role in dysregulation

of β-adrenergic signaling in T2D, although the mechanisms are not clear. Insulin-treated neonatal

mouse cultured cardiomyocytes exhibit G protein-coupled receptor kinase 2-mediated

phosphorylation and internalization of β2 receptors [101]. In contrast, numerous studies have

suggested that hyperinsulinemia promotes β-adrenergic signaling leading to hypertrophy and failure

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[102]. Together, these findings indicate a complex relationship between β-adrenergic and insulin

signaling pathways in the diabetic heart, with each pathway playing an integral role in the

development of the cardiac pathology [102, 103].

4. Perturbed cardiomyocyte fuel storage in diabetes

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4.1 Glycogen storage

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Disruption of metabolic signaling pathways, coupled with altered substrate accessibility and

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utilization is associated with irregular fuel storage in the diabetic heart. Glycogen is an important

fuel depot for glucose, well characterized in the liver and skeletal muscle, but its role in the heart is
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not well understood. Physiologically, glycogen is regulated via two key enzymes: glycogen

synthase which adds glucose monomers to glycogen chains, and glycogen phosphorylase which
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releases glucose-1-phosphate from glycogen [104]. In skeletal muscle, glycogen particles exhibit a

high surface area-to-volume ratio, thus facilitating rapid breakdown in situations where a swift
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glucose surge is required [105]. Experimentally, in response to fasting, glycogen content is depleted
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in skeletal muscle [106], but unchanged or even increased [17, 106, 107] in cardiac muscle. It has
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been suggested that nutrient deprivation may re-direct glucose to critical tissues such as the heart
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and brain for preservation of function in acute stress circumstances.

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In the diabetic context, insulin-sensitive tissues such as cardiac and skeletal muscle are subjected to

a state of glucose deprivation due to impaired glucose uptake induced by insulin deficiency (T1D)

or cellular insulin resistance (T2D). Thus, an expected decrease in skeletal muscle glycogen content

is evident in diabetic subjects [108, 109]. In contrast and paradoxically, increased glycogen in the

human diabetic heart is evident, first documented in early work by Warren (1930), and subsequent

studies in humans and rodents have mostly corroborated this finding [110-136]. However, some

reports of unchanged [129, 137-141] or decreased [140, 142, 143] glycogen are evident (Table 1).

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Interestingly, exercise training has been shown to both elevate [125] and attenuate [132] cardiac

glycogen accumulation in T2D rodents. These inconsistent findings may relate to differences in

diabetes duration, dose, species or age of the animal models. Aged animals appear to be more

susceptible to glycogen accumulation associated with T2D [144], and the dose of streptozotocin

administration is positively correlated with cardiac glycogen content in T1D rats [140]. It could be

expected that glycogen accumulation is a result of increased synthesis and/or decreased

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degradation. Yet it appears that cardiac glycogen accumulation in diabetes is not explained by

changes in glycogen regulatory enzymes - lower glycogen synthase activity and unchanged or

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increased activity of glycogen phosphorylase in various rodent models of T1D and T2D has been

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reported [113, 117, 121, 125, 145]. These findings suggest that glycogen accumulation may be

initiated early in disease progression, and snapshot measures at later time points may capture a later
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compensatory response, rather than an initial causative enzyme modulatory effect. Alternatively,
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other pathways involved in regulating glycogen content may play a role.

Electron micrographs have depicted glycogen in double-membrane phagosomal structures in the

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heart, indicative of an autophagy-mediated degradation breakdown process [146]. The process of

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glycogen autophagy (‘glycophagy’) has been identified in skeletal muscle, liver and the heart [147-
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149]. Glycophagy involves starch-binding domain-containing protein 1 (STBD1) tagging glycogen

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and recruiting it to the forming autophagosome via binding to γ-aminobutyric acid receptor-

associated protein-like 1 (GABARAPL1), an ATG8 homologue. The glycogen-containing

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autophagosome fuses with a lysosome where acid α-glucosidase (GAA) degrades glycogen to its

glucose monomers [150]. GAA deficiency in Pompe disease, and inherited lysosome-associated

protein (LAMP2) deficiency, result in severe myocardial glycogen accumulation and cardiac

dysfunction, thus highlighting the importance of lysosomal glycogen breakdown in the heart [151,

152]. In cultured primary cardiomyocytes, glycophagy is modulated by extracellular glucose and

insulin, coincident with glycogen accumulation [153]. Disturbances in glycophagy may play a role

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in mediating glycogen accumulation in the diabetic heart in vivo, and further investigation is

warranted.

4.2 Lipid storage

A shift in reliance on fatty acid oxidation in the diabetic heart is linked with increased fatty acid

availability driven by sarcolemmal fatty acid uptake [154]. Experimentally, diabetes-induced

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upregulation of genes involved in lipid metabolism is evident early in disease progression, detected

at 48 hours post-injection of streptozotocin to induce T1D [155]. Despite increased metabolism of

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lipids, cardiac accumulation of lipid stores has been reported in mouse models of T2D (db/db

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mouse) [156] and insulin resistance (high fat-fed mouse) [157], suggesting that upregulation of fatty
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acid oxidation is not sufficient to maintain intracellular lipid homeostasis in this setting of increased

fatty acid uptake. Lipid droplets consist of a neutral lipid core containing triacylglycerols and
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cholesteryl esters, surrounded by a monolayer of phospholipids serving as an anchor-point for

perilipins [158, 159]. Perilipin 5 is increased in the hearts of T2D (db/db) and T1D (STZ-treated
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and Akita) mice and cardiac triacylglycerol accumulation and reduced fractional shortening in STZ
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mice is abrogated by global perilipin5 knockout [160]. Interestingly, evidence of increased lipid
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droplets in T2D cardiomyocytes was observed prior to the onset of diastolic dysfunction in 12 week
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old male db/db mice [161]. Thus lipid accumulation may be an early, and perhaps primary,

manifestation of diabetic cardiomyopathy and further investigation into the relationship between
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excess lipids and diastolic functional pathology is now warranted.

Within the cardiomyocyte cytosol, lipid droplets increase in size via fusion, which is mediated by a

multitude of protein complexes, including membrane fusion proteins from the soluble NSF

attachment protein receptor (SNARE) family. In HL-1 cultured cardiomyocytes, oleic acid

incubation increases the lipid droplet-associated pool of the SNARE family protein, soluble NSF

attachment protein 23 (SNAP23), at the expense of the plasma membrane-associated SNAP23 pool

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[162]. Given that SNARE proteins are also involved in GLUT4 translocation, it has been suggested

that increased demand for lipid droplet fusion in diabetes may decrease the availability of SNARE

proteins for GLUT4 vesicle transport thus decreasing insulin sensitivity [162, 163]. Whether this

mechanism plays a role in the development of cardiac insulin resistance in vivo is yet to be

investigated. Fatty acid availability for ATP synthesis is driven by hydrolyzing stored lipids via

adipose triglyceride lipase (Atgl) and hormone sensitive lipase (Hsl) [164]. Cardiac-specific

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overexpression of Atgl in T1D (STZ) mice improved the cardiac lipid profile, restored glucose

oxidation and improved functional parameters, whereas Atgl knockdown exacerbated diabetic

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cardiac pathology [165]. Similarly, cardiac-specific overexpression of Hsl in T1D (STZ) mice

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prevented diabetes-induced lipotoxicity and cardiac fibrosis by facilitating lipid breakdown [166].

Bulk lipid droplet degradation via microtubule-associated protein light chain 3B (LC3B)-mediated
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autophagy has been identified in non-cardiac cell types [167], but the role of autophagy in lipid
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metabolism in the heart is not yet established. Interestingly, genetic knockout of the autophagy

regulator, forkhead box protein O1 (FoxO1), in T2D mice was associated with reduced lipid
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accumulation and lipotoxicity, preserved metabolic substrate selectivity and cardiac function, and
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improved mortality compared to wild-type T2D mice [168]. Although this study did not directly
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investigate autophagy-mediated lipid degradation, FoxO1 has been implicated as a key regulator of

transcription of autophagy proteins in the heart [169]. Given that autophagy plays an important role
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in energy stress responses in cardiomyocytes, further investigation into cardiomyocyte lipid-

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selective autophagy pathways in the diabetic setting would be informative.

5. Energy stress adaptations in the diabetic cardiomyocyte

5.1 Reactive oxygen species generation

Extracellular hyperglycemia, impaired glucose uptake, disturbed glycogen handling and metabolic

dysregulation creates an environment of energy stress in the diabetic cardiomyocyte. The

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augmented reliance on fatty acid oxidation evident in diabetic mouse hearts, is coincident with

increased myocardial oxygen consumption and reduced cardiac efficiency [170, 171]. Dysregulated

metabolism may be linked to increased production of reactive oxygen species (ROS) and

development of cardiac oxidative stress in diabetes [172]. A disruption in the homeostatic balance

of ROS management leads to oxidative damage of DNA, proteins and lipids as well as activating

stress-sensitive pathways. Cardiac oxidative stress has been observed in patients with diabetes [173]

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and diabetic animals models [68, 174, 175], and urinary levels of oxidation markers have been

proposed as potential biomarkers of micro- and macro-vascular complications in diabetic patients

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[173, 176]. Production of ROS in diabetic hearts and high glucose-incubated cultured

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cardiomyocytes has been largely attributed to increased activity of nicotin-amide adenine

dinucleotide phosphate (NADPH) oxidase [177-179] and uncoupling of the mitochondria and
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subsequent leakage of the mitochondrial electron transport chain, implicated in declining cardiac
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function [180-182]. Other sources of ROS production in the heart include uncoupling of nitric oxide

synthase, activation of protein kinase C, lipoxygenase, and xanthine oxidase [1, 183].
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Antioxidant treatment strategies have demonstrated some cardiac benefit in experimental animal
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models of diabetes. In obese insulin resistant mice, mitochondria-specific antioxidant treatment

reduced ROS and oxidation of cardiac myofilament proteins, leading to an improvement in cardiac
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diastolic function [182]. Similarly, systemic administration of the antioxidant coenzyme Q10 in

STZ-treated dominant-negative PI3K(p110α) transgenic mice with upregulated NADPH oxidase,

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limits diabetes-induced diastolic dysfunction, fibrosis and hypertrophy [175]. ROS production can

also mediate autophagy, a process of cellular ‘self-digestion’ implicated in the development of

diabetic cardiomyopathy. Induction of oxidative stress via hydrogen peroxide treatment in cultured

H9c2 cardiomyoblasts increases autophagy, an effect reversed by treatment with the antioxidant

resveratrol [184]. In the early phase of energy deprivation, cardiomyocyte autophagy induction has

been linked to NADPH oxidase isoform Nox4-derived ROS production in the endoplasmic

reticulum [185, 186]. Evidence suggests that autophagy induction may be a compensatory response
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to oxidative stress with a negative feedback action. Elevated myocardial ROS production and

autophagy activity with lipopolysaccharide (LPS)-induced sepsis in mice is attenuated by

antioxidant treatment, and conversely, activation of autophagy with rapamycin in this setting

attenuates ROS production [187]. Whether a similar mechanism is evident in diabetic hearts is yet

to be investigated.

5.2 Autophagic activity

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Autophagy is a highly conserved and tightly regulated homeostatic process vital for cell growth and

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survival. An excess level of autophagic activity has been linked to induction of programmed cell

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death [188]. Impaired autophagy is associated with an accumulation of dysfunctional mitochondria

and can trigger stress-response pathways and subsequent cell death via apoptosis [189, 190]. An
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understanding of cargo-selective autophagy is emerging - specific degradation processes targeting
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proteins (macrophagy), mitochondria (mitophagy), glycogen (glycophagy) and lipids (lipophagy) -

and may play an important role in diabetes-induced cardiac pathology [191]. Conflicting literature
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reports of increased, decreased and unchanged cardiac autophagy in rat and mouse models of
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diabetic cardiomyopathy have emerged, with no apparent consistency within species, strain,
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duration or type of diabetes (previously reviewed in [191]). For example, increased LC3B-positive

myocytes, LC3BII protein expression, phagosome number (from electron micrographs) and
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decreased p62 protein expression is evident in atrial biopsies from human T2D patients with
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ischemic heart disease [192]. In contrast, decreased Atg5 protein expression and no change in LC3B

is observed in T2D patients with coronary artery disease but without overt signs of cardiomyopathy

[193]. Interestingly, despite similar increases in autophagosome number and expression of

autophagy marker LC3BII, T1D rat hearts exhibit increased lysosome number, while T2D mouse

hearts (db/db) exhibit decreased lysosome number [194], suggesting that lysosomal availability

and/or function may play an important role. Inconsistent findings from animal models of diabetes

could be due to differences in severity and type of diabetes, but even highly constrained in vitro

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culture experiments where cardiomyocytes are exposed to high glucose (30mM) yield conflicting

results. Autophagy activation (via rapamycin) exacerbates cell death, and autophagy inhibition (via

3-methyladenine) attenuates cell death in high glucose primary neonatal rat cardiomyocytes [153,

195]. These findings suggest that suppression of autophagy in this context may be a favorable

adaptive response to preserve cell viability. In contrast, activation of autophagy (via metformin-

treatment) decreases high glucose-induced apoptosis in cultured H9c2 cardiomyoblasts [88]. The

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differences in these studies could be attributed to the different cell types used. The ‘control’ culture

conditions of ~5mM glucose (for comparison with 30mM glucose) are well-tolerated by primary

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neonatal rat ventricular myocytes but may pose a ‘starvation’ challenge to H9c2 cells routinely

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maintained in 25mM glucose.
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Interventions manipulating cardiac autophagy in experimental diabetic settings have also generated

contradictory findings on the role of autophagy in diabetic cardiomyopathy. Activation of

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autophagy via systemic administration of pharmacological agents (metformin [88], resveratrol

[194], fenofibrate [196]) has proved beneficial in ameliorating diabetes-induced cardiac

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dysfunction. But inhibition of autophagy via systemic administration of chloroquine [197], or

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global genetic knockdown of autophagy initiation proteins (Beclin 1 or Atg16L1 [198]) has also

improved cardiac dysfunction in diabetes. However, the cardiac effects observed in these studies
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may be secondary to the systemic impact of these interventions. Cardiac-specific Beclin 1

overexpression in T1D STZ-treated mice (via α-MHC cardiac promotor with tetracycline-controlled
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transactivator) increases autophagosomal clearance and exacerbates diabetes-induced cardiac

pathology [198]. Given that STZ-induced T1D increases lysosomal content [194], interventions

which upregulate early-autophagy proteins such as Beclin 1 could be detrimental in this disease

setting of heightened lysosomal throughput, but might prove therapeutic in type 2 diabetic db/db

mice where lysosomal availability is more limited. Understanding specific points of disruption in

the autophagic process and how they differ between T1D and T2D is an important priority.

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6. Conclusions

Diabetic cardiomyopathy is a distinct pathology independent of co-morbidities such as coronary

artery disease and hypertension. Experimental investigations of diabetic cardiomyopathy have

provided considerable mechanistic and molecular insight into disease characteristics. Diabetes-

related systemic disturbances in glucose, insulin and fatty acids, facilitate shifts in the cardiac

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metabolic profile and alterations in key cardiac signaling pathways. Decreased insulin signaling,

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and an aberrant response to β-adrenergic stimulation, impact on cardiomyocyte substrate utilization,

storage of glucose and lipids, and cell death via apoptosis and autophagy. AMPK signaling

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disturbance is evident but not well characterized in the diabetic heart. Disruptions in these signaling
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pathways, and energy stress responses such as oxidative stress and autophagy dysregulation, are

likely contributors to cardiomyocyte death and to impaired functional performance in the diabetic
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heart (Figure 1). Genetic manipulation of cardiac metabolic pathways, signaling intermediates, and

autophagy proteins have provided important insights into the molecular mechanisms of diabetic
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cardiac pathology. Despite these advances, an understanding of the etiology of diabetic

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cardiomyopathy has not yet been achieved. In some cases, pre-clinical findings have not translated
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to the human patient setting. Relative ischemic cardioprotection observed in some diabetic rodent
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models is not evident in diabetic patients where worse outcomes post-ischemia are evident [17].

Impaired cardiac glucose uptake and utilization, well-described in obese and lean diabetic rodents,
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are evident in T2D male but not female patients [199]. Interestingly, metformin treatment

exacerbated glucose handling abnormalities in male T2D patients, despite evidence of some

improvement in cardiac function [200]. Thorough investigation at the pre-clinical level

characterizing sex differences and interrogation of the translational value of rodent models of

disease is an important priority in this field.

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Figure Legends

Figure 1. Mechanisms of diastolic dysfunction in the diabetic heart. Diabetes is associated with

impaired glucose uptake and utilization, a shift towards fatty acid oxidation and accumulation of

glycogen and lipids. Metabolic dysregulation may trigger energy stress responses such as oxidative

stress and autophagy disturbance linked with increased cell death, fibrosis and ultimately leading to

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impaired cardiac relaxation and diastolic dysfunction in diabetes.

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Figure 2. Molecular mechanisms of diabetic cardiomyopathy. Diabetes is associated with

increased circulating fatty acids, increased glucose, either insulin deficiency (T1D) or
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hyperinsulinemia (T2D) and increased catecholamines from heightened sympathetic activity. These

systemic perturbations lead to increased cardiac fatty acid uptake, impaired glucose uptake and
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dysregulated insulin and β-adrenergic signaling. A shift towards fatty acid oxidation and

accumulation of glycogen and lipids are common observations in the diabetic heart and although
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inconsistent findings have been reported relating to AMPK and autophagic processes, disturbances
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in these pathways may exacerbate the metabolic mismanagement in the heart. ‘+’, upregulation; ‘-’,
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downregulation, ‘mTOR’ mammalian target of rapamycin.

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DIABETES
cardiac metabolic dysregulation:
• impaired glucose uptake & utilization
• shift towards fatty acid oxidation
• accumulation of fuel stores (glycogen and lipids)

energy stress responses:

• oxidative stress
• autophagy disturbance

impaired cardiac relaxation

• activation of cell death pathways
• fibrosis and ventricular stiffness

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Diastolic Dysfunction

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Figure 1. Mechanisms of diastolic dysfunction in the diabetic heart. Diabetes is associated with
impaired glucose uptake and utilization, a shift towards fatty acid oxidation and accumulation of
glycogen and lipids. Metabolic dysregulation may trigger energy stress responses such as oxidative
stress and autophagy disturbance linked with increased cell death, fibrosis and ultimately leading to

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impaired cardiac relaxation and diastolic dysfunction in diabetes.

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- insulin (T1D)
+ fatty acids + glucose + insulin (T2D) + catecholamines

- IR β1 +/- ?
- IRS1/2 cAMP
fatty acid - glycolysis
+ oxidation acute
glucose
- - PKA

cardiomyocyte
oxidation PI3K/Akt
lipid chronic
+ droplets + glycogen
mTOR Ca2+
regulation

AMPK autophagy
+/- ? +/- ?

cardiomyocyte death, fibrosis

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and cardiac dysfunction

Figure 2. Molecular mechanisms of diabetic cardiomyopathy. Diabetes is associated with increased circulating fatty
acids, increased glucose, either insulin deficiency (T1D) or hyperinsulinemia (T2D) and increased catecholamines from
heightened sympathetic activity. These systemic perturbations lead to increased cardiac fatty acid uptake, impaired

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glucose uptake and dysregulated insulin and β-adrenergic signaling. A shift towards fatty acid oxidation and accumulation
of glycogen and lipids are common observations in the diabetic heart and although inconsistent findings have been
reported relating to AMPK and autophagic processes, disturbances in these pathways may exacerbate the metabolic

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mismanagement in the heart. ‘+’, upregulation; ‘-’, downregulation, ‘mTOR’ mammalian target of rapamycin.

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Table 1. Reports of cardiac glycogen content in models of diabetes.
Cardiac glycogen Phenotype
Phenotype Species Model Reference
content duration
nr Human Diabetic Patient Chronic [162]
T1D Rat Alloxan inj Short term [77, 84]
T1D Rat Alloxan inj Chronic [82, 85]
T1D Rat STZ inj Short term [79, 87-89]
[78, 81, 87-89,
T1D Rat STZ inj Chronic
91, 164]
T1D Rat BB Wistar Chronic [86]
T1D Dog Alloxan inj Chronic [83]
↑ T1D Rabbit Alloxan inj nr [90]

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T2D Human T2D Patient Chronic [93]
T2D Rat ZDF Chronic [94, 99, 100]

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T2D (late) Rat ZDF Chronic [96]
T2D Rat STZ inj (low dose) Chronic [98]
T2D Mouse db/db Chronic [92, 95, 97]

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T2D Hamster CHAD strain Chronic [101]
Ins res +
Rat Glucosamine inj Short term [102]
obesity
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T1D Rat STZ inj nr [108]
T1D Swine STZ inj Chronic [105]
HFD + STZ inj
T2D Rat Chronic [107]
(mod dose)
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T2D Rat HFD + STZ inj Chronic [106]

↔ T2D (early) Rat ZDF Chronic [96]
Ins res +
Mouse MSG inj Chronic [104]
obesity
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Ins res +
Mouse HFD Chronic [103]
obesity
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HFD + STZ inj (low

T2D Rat Chronic [107] [165]
dose)
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↓ T2D Rat ZDF Chronic [109]

Ins res +
Rat HFD Short term [110]
obesity
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HFD + STZ inj (low

T2D Rat Chronic [166]
dose)
Ambiguous
Ins res +
Rat Hypercaloric diet# Chronic [111]
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Obesity
T1D, type I diabetes; T2D, type II diabetes; ins res, insulin resistance; inj, injection; STZ, streptozotocin;
HFD, high fat diet; ZDF, Zucker diabetic fatty; nr, not recorded. Short term  1 week. Chronic > 1 week.
For genetic models, duration of disease is equivalent to final age. #With 30% sucrose.

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Highlights
 Diabetes-related systemic disturbances facilitate alterations in cardiac metabolism.
 Dysregulation of cardiac insulin, AMPK and β-adrenergic signaling is well documented.
 Diabetes alters cardiac substrate utilization and storage of glucose and lipids.
 Cardiac energy stress responses in diabetes (eg. oxidative stress and autophagy) are linked with
cell death.
 Molecular disturbances ultimately culminate in impaired relaxation and functional performance
in the diabetic heart.

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