Kimberley M. Mellor

Myocardial autophagy activation and suppressed survival signaling is associated with insulin resistance in fructose-fed mice.

Fructose intake is linked with the increasing prevalence of insulin resistance and there is now evidence for a specific insulin-resistant cardiomyopathy. The aim of this study was to determine the cardiac-specific myocardial remodeling effects of high fructose dietary intake. Given the links between insulin signaling, reactive oxygen species generation and autophagy induction, we hypothesized that autophagy contributes to pathologic remodeling in the insulin-resistant heart, and in particular may be a feature of high fructose diet-induced cardiac phenotype. Male C57Bl/6 mice were fed a high fructose (60%) diet or nutrient-matched control diet for 12 weeks. Systemic and myocardial insulin-resistant status was characterized. Superoxide production (lucigenin) and cellular growth and death signaling pathways were examined in myocardial tissue. Myocardial structural remodeling was evaluated by measurement of heart weight indices and histological analysis of collagen deposition (picrosirius red). Fructose-fed mice exhibited hyperglycemia and glucose intolerance, but plasma insulin and blood pressure were unchanged. High fructose intake suppressed the myocardial Akt cell survival signaling coincident with increased cardiac superoxide generation (21% increase, p<0.05). Fructose feeding induced elevated autophagy (LC3B-II: LC3B-I ratio: 46% increase, p<0.05) but not apoptosis signaling (unchanged Bax-1:Bcl-2 ratio). Despite a 28% increase in interstitial fibrosis, no difference in heart weight was observed in fructose-fed mice. We provide the first evidence that myocardial autophagy activation is associated with systemic insulin resistance, and that high level fructose intake inflicts direct cardiac damage. Upregulated autophagy is associated with elevated cardiac superoxide production, suppressed cell survival signaling and fibrotic infiltration in fructose-fed mice. The novel finding that autophagy contributes to cardiac pathology in insulin resistance identifies a new therapeutic target for diabetic cardiomyopathy.

Reactive oxygen species and insulin-resistant cardiomyopathy.

1. The prevalence of insulin resistance has increased markedly in the past decade and is known to be associated with cardiovascular risk. Evidence of an insulin‐resistant cardiomyopathy, independent of pressure or volume loading influences, is now emerging.

High-fructose diet elevates myocardial superoxide generation in mice in the absence of cardiac hypertrophy

OBJECTIVE Dietary fructose intake has increased considerably in recent decades and this has been paralleled by an increase in the incidence of insulin resistance, especially in children and adolescents. The impact of a high-fructose diet on the myocardium is not fully understood. The aims of this study were to characterize the murine metabolic and cardiac phenotypes associated with a high-fructose diet and to determine whether this diet imparts differential effects with age. METHODS Juvenile (4 wk) and adult (14 wk) C57Bl/6 mice were fed a 60% fructose diet or isoenergetic control (starch) diet for 6 wk. RESULTS At completion of the dietary intervention (at ages 10 and 20 wk), fructose-fed mice were normotensive; hyperinsulinemia and cardiac hypertrophy were not evident. Interestingly, fructose-fed mice exhibited lower blood glucose levels (10 wk: 4.81+/-0.28 versus 5.42+/-0.31 mmol/L; 20 wk: 4.88+/-0.30 versus 5.96+/-0.42 mmol/L, P<0.05) compared with controls. Nicotinamide adenosine dinucleotide phosphate-driven myocardial superoxide production was significantly increased in fructose-fed mice at both ages (by approximately 29% of control at 10 wk of age and 16% at 20 wk, P<0.01). No increase in aortic superoxide production was observed. Fructose feeding did not alter gene expression of the antioxidant thioredoxin-2, suggesting an imbalance between myocardial reactive oxygen species generation and antioxidant induction. CONCLUSION These findings indicate that increased myocardial superoxide production may represent an early and primary cardiac pathologic response to the metabolic challenge of excess dietary fructose in juveniles and adults that can be detected in the absence of cardiac hypertrophy and hypertension.

Fructose diet treatment in mice induces fundamental disturbance of cardiomyocyte Ca2+ handling and myofilament responsiveness

High fructose intake has been linked to insulin resistance and cardiac pathology. Dietary fructose-induced myocardial signaling and morphological alterations have been described, but whether cardiomyocyte function is influenced by chronic high fructose intake is yet to be elucidated. The goal of this study was to evaluate the cardiomyocyte excitation-contraction coupling effects of high dietary fructose and determine the capacity for murine cardiomyocyte fructose transport. Male C57Bl/6J mice were fed a high fructose diet for 12 wk. Fructose- and control-fed mouse cardiomyocytes were isolated and loaded with the fura 2 Ca(2+) fluorescent dye for analysis of twitch and Ca(2+) transient characteristics (4 Hz stimulation, 37°C, 2 mM Ca(2+)). Myocardial Ca(2+)-handling protein expression was determined by Western blot. Gene expression of the fructose-specific transporter, GLUT5, in adult mouse cardiomyocytes was detected by real-time and conventional RT-PCR techniques. Diastolic Ca(2+) and Ca(2+) transient amplitude were decreased in isolated cardiomyocytes from fructose-fed mice relative to control (16 and 42%, respectively), coincident with an increase in the time constant of Ca(2+) transient decay (24%). Dietary fructose increased the myofilament response to Ca(2+) (as evidenced by a left shift in the shortening-Ca(2+) phase loop). Protein expression of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a), phosphorylated (P) phospholamban (Ser(16)), and P-phospholamban (Thr(17)) was reduced, and protein phosphatase 2A expression increased, in fructose-fed mouse hearts. Hypertension and cardiac hypertrophy were not evident. These findings demonstrate that fructose diet-associated myocardial insulin resistance induces profound disturbance of cardiomyocyte Ca(2+) handling and responsiveness in the absence of altered systemic loading conditions.

Heritable pathologic cardiac hypertrophy in adulthood is preceded by neonatal cardiac growth restriction

The identification of genetic factors influencing cardiac growth independently of increased load is crucial to an understanding of the molecular and cellular basis of pathological cardiac hypertrophy. The central aim of this investigation was to determine how pathological hypertrophy in the adult can be linked with disturbances in cardiomyocyte growth and viability in early neonatal development. The hypertrophic heart rat (HHR) model is derived from the spontaneously hypertensive rat and exhibits marked cardiac hypertrophy, in the absence of a pressure load at maturity. Hearts were harvested from male HHR, and control strain normal heart rats (NHR), at different stages of postnatal development [neonatal (P2), 4 wk, 6 wk, 8 wk, 12 wk, 20 wk]. Isolated neonatal cardiomyocytes were prepared to evaluate cell size, number, and binucleation. At postnatal day 2, HHR hearts were considerably smaller than control NHR (4.3 +/- 0.2 vs. 5.0 +/- 0.1 mg/g, P < 0.05). Cardiac growth restriction in the neonatal HHR was associated with reduced myocyte size (length and width) and an increased proportion of binucleated cardiomyocytes. Furthermore, the number of cardiomyocytes isolated from HHR neonatal hearts was significantly less ( approximately 29%) than NHR. We also observe that growth stress in the neonate is associated with accentuated PI3K and suppressed MAPK activation, although these signaling pathways are normalized in the adult heart exhibiting established hypertrophy. Thus, using the HHR model, we identified novel molecular and cellular mechanisms involving premature exit from the cell cycle, reduced cardiomyocyte endowment, and dysregulated trophic signaling during early development, which are implicated in the etiology of heritable cardiac hypertrophy in the adult.

Aromatase Deficiency Confers Paradoxical Postischemic Cardioprotection

The conventional view is that estrogen confers female cardioprotection. Estrogen synthesis depends on androgen availability, with aromatase regulating conversion of testosterone to estradiol. Extragonadal aromatase expression mediates estrogen production in some tissues, but a role for local steroid conversion has not yet been demonstrated in the heart. This studys goal was to investigate how aromatase deficiency influences myocardial function and ischemic resilience. RT-PCR analysis of C57Bl/6 mouse hearts confirmed cardiac-specific aromatase expression in adult females. Functional performance of isolated hearts from female aromatase knockout (ArKO) and aromatase wild-type mice were compared. Left ventricular developed pressures were similar in aerobic perfusion, but the maximal rate of rise of ventricular pressure was modestly reduced in ArKO hearts (3725 ± 144 vs. 4272 ± 154 mm Hg/sec, P < 0.05). After 25 min of ischemia, the recovery of left ventricular developed pressure was substantially improved in ArKO (percentage of basal at 60 min of reperfusion, 62 ± 8 vs. 30 ± 6%; P < 0.05). Hypercontracture was attenuated (end diastolic pressure, 25 ± 5 vs. 51 ± 1 mm Hg; P < 0.05), and lactate dehydrogenase content of coronary effluent was reduced throughout reperfusion in ArKO hearts. This was associated with a hyperphosphorylation of phospholamban and a reduction in phosphorylated Akt. Immediately after reperfusion, ArKO hearts exhibited increased incidence of ventricular premature beats (194 ± 70 vs. 46 ± 6, P < 0.05). These observations indicate more robust functional recovery, reduced cellular injury, and modified cardiomyocyte Ca(2+) handling in aromatase-deficient hearts. Our findings indicate that androgen-to-estrogen conversion may be of pathophysiologic importance to the heart and challenge the notion that estrogen deficiency is deleterious. These studies suggest the possibility that aromatase suppression may offer inotropic benefit in the acute ischemia/reperfusion setting with appropriate arrhythmia management.

Myocardial autophagic energy stress responses—macroautophagy, mitophagy, and glycophagy

An understanding of the role of autophagic processes in the management of cardiac metabolic stress responses is advancing rapidly and progressing beyond a conceptualization of the autophagosome as a simple cell recycling depot. The importance of autophagy dysregulation in diabetic cardiomyopathy and in ischemic heart disease - both conditions comprising the majority of cardiac disease burden - has now become apparent. New findings have revealed that specific autophagic processes may operate in the cardiomyocyte, specialized for selective recognition and management of mitochondria and glycogen particles in addition to protein macromolecular structures. Thus mitophagy, glycophagy, and macroautophagy regulatory pathways have become the focus of intensive experimental effort, and delineating the signaling pathways involved in these processes offers potential for targeted therapeutic intervention. Chronically elevated macroautophagic activity in the diabetic myocardium is generally observed in association with structural and functional cardiomyopathy; yet there are also numerous reports of detrimental effect of autophagy suppression in diabetes. Autophagy induction has been identified as a key component of protective mechanisms that can be recruited to support the ischemic heart, but in this setting benefit may be mitigated by adverse downstream autophagic consequences. Recent report of glycophagy upregulation in diabetic cardiomyopathy opens up a novel area of investigation. Similarly, a role for glycogen management in ischemia protection through glycophagy initiation is an exciting prospect under investigation.

Myocardial stress and autophagy: mechanisms and potential therapies

Autophagy is a ubiquitous cellular catabolic process responsive to energy stress. Research over the past decade has revealed that cardiomyocyte autophagy is a prominent homeostatic pathway, important in adaptation to altered myocardial metabolic demand. The cellular machinery of autophagy involves targeted direction of macromolecules and organelles for lysosomal degradation. Activation of autophagy has been identified as cardioprotective in some settings (that is, ischaemia and ischaemic preconditioning). In other situations, sustained autophagy has been linked with cardiopathology (for example, sustained pressure overload and heart failure). Perturbation of autophagy in diabetic cardiomyopathy has also been observed and is associated with both adaptive and maladaptive responses to stress. Emerging research findings indicate that various forms of selective autophagy operate in parallel to manage various types of catabolic cellular cargo including mitochondria, large proteins, glycogen, and stored lipids. In this Review, induction of autophagy associated with cardiac benefit or detriment is considered. The various static and dynamic approaches used to measure autophagy are critiqued, and current inconsistencies in the understanding of autophagy regulation in the heart are highlighted. The prospects for pharmacological intervention to achieve therapeutic manipulation of autophagic processes are also discussed.

Autophagy anomalies in the diabetic myocardium.

The prominent occurrence of autophagy in fetal/neonatal myocardial tissue has been recognized for more than three decades as a key process in managing the period of perinatal nutrient deprivation. Fasting-induced autophagy has similarly been characterized as an expedient short-term cardiomyocyte response to nutrient restriction. Discerning how autophagy operates in the heart in disease contexts of substrate dysregulation is proving to be a much more complex challenge. Recent studies relating to insulin signaling and cardiac autophagy activation have provided new insights—and generated new contradictions. We highlight several anomalies and pose a number of questions, which emerge from these studies. How can myocardial autophagy induction be associated with both PtdIns3K-Akt activation (in ischemia) and suppression (in insulin resistance)? What is the explanation for the contrasting findings that myocardial autophagy is elevated in a murine model of type 2 diabetes, yet suppressed in the type 1 diabetic state? And finally, in the type 1 diabetic setting, what could be the basis for downregulated cardiac AMP-activated protein kinase (AMPK)-driven autophagic activity, when activation of this ‘energy stress’ kinase is usually integral to the cellular response to glucose deficit? We summarize and discuss these interesting ambiguities of myocardial autophagy regulation.

Fructose Modulates Cardiomyocyte Excitation-Contraction Coupling and Ca2+ Handling In Vitro

Background High dietary fructose has structural and metabolic cardiac impact, but the potential for fructose to exert direct myocardial action is uncertain. Cardiomyocyte functional responsiveness to fructose, and capacity to transport fructose has not been previously demonstrated. Objective The aim of the present study was to seek evidence of fructose-induced modulation of cardiomyocyte excitation-contraction coupling in an acute, in vitro setting. Methods and Results The functional effects of fructose on isolated adult rat cardiomyocyte contractility and Ca2+ handling were evaluated under physiological conditions (37°C, 2 mM Ca2+, HEPES buffer, 4 Hz stimulation) using video edge detection and microfluorimetry (Fura2) methods. Compared with control glucose (11 mM) superfusate, 2-deoxyglucose (2 DG, 11 mM) substitution prolonged both the contraction and relaxation phases of the twitch (by 16 and 36% respectively, p<0.05) and this effect was completely abrogated with fructose supplementation (11 mM). Similarly, fructose prevented the Ca2+ transient delay induced by exposure to 2 DG (time to peak Ca2+ transient: 2 DG: 29.0±2.1 ms vs. glucose: 23.6±1.1 ms vs. fructose +2 DG: 23.7±1.0 ms; p<0.05). The presence of the fructose transporter, GLUT5 (Slc2a5) was demonstrated in ventricular cardiomyocytes using real time RT-PCR and this was confirmed by conventional RT-PCR. Conclusion This is the first demonstration of an acute influence of fructose on cardiomyocyte excitation-contraction coupling. The findings indicate cardiomyocyte capacity to transport and functionally utilize exogenously supplied fructose. This study provides the impetus for future research directed towards characterizing myocardial fructose metabolism and understanding how long term high fructose intake may contribute to modulating cardiac function.