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Aging is a major risk factor for cardiovascular disease. It has previously been shown that protein levels of cathepsin K, a lysosomal cysteine protease, are elevated in the failing heart and that genetic ablation of cathepsin K protects against pressure overload-induced cardiac hypertrophy and contractile dysfunction. Here we test the hypothesis that cathepsin K knockout alleviates age-dependent decline in cardiac function. Cardiac geometry, contractile function, intracellular Ca(2+) properties, and cardiomyocyte apoptosis were evaluated using echocardiography, fura-2 technique, immunohistochemistry, Western blot and TUNEL staining, respectively. Aged (24-month-old) mice exhibited significant cardiac remodeling (enlarged chamber size, wall thickness, myocyte cross-sectional area, and fibrosis), decreased cardiac contractility, prolonged relengthening along with compromised intracellular Ca(2+) release compared to young (6-month-old) mice, which were attenuated in the cathepsin K knockout mice. Cellular markers of senescence, including cardiac lipofuscin, p21 and p16, were lower in the aged-cathepsin K knockout mice compared to their wild-type counterpart. Mechanistically, cathepsin K knockout mice attenuated an age-induced increase in cardiomyocyte apoptosis and nuclear translocation of mitochondrial apoptosis-inducing factor (AIF). In cultured H9c2 cells, doxorubicin stimulated premature senescence and apoptosis. Silencing of cathepsin K blocked the doxorubicin-induced translocation of AIF from the mitochondria to the nuclei. Collectively, these results suggest that cathepsin K knockout attenuates age-related decline in cardiac function via suppressing caspase-dependent and caspase-independent apoptosis.

Background/Aims: Anion channels such as chloride channel are known to participate in the regulation of a wide variety of cellular processes including development, differentiation, proliferation, apoptosis and regeneration. This study was designed to examine the effect of the non-selective anion channel blocker 4,4'-Diisothiocyanostilbene-2, 2'-disulfonic acid (DIDS) on cardiac function and apoptosis using a rat model of ischemia/reperfusion (I/R). Methods: Fifty male SD rats were randomly divided into the following groups including sham, I/R and I/R+DIDS (7, 14 or 28 mg/kg). In DIDS group, rats received DIDS treatment (4 ml/kg/hr) at the beginning of reperfusion for 2 hrs using a programmed micro-pump. Cardiac function was evaluated including left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP) as well as positive and negative maximal derivatives of left ventricular pressure (± dP/dtmax). Myocardial infarct size was detected using the double staining with 2, 3, 5-triphenyl-2H-tetra-zolium chloride (TTC) and Evan's blue dye. DNA ladder, TUNEL assay, Bax and Bcl-2 protein levels were evaluated. Levels of ROS and Akt phosphorylation were detected. Results: I/R injury compromised cardiac function as manifested by reduced LVSP and ± dP/dtmax as well as pronounced apoptosis. I/R-induced cardiac anomalies were markedly ameliorated by DIDS. DIDS retarded I/R-induced myocardial infarct and apoptosis. In addition, DIDS ameliorated I/R-induced ROS production and Akt dephosphorylation in the heart. Conclusion: Taken together, our data revealed that DIDS may protect cardiomyocytes against I/R injury as evidenced by improved cardiac function, Bcl-2, Akt phosphorylation, and reduced myocardial apoptosis, Bax expression, ROS production and myocardial infarct size.

Alcohol intake is associated with myocardial contractile dysfunction and apoptosis although the precise mechanism is unclear. This study was designed to examine the effect of the cytochrome P450 enzyme CYP2E1 inhibition on ethanol-induced cardiac dysfunction. Adult male mice were fed a 4% ethanol liquid or pair-fed control diet for 6weeks. Following 2weeks of diet feeding, a cohort of mice started to receive the CYP2E1 inhibitor diallyl sulfide (100mg/kg/d, i.p.) for the remaining feeding duration. Cardiac function was assessed using echocardiographic and IonOptix systems. Western blot analysis was used to evaluate CYP2E1, heme oxygenase-1 (HO-1), iNOS, the intracellular Ca(2+) regulatory proteins sarco(endo)plasmic reticulum Ca(2+)-ATPase, Na(+)Ca(2+) exchanger and phospholamban, pro-apoptotic protein cleaved caspase-3, Bax, c-Jun-NH(2)-terminal kinase (JNK) and apoptosis signal-regulating kinase (ASK-1). Ethanol led to elevated levels of CYP2E1, iNOS and phospholamban, decreased levels of HO-1 and Na(+)Ca(2+) exchanger, cardiac contractile and intracellular Ca(2+) defects, cardiac fibrosis, overt O(2)(-) production, and apoptosis accompanied with increased phosphorylation of JNK and ASK-1, the effects were significantly attenuated or ablated by diallyl sulfide. Inhibitors of JNK and ASK-1 but not HO-1 inducer or iNOS inhibitor obliterated ethanol-induced cardiomyocyte contractile dysfunction, substantiating a role for JNK and ASK-1 signaling in ethanol-induced myocardial injury. Taken together, these findings suggest that ethanol metabolism through CYP2E1 may contribute to the pathogenesis of alcoholic cardiomyopathy including myocardial contractile dysfunction, oxidative stress and apoptosis, possibly through activation of JNK and ASK-1 signaling.

Background Macrophage migration inhibitory factor (MIF) plays a role in the development of obesity and diabetes. However, whether MIF plays a role in fat diet-induced obesity and associated cardiac anomalies still remains unknown. The aim of this study was to examine the impact of MIF knockout on high fat diet-induced obesity, obesity-associated cardiac anomalies and the underlying mechanisms involved with a focus on Akt-mediated autophagy. Methods Adult male wild-type (WT) and MIF knockout (MIF−/−) mice were placed on 45% high fat diet for 5 months. Oxygen consumption, CO2 production, respiratory exchange ratio (RER), locomotor activity, and heat generation were measured using energy calorimeter. Echocardiographic, cardiomyocyte mechanical and intracellular Ca2+ properties were assessed. Apoptosis was examined using TUNEL staining and western blot analysis. Akt signaling pathway and autophagy markers were evaluated. Cardiomyocytes isolated from WT and MIF−/− mice were treated with recombinant mouse MIF (rmMIF). Results High fat diet feeding elicited increased body weight gain, insulin resistance, and caloric disturbance in WT and MIF−/− mice. High fat diet induced unfavorable geometric, contractile and histological changes in the heart, the effects of which were alleviated by MIF knockout. In addition, fat diet-induced cardiac anomalies were associated with Akt activation and autophagy suppression, which were nullified by MIF deficiency. In cardiomyocytes from WT mice, autophagy was inhibited by exogenous rmMIF through Akt activation. In addition, MIF knockout rescued palmitic acid-induced suppression of cardiomyocyte autophagy, the effect of which was nullified by rmMIF. Conclusions These results indicate that MIF knockout preserved obesity-associated cardiac anomalies without affecting fat diet-induced obesity, probably through restoring myocardial autophagy in an Akt-dependent manner. Our findings provide new insights for the role of MIF in obesity and associated cardiac anomalies.

Adiponectin (APN), an adipose-derived adipokine, offers cardioprotective effects although the precise mechanism of action remains unclear. This study was designed to examine the role of APN in high fat diet-induced obesity and cardiac pathology. Adult C57BL/6 wild-type and APN knockout mice were fed a low or high fat diet for 22weeks. After 40day feeding, mice were treated with 2mg/kg rapamycin or vehicle every other day for 42days on respective fat diet. Cardiomyocyte contractile and Ca2+ transient properties were evaluated. Myocardial function was evaluated using echocardiography. Dual energy X-ray absorptiometry was used to evaluate adiposity. Energy expenditure, metabolic rate and physical activity were monitored using a metabolic cage. Lipid deposition, serum triglyceride, glucose tolerance, markers of autophagy and fatty acid metabolism including LC3, p62, Beclin-1, AMPK, mTOR, fatty acid synthase (FAS) were evaluated. High fat diet intake induced obesity, systemic glucose intolerance, cardiac hypertrophy, dampened metabolic ability, cardiac and intracellular Ca2+ derangements, the effects of which were accentuated by APN knockout. Furthermore, APN deficiency augmented high fat diet-induced upregulation in the autophagy adaptor p62 and the decline in AMPK without affecting high fat diet-induced decrease in LC3II and LC3II-to-LC3I ratio. Neither high fat diet nor APN deficiency altered Beclin-1. Interestingly, rapamycin negated high fat diet-induced/APN-deficiency-accentuated obesity, cardiac hypertrophy and contractile dysfunction as well as AMPK dephosphorylation, mTOR phosphorylation and p62 buildup. Our results collectively revealed that APN deficiency may aggravate high fat diet-induced obesity, metabolic derangement, cardiac hypertrophy and contractile dysfunction possibly through decreased myocardial autophagy.