Role Of Calpain In Lipotoxic Cardiomyopathy And Protective Effect Of Exercise

Background:Since obesity and type 2 diabetes increased year by year, lipotoxicity induced cardiovascular diseases are obtained more widespread concern. Lipotoxicity related cardiomyopathy is a chronic and pathological changes. However, the underlying of its pathogenesis remains to be elucidated. In this study, we focus on exploring the role of calpain in cardiomyopathy induced by lipotoxicity. We analysis endoplasmic reticulum stress(ER stress), cardiomyocyte apoptosis and pro-inflammatory cytokines after calpain inhibition, in order to discuss the mechanism of lipotoxicity induced injury. We further to confirm that whether exercise could attenuate lipotoxicity cardiac dysfunction through inhibiting calpain activity. The study also further explore the movement to improve lipotoxicity heart dysfunction by influencing the activity of calpain and the protection of benefits, and provide theoretical and experimental basis for the prevention and treatment of lipotoxicity cardiomyopathy. This work may contribute to design the therapeutic target for lipotoxicity injury in heart.Methods:Cardiomyocyte-specific knockout capn4(capn4 KO) and their littermates wild type(WT) mice(n=6)were fed a high fat diet(HFD)(60% calorie from fat)or normal diet(ND) for 20 weeks. Glucose tolerance test(GTT) and insulin resistance/tolerance test(ITT) were conducted at the end of the animal procedure for insulin sensitivity. Cardiac function including systolic and diastolic function was assayed by echocardiography. The level of triglyceride(TG), total cholesterol(TC), low density lipoprotein(LDL) and high density lipoprotein(HDL) were checked from mouse plasma. The value of heart weight / tibia length was calculated representing for myocardial hypertrophy.Myocardial tissues were embedded and sectioned,stained with sirius red-saturated picric acid,then observed collagen deposition; WGA-Hochest fluorescent staining to measure cardiomyocyte-cross area. RT-PCR was carried out to detect mRNA levels in heart,including the fibrotic character genes collagen 1 and collagen 3, hypertrophic character genes ANP and β-MHC, and pro-imflammatory cytokines TNF-α and IL1β. Caspase-3 activity was analyzed as a apoptosis index. ER stress related protein CHOP, BIP and phosphate –JNK were analyzed by western blot.Cultured cardiomyocyte were treated with palmitic acid(0.2 mM) for 24 h as a lipotoxicity model in vitro. The levels of DNA fragmentation and caspase-3 activity were assessed for apoptosis. Calpain inhibitor-III, siRNA of capn1 and capn2 were used to inhibit calpain activity or reduce protein levels. ER stress inhibitor was also used to investigate role of calpain in apoptosis induced by lipotoxicity. Proinflammatory cytokines were detected by RT-PCR. The expression of ER stress related proteins were detected by western blot.C57 mice(male, 6 weeks old,n=6) were divided into four groups: HFDsedentary group, HFD-exercise group(HFD 20 weeks+ 8 weeks swimming), NDsedentary group and ND-exercise group(ND 20 weeks+ 8 weeks swimming). The exercise training procedure was conducted after 12 weeks. Mouse in trained group were subjected swimming for 30 minutes one time, 5 times one week, and last for 8 weeks. The changes of serum lipid, cardiac function and calpain activity were analyzed after 8 weeks exercise training.Results:1 Plasma lipid profiles were increased induced by HFD, including levels of TG, TC, LDL-C and HDL-C in blood. Body weight was also increased in HFD mice. Glucose tolerance was impaired in both capn4 KO and WT mice after HFD.2 Systolic function was decreased as EF and FS reduced in HFD induced mouse hearts compared with the ND mouse hearts. Capn4 KO restored the systolic function in HFD-induced mice. Similarly, Tissue Doppler showed that the E/A ratio was significantly decreased in HFD-induced mice, indicated that diastolic dysfunction. Capn4 KO protected heart from lipotoxic cardiomyopathy. Calpain activity was increased in HFD WT mice heart, which was blocked in capn4 KO heart.3 The ratio of heart weight to tibia length was increased in lipotoxic mice compared with normal mice. Histological analysis of cardiomyocytes cross-sectional areas found that cardiomyocyte size was increased in HFD group, compared with ND group. These effects of cardiac hypertrophy were attenuated by inhibiting calpain. In addition, capn4 KO namalized the mRNA expression of hypertrophic genes( ANP and β-MHC) in HFD-induced hearts. Consistently, total collagen, collagen I and collagen III were increased in lipotoxci-induced heart compared with controlled hearts. Collagen deposition reduced significantly in HFD-induced hearts from capn4 KO mice. Consistant with these changes, inhibition of calpain activation reduced fibrosis related genes(collagen I and collagen III) mRNA expression.4 Lipotoxic-induced an increased in ER stress in WT mice as determined by increases in BIP, CHOP and phosphorylated JNK 1/2; However,the respondence of ER stress was significantly attenuated in capn4 KO mice.5 In vitro study confirmed that palmitate induced calpain activity, caspase-3 activity and DNA fragmentation increased significantly. Calpain activity contributed to ER stress, as showed protein expression of BIP, CHOP and phosphorylated JNK 1/2 were increased in cardiomyocytes after palmitate induction. Importantly, Downregulation of calpain activity both by calpain inhibitor- III and capn1 siRNA prevented ER stress and apoptosis.6 Body weight, Lee’s index, TG and TC were increased in HFD-induced mice. These effects were attenuated by swim training.7 Calpain activity reduced in HFD mouse hearts after swimming. What’s more, exercise training also attenuated HFD-induced cardiac dysfunction.Conclusion:1 HFD induce animal model of lipotoxicity cardiomyopathy. Lipotoxic injury is attenuated by inhibiting calpain activity, which is independent from metabolic pathway.2 Inhibition of calpain activity through attenuating endoplasmic reticulum stress and inflammatory pathway, reduce cardiomyocytes apoptosis in lipotoxicity mouse heart, ultimately protect cardiac function.3 Inhibition of calpain-1 but not calpain-2, through ER stress pathway, can reduce palmitate induced apoptosis.4 Exercise training attenuate cardiac dysfunction in lipotoxic injury by reducing calpain activity.