The Role Of Endothelial Cell Calpain In The Pathogenesis Of Diabetic Cardiomyopathy And Its Underlying Mechanisms

In 2016,the world Health Organization(WHO)reported that 422 million adults live with diabetes in the world,mainly in developing countries.In the past thirty years,the prevalence of diabetes in China has increased by 17%.Without any intervention,China will be the country with the highest number of patients with diabetes.Diabetic patients are at increased risk of developing cardiovascular diseases,which account for ?80% of diabetes-related deaths.Both type-1 and type-2 diabetes can affect cardiac structure and function independent of vascular defects and hypertension,a condition termed diabetic cardiomyopathy.Diabetic cardiomyopathy is characterized by early diastolic dysfunction and later systolic dysfunction and heart failure.The pathogenesis of diabetic cardiomyopathy remains incompletely understood and effective therapies are limited.Therefore,it is urgently needed to further understand the molecular mechanisms underlying diabetic cardiac complications and determine effective therapeutic targets for developing new drugs.Calpains belong to a family of calcium-dependent cysteine protease.Among 15 family members,only calpain-1 and calpain-2 are expressed in the endothelial cells.Calpain-1 and calpain-2 are heterodimers consisting of a distinct catalytic subunit called CAPN1 or CAPN2,and a common small regulatory subunit(encoded by capns1).The small subunit is indispensable for stability and activity of calpain-1 and calpain-2.Thus,deletion of capns1 disrupts calpain-1 and calpain-2.Both calpain-1 and calpain-2 activities are negatively regulated by calpastatin,an endogenous calpain inhibitor.We have recently reported that cardiomyocyte-specific deletion of capns1 reduces adverse cardiac changes in mouse models of pre-diabetes and type-1 diabetes.These findings indicate a critical role of cardiomyocyte calpain in diabetic cardiomyopathy.Whereas dysfunction of the cardiomyocytes plays a central role in diabetic cardiomyopathy,non-cardiomyocytes in the heart,such as endothelial cells are important in maintaining coronary vessel function,ventricular homeostasis and proper cardiac function.Indeed,calpain activation has been implicated in endothelial dysfunction and inflammation under diabetic conditions.This raises an intriguing possibility that endothelial cell calpain may mediate adverse microvascular changes in diabetic hearts,which may contribute to diabetic cardiomyopathy.[Aims]1.To determin the roles of endothelial cell calpain in diabetic cardiomyopathy.2.To explore the molecular mechanisms by which endothelial cell calpain contributes to diabetic cardiomyopathy by focusing on neovascularization and fibrosis.[Methods]1.A novel line of endothelial cell-specific capns1 knockout mice was generated.Pre-diabetes,type-1 and type-2 diabetes were induced in endothelial cell-specific capns1 knockout mice and their wild-type littermates.Myocardial function and coronary flow reserve were assessed by echocardiography.Histological analyses were performed to determine capillary density,cardiomyocyte size and fibrosis in the heart.Isolated aortas were assayed for neovascularization.2.Cultured cardiac microvascular endothelial cells were stimulated with high palmitate.Calpain was inhibited by over-expression of calpastatin or chemical inhibitor of calpain.?-catenin expression was up-regulated or down-regulated by transfecting ?-catenin plasmid or crispr/cas9 KO plasmid,respectively.Angiogenesis and apoptosis were analyzed.3.The m RNA levels of TGF-?1 and IL-6 were analyzed in pre-diabetic mouse hearts and cultured cardiac microvascular endothelial cells stimulated with high palmitate.Endothelial to mesenchymal transition was induced in endothelial cells by recombinant IL-6 and determined by measuring ?-SMA expression.The protein levels of I?B were determined in cardiac microvascular endothelial cells treated with high palmitate for 6h,12 h and 24 h.[Results]1.Endothelial cell-specific deletion of capns1 disrupted calpain-1 and calpain-2 in endothelial cells;however,disruption of endothelial cell calpain did not affect normal physiological function in mice.2.Deletion of endothelial cell calpain reduced cardiac fibrosis and hypertrophy,and alleviated myocardial dysfunction in mouse models of diabetes without significantly affecting systemic metabolic parameters.These protective effects of calpain disruption in endothelial cells were associated with an increase in myocardial capillary density and coronary flow reserve.3.Ex vivo analysis of neovascularization revealed more endothelial cell sprouts from aortic rings of pre-diabetic and diabetic capns1 knockout mice compared with their wild-type littermates.4.In cultured cardiac microvascular endothelial cells,inhibition of calpain improved angiogenesis and prevented apoptosis under metabolic stress.Mechanistically,deletion of capns1 elevated the protein levels of ?-catenin in endothelial cell capns1 knockout mice and constitutive activity of calpain-2 suppressed ?-catenin protein expression in cultured endothelial cells.Up-regulation of ?-catenin promoted angiogenesis and inhibited apoptosis whereas knockdown of ?-catenin offset these protective effects of calpain inhibition in endothelial cells under metabolic stress.5.The m RNA levels of IL-6,but not TGF-?1,were significantly increased in prediabetic mice and cardiac microvascular endothelial cells under metabolic stress.Inhibition of calpain by capns1 deletion,calpastatin or calpain inhibitor decreased IL-6 expression in diabetic hearts and in cardiac microvascular endothelial cells.Furthermore,IL-6 directly induced ?-SMA expression,a specific marker of interstitial cells,indicative of endothelial to mesenchymal transition.[Conclusions]The present study delineates a primary role of endothelial cell calpain in the pathogenesis of diabetic cardiomyopathy.On the one hand,endothelial cell calpain induced cardiac endothelial injury and impaired neovascularization via suppression of ?-catenin;on the other hand,endothelial cell calpain increased the expression of IL-6 via down-regulation of I?B,leading to cardiac fibrosis.Both coronary endothelial cell injury,impaired neovascularization and cardiac fibrosis directly contribute to diabetic cardiomyopathy.Given the contributing role of cardiomyocyte calpain in diabetic cardiomyopathy,the present study provides further evidence to support that calpain is a promising therapeutic target to prevent diabetic cardiac complications.