Endothelial Cell-specific Calpain Regulates Cardiac Fibrosis Via The Endothelial-Mesenchymal Transition

Objective Cardiac fibrosis is a scar formation process in the heart,which is characterized by type I collagen deposition increasing,activation and differentiation of cardiac fibroblasts into collagen-producing myofibroblasts,increasing ventricular stiffness,hardness and diastolic dysfunction.Therefore,it is of great significance to prevent and reverse cardiac fibrosis.Coronary Microvascular Disease refers that it affects the structure and function of the coronary microcirculation,which occurs in patients with a variety of cardiovascular risk factors,associating with adverse event risk.Coronary microvascular abnormalities,endothelial cell inflammation,and increased oxidative stress promote the Cardiac Fibrosis.In particular,cardiac microvascular endothelial cells play an important role in maintaining cardiac function and cardiac remodeling;While cardiac microvascular endothelial cells are the most abundant cell type in adult myocardium,and also are closely related to adjacentmyocardial cells and fibroblasts which are in direct contact.In clinical cases,coronary macrovascular disease is emerging more and more,and there are currently no targeted treatments for patients.So clearly,the mechanism and target of intervention of myocardial fibrosis can bring targeted prevention and treatment measures for CMD.Calpain-1 and Calpain-2 are heterodimers and contain specific subunits encoded by Capn1 and Capn2(80 kDa)and the common subunit controlled by Capn4(28 kDa),a gene that regulates calpain activity.Knocking out Capn4 will suppress both calpain-1 and calpain-2 activity;Meanwhile,intracellular calcium concentration and specific endogenous calpastatin can also adjust calpain activity.Increasing evidence suggest that Epithelial-Mesenchymal Transition(EMT)is involved in tissue fibrotic lesions,manifested as representing markers of fibroblasts or myofibroblasts.Myofibroblasts have been identified as cells being responsible for the progression of fibrosis,including tissue internal resting fibroblasts,circulating CD34 positive fibroblasts,and myofibroblasts transformed and activated by various cell types.Endothelial cells can eventually undergo fibroblasts and/or myofibroblasts through the process of Endothelial-Mesenchymal Transition(EndMT),which is of great significance for cardiac fibrosis.Endothelial cells(ECs)experience mesenchymal transition process,turning into fibroblasts and/or myofibroblasts that secreting collagen caused cardiac fibrosis eventually.It can also block cardiac microcirculation,and destroy normal myocardial structure,contributing to cardiac remodeling eventually.ECs are the most abundant in the heart.This study indicates whether endothelial cell-specific calpain is involved in EndMT and fibroblasts proliferation,ultimately leading to cardiac fibrosis.The role of non-cardiomyocytes in cardiac remodeling and fibrosis has not been totally understood until now.This study investigated if EC-specific calpain participates in myocardial endothelial injury via EndMT and in cardiac fibroblasts during cell proliferation,thereby contributing to cardiac fibrosis.Aims To explore the role of endothelial cell-specific calpain in cardiac fibrosis via HSP90/Akt signaling way in the Endothelial-Mesenchymal Transition,it represents that there is a new specific target for treating patients with cardiac fibrosis clinically.Methods In vitro cultured mouse cardiac ECs were induced with transforming growth factor(TGF)-?1(10 ng/ml)and calpain inhibitor III(20?M)or Akt inhibitor(LY294002,20?M).Isolated cardiac fibroblasts were induced by TGF-?1 and an HSP90 inhibitor(17AAG,20?M),and EndMT were analysed.Capn4-knockout(KO)specific toECs of mice was generated.We induced the pathological process mimicking cardiac hypertrophy and fibrosis in both Capn4-KO mice and their wild-type littermates.The histological analysis was used to measure cardiomyocyte size and collagen contained in the heart.The immunofluorescence analysis was performed to demonstrate that the ECs went through the EndMT,transforming mesenchymal cells into fibroblasts and myofibroblasts.Results Capn4 deletion specific to ECs abrogated activity of both calpain1 and calpain 2 in ECs,lowered the volume of cardiac collagen and cardiomyocytes size.An ex vivo analysis of cardiomyocytes by Evans Blue staining revealed that isoproterenol increased cell death compared with the control,and Capn4-KO alleviated this result.Inhibiting calpain in cultured cardiac microvascular endothelial calpastatin decreased the pathological EndMT process,showing that the cultured MCECs have more mesenchymal markers,such as ?-smooth muscle actin(SMA),and fewer endothelial markers,such as VE-cadherin.Activating calpain elevated phosphorylated Akt in mice cultured ECs,and inhibiting calpain decreased phosphorylated Akt.Upregulation of p-Akt by calpain promoted the EndMT,whereas inhibiting calpain switched on the protective mechanism during the EndMT via the heat shock protein(HSP)90/Akt signaling way in cultured ECs.Conclusion This study demonstrated a vital role of calpain in ECs for inducing myocardiocyte hypertrophy,cell death and the EndMT via the HSP90/Akt signaling pathway,thereby promoting cardiac fibrosis.The results indicate that inhibiting ECs calpain is a novel therapeutic target to retard cardiac fibrosis and has positive effects on heart failure.