Role And Mechanism Study Of Calpain1 And Calpain2 In CVB3-induced Viral Myocarditis

Viral myocarditis(VMC)represents local or diffuse myocardial inflammation,and it can be developed as acute or chronic disease process.The majority of VMC patients have a good prognosis,while about 10-15% of patients develop dilated cardiomyopathy(DCM)due to persistent inflammation and repeated disease.Viral infections exsist in up to 60% of patients with primary dilated cardiomyopathy,suggesting that it is one of the important triggers of myocarditis.More than 20 viruses are known to cause viral myocarditis,including coxsackie virus,adenovirus,influenza virus,and HIV.The coxsackievirus group B type 3(CVB3)is the most common.Therefore,exploring the target of inhibiting CVB3 and looking for possible interventions for viral myocarditis have important clinical significance for VMC therapy.There are several roles of autophagy in heart disease: Firstly,it was found that autophagy is essential to maintain the function and viability of cardiomyocytes under normal physiology;Secondly,it was found that the basal level of autophagy can prevent cardiac hypertrophy,and blocking autophagy in the mouse heart induce myocardial hypertrophy;In streptozotocin(STZ)-induced type 1 diabetic mice,inhibition of autophagy will exacerbate myocardial injury.In summary,it is known that autophagy plays an important role in maintaining normal heart physiology and improving cardiac pathology.A growing number of studies have shown that the process of cell autophagy promotes the replication of CVB3 in vitro,and studies in vivo have also shown that the reduction of autophagy in pancreatic acinar cells can also improve pancreatitis.As a cardiotropic virus,CVB3 causes the most harmful disease VMC.However,the effects of autophagy on CVB3-induced VMC have not been reported.As a calcium-dependent activation cysteine protease,calpain can hydrolyze many substrates and play important physiological functions after its activation.The function of calpain 1 and 2 in the heart has been extensively studied and are involved in various cardiovascular diseases.we have demonstrated that downregulation of calcium channel proteins leads to an excessive intracellular calcium ion concentration in cardiac myocytes of VMC,thereby activating intracellular calpain.The calpain protease activity was activated in CVB3-infected cells in vitro,suggesting that calpain plays an important role in the pathogenesis of VMC.Therefore,we first inhibited the activity of calpain in cells,and the results showed that the CVB3 replication was significantly inhibited.Further results showed that inhibition of calpain activity significantly blocked autophagy and thus inhibited virus replication.Calpain4,as a small subunit of calpain1 and calpain2,can stabilize protein structure and regulate the activity of large subunits,when capn4 knockout largely inhibits the activity of calpain1 and calpain2 large subunits.Therefore,cardiac-specific knockout capn4 mice are commonly used as Calpain-functional mice.We applied cardiac capn4 specific knockout mice to explore the effects of calpain1 and 2 function on CVB3-induced viral myocarditis.Unlike the cellular results,our results suggest that capn4 KO mice have more severe myocarditis and higher viral loads in the heart.ATG7 cardiac specific knockout mice and their littermates were infected with CVB3 virus.Our results were consistent with that of the capn4 knockout mice: the mice in the KO group had significantly aggravated myocarditis.Therefore,we believe that inhibiting the calpain activity in cardiomyocytes in VMC mice may aggravate viral myocarditis.Taken together,this study indicates that inhibition of calpain protease activity can inhibit CVB3 viral replication at the cellular level,but it can promote disease progression in VMC mice,implying that calpain protease may not be a good target for the VMC treatment in clinical.