Research On The Mechanism Of Orai1Calcium Channel Regulating Mitochondrial Fission In Diabetic Cardiomyocytes

Objective:Abnormal cellular energy metabolism leading to cardiomyocyte hypertrophy is an important factor of cardiac dysfunction in patients with diabetic cardiomyopathy(DCM)Abnormal mitochondrial fission and fusion lead to its dynamic imbalance,which is the main part of abnormal energy metabolism of diabetic cells.Currently,changes in the phosphorylation level of mitochondrial dynamic relative protein 1(Drp1)in DCM myocardial tissue can lead to the imbalance of mitochondrial dynamics,but the mechanism is unknown.Our previous study found that abnormal regulation of Ca2+signaling pathway in cardiomyocytes promoted the occurrence and development of DCM,and the Orail calcium channel is an important pathway for the increase of Ca2+ entry in cardiomyocytes.This study intends to investigate whether Orail calcium signal can induce mitochondrial dynamic abnormalities in DCM cardiomyocytes by regulating the phosphorylation level of DrplMethods:(1)The rat model of type 2 diabetes mellitus was established by high-fat diet,and the cardiac function indexes were detected by echocardiography.(2)Neonatal rat cardiomyocytes(NRCMs)were cultured with medium containing 33 mmol/L high glucose(HG)for establishing DCM cell model.(3)NRCMs surface area was determined by WGA staining.Calcium ion indicator Fluo4/AM loading NRCMs was used to measure the change of intracellular calcium ion concentration.Mitochondrial morphology of NRCMs was detected by mito-tracker staining.The changes of NRCMs mitochondrial membrane were detected by JC-1 probe.(4)Western blot was used to detect the expression levels of hypertrophy related proteins ANP and β-MHC,mitochondrial fission related proteins Drpl and its phosphorylated proteins,mitochondrial fusion related proteins Mfn2 and Opal,Orail,and downstream kinases ERK1/2 and phosphatase CnA in ventricular tissues of type 2 diabetic rats and NRCMsResults:(1)In the animal model of type 2 diabetes,the blood glucose level of ZDF rats was significantly increased.The results of echocardiography showed that the diastolic function was weakened and the ventricular wall was thickened.The expression of ventricular hypertrophy related proteins ANP and β-MHCwere increased.The expression levels of Orail,CnA and p-ERK1/2 were significantly increased in hypertrophic ZDF rat ventricular tissues.Drp1 activation,that is,the expression of p-Drp1S616 increased and the expression of p-Drp1S637 decreased.The expression of Opal and Mfn2 were decreased(2)In DCM cell model,HG-induced NRCMs surface area increased,mitochondrial morphology fragmentation,mitochondrial membrane potential decreased,and the expression of ANP and β-MHC proteins significantly increased.Mitochondrial fission related protein Drp1 was activated,p-Drp1S616 expression was up-regulated and p-Drp1S637 expression was down-regulated.Decreased expression of Opal and Mfn2 of mitochondrial fusion related proteins.Orail protein expression increased and orail-mediated Ca2+ entry increased(3)In the DCM cell model,the Drpl inhibitor mdivi-1 inhibited the surface area increase of cardiomyocytes induced by high glucose,and inhibited the expression increase of ANP and β-MHC.By regulating the phosphorylation of Drpl,mdivi-1 can improve the mitochondrial morphological and functional abnormalities induced by high glucose.By regulating the phosphorylation of Drpl,Orail channel inhibitor BTP2 inhibits the mitochondrial fission induced by high glucose,and reduces the protein expression of ANP and β-MHC induced by high glucose,as well as the protein expression of CnA and p-ERK1/2.p-ERK1/2 inhibitor U0126 significantly inhibited the cardiac hypertrophy induced by high glucose,inhibited phosphorylation of p-Drp1S616,and reduced mitochondrial fission.CnA inhibitor CsA significantly inhibited the up-regulation of ANP expression induced by high glucose,promoted phosphorylation of p-Drp1S637,and reduced mitochondrial fission.Down-regulation of Orail expression can decrease the expression of p-Drp1S616,increase the expression of p-Drp1S637,then inhibit the Drpl-mediated mitochondrial fission,and then inhibit the expression of ANP and β-MHC in NRCMs induced by HGConclusion:This study reveals a new mechanism for hyperglycemia to induce cardiac hypertrophy and dysfunction.High glucose promotes Orail-mediated Ca2+ entry,activates the downstream CnA/ERK pathway,and regulates the phosphorylation of Drp 1,thereby promoting the fission of Drpl-dependent mitochondria,leading to cardiomyocyte hypertrophy.Therefore,Orai1-Drp1 signaling pathway may be a key link in hyperglycemia induced cardiac hypertrophy and a potential therapeutic target.