Effect Of TRPC3 And TRPC6 Channels On Myocardial Ischemia Reperfusion Injury And Underlying Mechanism

Myocardial ischemia-reperfusion injury(MIRI)refers to restoring blood flow on the basis of ischemic cardiac actually worsen or even leading to irreversible damage phenomenon.It is common in surgical thrombolytic therapy,cardiopulmonary bypass,coronary artery bypass surgery,angioplasty,valve replacement surgery,etc.after reperfusion,lower systolic and diastolic function,arrhythmias,disorder of myocardial energy metabolism may appear,Furthmore ultrastructural changes and vascular no-reflow phenomenon.Finally myocardial cells dysfunction,ventricular systolic and diastolic function disorders may occur.So far accepted MIRI pathological mechanisms include:oxidative stress,intracellular calcium overload,energy loss,cardiac myocyte apoptosis,and activation of neutrophil inflammatory reaction.Calcium is an endogenous messenger;intracellular calcium homeostasis can maintain and regulate cell function,Intracellular and mitochondrial calcium overload has already begun during myocardial ischemia period.Calcium overload has become more seriously at the beginning of reperfusion,the sarcoplasmic reticulum damage and mitochondrial charge redistribution may occur,followed by induction of mitochondrial permeability transition pore(mPTP)opening,resulting in myocardial cell Ca2+ disorders,accelerating Ca2+ influx and Ca2+ separation barrier.In addition,ATP activity may recover gradually during reperfusion,which can activate the sarcoplasmic reticulum Ca2+ cycling,followed by the spread of cytosolic calcium oscillations and calcium waves.Calcium overload and the activation of proteases,enzymes phospholipase and endonuclease may lead to DNA breakdown,apoptosis and irreversible damages of tissue.Intracellular Calcium overload may be one of the key factors which initiate myocardial ischemia-reperfusion injury,therefore,it is essential to reduce intracellular Ca2+ overload during reperfusion which could protect myocardium from MIRI.Classical transient receptor potential(TRPC)channel are important non-selective cation channels located on the plasma membrane,with the selective permeability of sodium ions and calcium ions.TRPCs have seven subfamily members,according to the differences of amino acid sequences;they were being divided into two subclasses of TRPC1/2/4/5 and TRPC3/6/7.Cell functions are closely related to the channel activation,such as cell growth and contraction,proliferation,motility,secretion,synaptic potentials and gene expression.The abnormalities of expression and function of TRPC channels may be associated with various diseases.Recent datas have shown that TRPC3,TRPC4 and TRPC6 played important roles in cardiac hypertrophy and heart failure,the roles and mechanisms have been thoroughly researched.However,the relationship between TRPCs and MIRI has not been reported yet.For this study,we built I/R models with TRPC3/6/7 knockout mice,and systematically explored the roles of TRPC3 and TRPC6 in MIRI In vivo and in vitro.The results showed that:TRPC3/6 knockout can reduce the level of NFAT,thereby reducing calcium overload during reperfusion,the infarcted area and the rate of apoptosis was reduced by raising the levels of p-AKT and p-ERK1/2.TRPC inhibitor SKF96365 inhibit myocardial apoptosis during the process of hypoxia/reoxygenationTo investigate the effect of TRPCs during hypoxia/reoxygenation process,the inhibitor SKF96365 of TRPCs was used;the first application of calcium imaging technology was performed to probe CPA-induced calcium changes of normal H9C2 and primary cardiomyocytes,the results showed:TRPCs channel inhibitor SKF96365;TRPC3 specific inhibitor Pyr3 could reduce CPA-induced SOCE.To further explore the effect of TRPCs during hypoxia/reoxygenation process,we performed Western blotting analysis in hypoxia/reoxygenation H9C2 cells,the results showed that time,BAX/BCL2 ratio decreased significantly with accession of SKF96365.To investigate whether TRPC channels influence on mitochondrial membrane potential changes during hypoxia/reoxygenation,JC-1 fluorescent probe was used,mitochondrial membrane potential was improved with different concentrations of SKF96365.These results indicated that TRPCs inhibitor-SKF96365 could lower mitochondrial membrane potential and ameliorate apoptosis by reducing calcium influx.TRPC3/6 upregulated during I/R processStudies have shown that TRPCs play roles during hypoxia/reoxygenation process,we performed RT-PCR assay and immunohistochemical method to investigate the TRPCs expression levels in cardiomyocytes,RT-PCR showed that:TRPC 1,2,3,4,6 were expressed at the mRNA level,TRPC5,7 had no expression.Immunohistochemistry analysis observed that TRPC3,6 expressed in mice primary cardiomyocytes,TRPC7 had no expression.In order to clarify whether TRPC3/6 plays roles in the MIRI process or not,we built I/R models.Western blots analysis showed that the protein expression levels of TRPC3,TRPC6,CaNa,CaN?,and NFATc3 increased significantly after I/R compared with sham mice.Calcium imaging technology showed OAG increased H9C2 cells calcium influx by TRPC3/6 channels after hypoxia/reoxygenation.These results revealed that TRPC3/TRPC6 increased calcium influx significantly;Ca2 +-Calcineurin-NFAT signaling pathway was activated while MIRI.TRPC3/6/7 knock out ameliorate ischemia-reperfusion injuryTRPC3 and TRPC6 have been shown to activate the calcium-Calcineurin-NFAT signaling pathway.To further investigate the roles of TRPC3/6/7 gene knockout on MIRI,Evans-Blue/TTC staining method was used to evaluate the myocardial infarct size(IS),the results showed that,IS of TRPC3/6/7-/-mice decreased significantly compared with WT mice.TUNEL staining method illustrated that the myocardial apoptosis rate of TRPC3/6/7-/-mice decreased significantly compared with WT mice after I/R.To further explore the cardiomyocytes SOCE characteristics of TRPC3/6/7-/-knockout mice,calcium imaging technology was adopted to detect SOCE of neonatal cardiomyocytes of TRPC3/6/7-/-and WT mice,calcium imaging results indicated that the CPA-induced SOCE was significantly smaller than that of WT neonatal cardiomyocytes.To further explore the molecular mechanisms of reducing infarct size and apoptosis rate while TRPC3/6/7 knockout,we performed western blotting analysis in the myocardium after I/R,western blotting analysis showed that,the protein expression of CaNa,CaN?,NFATc3,BAX,BCL2,Cleaved Caspase 3,reduced significantly compared with WT mice,while the phosphorylation level of p-AKT,p-ERK1/2 were significantly increased.All these results suggest that TRPC3/6 knock out ameliorate MIRI.The results of this study showed that,TRPC3/6 increased calcium influx through the TRPC3/6-Ca2+-Calcineurin-NFAT positive feedback loop during I/R process,resulted in the increased expression levels of TRPC3/6,increased calcium influx,the aggravated calcium overload of myocardial endoplasmic reticulum and mitochondrial.TRPC3/6/7 knockout couldreduce myocardial intracellular calcium overload by down-regulating the activity of Ca2+-Calcineurin-NFAT pathway,increase p-AKT,p-ERK1/2 expression level,which could decrease myocardial infarct size and reduce the myocardial apoptosis rate.Therefore,the present study provides a new experimental basis to reveal the pathogenesis mechanism of MIRI.