Study On The Mechanism Of L-type Calcium Channel Autoinhibition Function Weakening And Accelerating Cell Death During Myocardial Ischemia-reperfusion Injury

Objective:In the process of myocardial ischemia reperfusion（I/R）injury,many factors are involved in the final process of myocardial infarction,among which calcium overload is one of the important causes of cell death.At present,L-type voltage-dependent calcium channel（L-VDCC）involved in the formation of calcium overload during myocardial I/R has been widely recognized,but the internal mechanism of channel function change has not been studied clearly.Therefore,the purpose of this study is to confirm that myocardial I/R can lead to changes in L-VDCC activity,to explore whether the changes in L-VDCC activity caused by myocardial I/R are related to the weakening of autoinhibition function.Methods:1.Detection of isolated heart function in rats:30 SD rats were randomly divided into 3 groups,10 rats in each group,which were Ischemia 0min group（continuous perfusion for 100min）,Ischemia 30min group and Ischemia 60min group.The isolated rat hearts were suspended in Langendorff perfusion system to simulate ischemia-reperfusion.The cardiac status was monitored by BL-420 N biological signal acquisition and analysis system,and the cardiac contractility and surface electrocardiogram were recorded.2.Acute isolation of rat cardiomyocytes and grouping treatment:The cell suspension containing rat cardiomyocytes isolated by Langendorff isolated heart perfusion system was divided into the following three groups:Hypoxia 0h group（normal culture 24h）,Hypoxia 6h group,Hypoxia 12h group,each group was reoxygenated for 12h.Acutely isolated rat cardiomyocytes were treated with Co Cl₂ to construct an in vitro hypoxia-reoxygenation model.3.Detection indexes and methods of each group:The changes of I_Ca-L in rat cardiomyocytes were detected by patch clamp technique.The survival rate of cardiomyocytes was counted under the microscope;co-IP was used to detect the changes of Cav1.2 binding DCT,Ca M and the expression level of t-Cav1.2;the protein expression levels of p-Cav1.2,PKA,PKG,Ca MKII and Calpain-1 were detected by Western Blot.4.Statistical methods:SPSS 21.0 and Graph Pad 8.0 were used for statistical analysis and mapping.The experimental data were expressed as mean±SEM.For the measurement data that conformed to the normal distribution and homogeneity of variance,One-way ANOVA was used for multi-group comparison.Kruskal-Wallis test was used for non-normal distribution and homogeneity of variance.P<0.05 indicated that the difference was statistically significant.Results:1.With the prolongation of myocardial ischemia time,the cardiac contractility and myocardial electrical activity decreased gradually after reperfusion.The isolated heart function monitoring of rats showed that the cardiac contractility（g）and the peak value of R wave（m V）in the ischemic period and reperfusion period of Ischemia 0min group,Ischemia 30min group and Ischemia 60min group were significantly lower than those in the equilibrium period（P<0.01）,and the reperfusion period was significantly higher than that in the ischemic period（P<0.01）.Compared with the Ischemia 0 min group,the cardiac contractility and R wave peak during reperfusion in the Ischemia 30 min group and the Ischemia 60 min group were significantly decreased（P<0.05）,and the Ischemia 60 min group was more obvious（P<0.01）.2.Myocardial ischemia-reperfusion can promote the activation of L-VDCC in cardiomyocytes,resulting in increased ICa-L,and with the prolongation of hypoxia time,cell mortality gradually increased.Compared with Hypoxia 0h group,the peak value of I-V curve of L-VDCC in Hypoxia 6h group and Hypoxia 12h group increased gradually,and the peak value of Hypoxia 12h group increased more significantly（P<0.01）.At the same time,the steady-state activation curve gradually shifted to the left,the channel half activation voltage(V_1/2)gradually increased,and the Ka value of the steady-state activation curve did not change significantly in the Hypoxia 6h group（P>0.05）,but decreased significantly in the Hypoxia 12h group（P<0.01）.The results of cell counting under microscope showed that compared with Hypoxia 0h group,the survival rate of cardiomyocytes in Hypoxia 6h group and Hypoxia 12h group decreased significantly with the prolongation of ischemia time（P<0.01）.3.With the prolongation of hypoxia time,the expression of Calpain-1 decreased,and the amount of Cav1.2 binding DCT and Ca M decreased gradually.WB was used to detect the expression level of Calpain-1.It was found that compared with the Hypoxia 0h group,the expression of Calpain-1 in the Hypoxia 6h group and the Hypoxia 12h group decreased（P<0.05）,and the Hypoxia 12h group down-regulated more significantly（P<0.01）..At the same time,the results of Co-IP showed that the amount of Cav1.2 binding DCT and Ca M in Hypoxia 6h group and Hypoxia 12h group was significantly lower than that in Hypoxia 0h group（P<0.05）.There was no significant difference in the amount of Cav1.2 binding DCT and Ca M between Hypoxia 12h group and Hypoxia 6h group（P>0.05）;there was no significant change in the expression of t-Cav1.2 in each group（P>0.05）.4.There was no significant change in the expression of exogenous regulatory proteins during hypoxia-reoxygenation of cardiomyocytes.The results of WB showed that the expression levels of p-Cav1.2,PKA and PKG in Hypoxia 6h group and Hypoxia 12h group were not significantly different from those in Hypoxia 0h group（P>0.05）,and the expression level of Ca MKII was significantly increased（P<0.01）.There was no significant difference in the expression of each protein between Hypoxia 6h group and Hypoxia 12h group.Conclusion:Myocardial I/R injury can lead to the decrease of L-VDCC autoinhibition,the increase of channel current and Ca²⁺influx,resulting in calcium overload and promoting cardiomyocyte death.