CaMKⅡ Regulating The Role Of TPM1 And MYOM2 In Diacetylmorphine-induced Arrhythmia

Objective:To investigate the roles of Ca²⁺/calmodulin-dependent protein kinaseⅡ（Ca MKⅡ）,tropomyosin1（TPM1）and Myomesin2（MYOM2）in diacetylmorphine-induced arrhythmias.Method:（1）Tandem mass spectrometry labeling of relative quantitative proteomics techniques（TMT）screening differentially expressed proteins,GO,KEGG function enrichment analysis of differentially expressed protein function.（2）Cell level:primary lactating mouse cardiomyocytes were cultured,and the survival rate of cardiomyocytes under different diacetylmorphine concentrations was detected by CCK-8.Primary cardiomyocytes were divided into:control group（Control group）,diacetylmorphine treatment group（Drug group）,diacetylmorphine+KN-93treatment group（Drug+KN-93 group）,diacetylmorphine+KN-92 treatment group（Drug+KN-92 group）.Fluorescence inverted microscopy to observe the rhythm of contraction frequency of primary cardiomyocytes;Flow cytometry to detect changes in primary cardiomyocytes[Ca²⁺]_i;Laser confocal microscopy to detect mitochondrial membrane potential changes in primary cardiomyocytes;Microplate reader to detect myocardial injury markers GOT and LDH activity;WB detected Ca MKⅡδ,p-Ca MKⅡ（T287）,TPM1,MYOM2 protein expression levels.（3）Animal level:a model of diacetylmorphine addiction in SD rats was established and treated with Ca MKⅡinhibitor KN-93,and ECG changes in SD rats were observed by BL-420 system;WB detected the expression levels of p-Ca MKⅡ（T287）and TPM1 and MYOM2 proteins in myocardial tissues.Result:（1）Proteomics results showed that a total of 784 differentially expressed proteins were screened in the Drug vs Con group and 346 differentially expressed proteins were screened in the Drug+KN-93 vs Drug group.The results of GO enrichment showed that the differentially expressed proteins in the Drug vs Con group participated in important biological processes such as myocardial tissue development and cardiac process,and participated in NADH dehydrogenase activity,oxidoreductase activity and other molecular functions,while the proteins of muscle segment,myofibril,and I band changed significantly.The enrichment results of KEGG pathway showed that the myocardial contraction pathway and calcium signaling pathway changed significantly.（2）Cell level:primary cardiomyocytes were treated with different diacetylmorphine concentrations,and with the increase of concentration,the survival rate of primary cardiomyocytes decreased significantly,and the difference was statistically significant（P<0.05）;Compared with the Control group,the average pulsation frequency of primary cardiomyocytes in the Drug group was significantly reduced（P<0.05）,LDH and GOT activities were significantly increased（P<0.05）,mitochondrial membrane potential was significantly reduced（P<0.05）,and cell[Ca²⁺]_iwas significantly increased（P<0.05）.Compared with the Drug group,the spontaneous pulsation frequency of primary cardiomyocytes in the Drug+KN-93 group was significantly increased（P<0.05）,the LDH and GOT activities were significantly reduced（P<0.05）,the mitochondrial membrane potential was significantly increased（P<0.05）,and the cell[Ca²⁺]_iwas significantly decreased（P<0.05）;Compared with the Drug group,there was no significant difference in spontaneous beating frequency,LDH,GOT activity,mitochondrial membrane potential,and cell[Ca²⁺]_iin the Drug+KN-92group,and compared with the Con group,the expression of p-Ca MKⅡ（T287）protein,TPM1 protein and MYOM2 protein in the Drug group was significantly increased（P<0.05）,and compared with the Drug group,the p-Ca MKⅡ（T287）expression in the Drug+KN-93 group was significantly increased（P<0.05）The expression of protein,TPM1 protein and MYOM2 protein was significantly reduced（P<0.05）,and there were no significant differences in the expression of p-Ca MKⅡ（T287）protein,TPM1 protein and MYOM2 protein in the Drug+KN-92 group compared with the Drug group（P>0.05）.（3）Animal level:the score results of withdrawal symptoms showed that the score of Drug group was significantly higher than that of Vehicle group（P<0.05）;ECG results showed that there was no abnormality in ECG in Vehicle group,while SD rats in Drug group had various types of arrhythmic ECG performance,and rats in DM+KN-93 group had arrhythmias,but the frequency was reduced compared with the Drug group（P<0.05）.The WB results showed that compared with the Vehicle group,the expression of p-Ca MKⅡ（T287）,TPM1 and MYOM2 proteins in the Drug group was increased（P<0.05）,and the expression of p-Ca MKⅡ（T287）,TPM1 and MYOM2 proteins in the Drug+KN-93group was lower than that in the Drug group（P<0.05）.Conclusion:Diacetylmorphine can cause abnormal changes in the rhythm of primary cardiomyocytes,which can cause arrhythmia in SD rats,which may be related to the upregulation of myocardial contraction-related proteins TPM1 protein and MYOM2 protein after Ca MKⅡactivation.