The Transcriptional Regulation Of L-Ca²⁺ Channel Mediated By Myocardin

Objective: The transcriptional regulation of L-~Ca(2+) channel mediated by Myocardin.Methods:1.Demonstrating whether the activation of Cav1.2 LTCC gene mediated by Myocardin would affect the Cardiomyocyte membrane ~Ca(2+) current:Primary cardiocytes were infected with LV –Myocardin lentiviral plasmid to over-express Myocardin or LV-sh-Myocardin lentiviral plasmid to interfer Myocardin or blank lentivirus plasmid PTK642 as control.48 h later,Cardiomyocyte membrane ~Ca(2+) current was detected by electrophysiological patch clamp technique.2.Proving that Myocardin can activate expression of the LTCC gene:Cardiomyocyte H9C2 was infected with LV-Myocardin lentiviral plasmid to over-express Myocardin.48 h later,we used RT-PCR and real-time PCR to detect the level of the LTCC mRNA;The protein expression level of LTCC was detected by Western blotting;The distribution of LTCC protein was detected by immune fluorescence.3.Proving that the interference of Myocardin can inhibit the expression of LTCC Cav 1.2 gene:Cardiomyocyte H9C2 was infected with LV-sh-Myocardin lentiviral plasmid to interfer Myocardin.48 h later,we used RT-PCR and real-time PCR to detect the level of LTCC m RNA;The protein expression level of LTCC was detected by Western blotting;The distribution of LTCC protein was detected by immune fluorescence.4.Proving that Myocardin can activate the transcription of the LTCC gene:Co-transfect Myocardin and Cav1.2 luciferase reporter plasmid into cultured 293-T cells,using luciferase assay to detect the promoter activity of Cav1.2.5.Demonstrating whether the activation of LTCC Cav1.2 gene mediated by Myocardin is dependent on its CarGbox:Using WT-Cav1.2-luc,mut-CarG box-Cav1.2-luc(mutant Car G box),cutdownCarG box-Cav1.2-luc(cutdown CarG box)plasmid to detect promoter activity of Cav1.2-Luc.We further confirm the binding site of Myocardin on Cav1.2 gene promoter by chromatin immunoprecipitation assay(ChIP).Results:1.Myocardin up-regulate the Cardiomyocyte membrane ~Ca(2+) current:Compared with the control,the Cardiomyocyte membrane ~Ca(2+) current was significantly enhanced after overexpression of the Myocardin,interference of endogenous Myocardin is opposite.2.Myocardin activates LTCC Cav1.2 gene transcription and expression:Over expression of Myocardin activate Cav1.2-luci promoter activity,mRNA and protein expression levels of LTCC gene were significantly enhanced;Immune fluorescence demonstrated that the expression of LTCC Cav1.2 protein is increased compare with the control,especially in the nucleus and the cytoplasm;while,interference of endogenous Myocardin is opposite.3.Myocardin activation of LTCC Cav1.2 gene transcription depends on its CarGbox on the promoter.Mutation of the CarGbox result in low activity of the promoter,and chromatin immunoprecipitation assay(ChIP)further confirmed the binding site of Myocardin on Cav1.2 gene promoter.Conclusions:Myocardin activates LTCC Cav1.2 gene transcription and expression by binding on the CarGbox of its promoter,which promote the transportion of calcium ion channel protein from nucleus to membrane,enhance the flow of ~Ca(2+),and up-regulate the Cardiomyocyte membrane ~Ca(2+) current.