Cardiomyopathy-Associated Gene 1-Sensitive PKC-Dependent Connexin 43 Expression and Phosphorylation in Left Ventricular Noncompaction Cardiomyopathy
Cellular Physiology and Biochemistry
Key Words Left ventricular noncompaction cardiomyopathy (LVNC) • Cardiomyopathy-associated gene 1 (CMYA1) • Connexin 43 (Cx43) • PKC signaling pathway Abstract Background/Aims: Cardiomyopathy-associated gene 1 (CMYA1) plays an important role in embryonic cardiac development, postnatal cardiac remodeling and myocardial injury repair. Abnormal CMYA1 expression may be involved in cardiac dysplasia and primary cardiomyopathy. Our study aims to establish the relationship between CMYA1 and Left
... MYA1 and Left ventricular noncompaction cardiomyopathy (LVNC) pathogenesis. Methods: We explored the effects of CMYA1 on connexins (Cx), which contribute to gap junction intercellular communication (GJIC), and the underlying signaling pathway in human normal tissues, LVNC myocardial tissues and HL1 cells by means of western blotting, RT-qPCR, immunohistochemistry, immunofluorescence, co-immunoprecipitation and scrape loading-dye transfer. Results: CMYA1 expression was inversely associated with Cx43 and Cx40 expression, as determined by gap junction PCR array analysis. An increased expression and disordered distribution of CMYA1 at the intercalated discs in LVNC myocardial tissue was also observed. CMYA1 and Cx43 are co-expressed and interact in myocardial cells. CMYA1 expression was positively correlated with p-Cx43 (S368) via the Protein kinase C (PKC) signaling pathway in myocardial tissue and HL1 cells. The diffusion distance of Lucifer Yellow in the HL1 cells in which CMYA1 was over-expressed or knocked down was significantly less or more than that of the control group, respectively. Conclusion: Abnormal CMYA1 expression affects the expression and phosphorylation of Cx43 through the PKC signaling pathway, which is involved in the regulation of GJIC. CMYA1 participates in the molecular mechanism of LVNC pathogenesis. Xie et al.: Effects of CMYA1 On Cx43 via the PKC Signaling Pathway to the ICDs and, consequently, the noncompaction phenotype  . ICDs are part of the cardiac muscle sarcolemma, as they contain gap junctions (GJs) and desmosomes  . GJs are important determinants of cardiac conduction, and evidence has recently emerged that altered distribution of these junctions and altered expression of their constituent connexins (Cx) may lead to abnormal coupling between cardiomyocytes, likely contributing to myocardial diseases  . Twenty-one connexins have been identified in humans. Cx43 is the major subtype of ventricular myocardium  . Disorganization of GJ distribution and down-regulation of Cx43 are typical features of myocardial remodeling and may play an important role in the development of arrhythmogenic substrates in human cardiomyopathies  . The purpose of this study is to clarify the effects of CMYA1 on the gap junction intercellular communication (GJIC) of cardiomyocytes and the underlying signaling pathway. Materials and Methods Cell culture and treatment HL1 cells were cultured in Dulbecco's Modified Eagle's Medium supplemented with 10% fetal bovine serum (GIBCO) at 37°C and 5% CO2 in a humidified incubator, and the medium was changed every two days. Cells were serum-starved for 24 h once they reached 70% confluency. They were then either infected with lentiviral particles or transfected with shRNA-CMYA1 plasmids. After treatment with the lentiviral particles or plasmids for 24, 48, or 72 h, the HL1 cells were harvested for the experiments (co-immunoprecipitation, western blotting, RT-qPCR, immunocytochemistry, and scrape loading-dye transfer). The HL1 cells were cultured in serum-free medium for 12 h before being exposed to the protein kinase C (PKC) inhibitor GF109203X or the PKC activator phorbol 12-myristate 13 acetate (PMA). Tissue samples Myocardial samples were taken from the LVs of 12 terminal cardiomyopathy patients exhibiting abnormal trabeculation morphology from our heart transplantation program. Non-cardiomyopathy Expression and localization of CMYA1, Cx43 and Cx40 To investigate the relationship between the CMYA1 protein and the identified connexins (Cx43 and Cx40), we performed immunocytochemistry, western blot analysis and RT-qPCR in the human cardiac tissues and HL1 cells. Cx43 and Cx40 were localized to the cytoplasm and membranes in the cardiac tissues and HL1 cells ( Fig. 2A and Fig. 3A ).