Increased Connexin 43 expression in AMPK deficient cardiomyocytes in response to Doxorubicin treatment

Abstract

ContextDoxorubicin (DOX) is an anthracycline anticancer drug that can cause dose‐dependent cardiomyopathy and chronic heart failure. DOX cardiotoxicity has been attributed to excess production of ROS and genotoxicity that leads to cell death. AMPK is a kinase composed of α, β and γ subunits. AMPK has been proposed to modulate DOX‐induced cardiotoxicity, but the downstream mediators or effectors remain unclear. Connexin 43 (Cx43) is a gap junction protein abundantly expressed in the cardiomyocyte and is essential for heart function. Studies have suggested a role for Cx43 in DOX cardiotoxicity, but it remains unknown whether AMPK interacts with Cx43 to regulate cardiac response to DOX chemotherapy.ObjectiveTo determine the role of AMPK in the regulation of the Cx43 in response to DOX‐induced cardiotoxicity.MethodsH9c2 rat cardiomyocytes were seeded on 6‐well plates and cultured in DMEM with 6.5% fetal bovine serum. To investigate the role of AMPK, the cells were transfected with siRNAs targeting different AMPK isoforms using Lipofectamine RNAiMAX and Opti‐MEM I reduced serum medium. After 48 hours of transfection, half of the wells received 0.5 µM DOX treatment, and the other half received equal amounts of saline. Protein samples were collected 16 hours later using the Cell lysis buffer. Western blot analysis was performed to confirm the knockdown of AMPK isoforms and to determine the protein expression levels of Cx43.ResultsOur findings showed that siRNA transfections effectively reduced the protein levels of AMPK isoform a1 and a2, respectively, as indicated by the Western blot analysis. DOX treatment increased the expression levels of the Cx43 in cardiomyocytes. Also, knockdown (KD) of AMPKα1 isoform resulted in a similar increase in Cx43 expression levels as did DOX, but AMPKα2 KD did not significantly alter Cx43 levels, suggesting an AMPKa1 specific effect on Cx43 expression. Interestingly, AMPKα1 KD, together with DOX treatment, did not produce a synergistic effect to increase Cx43 expression levels further, suggesting the possibility that DOX might have elevated the Cx43 expression through its effect on AMPK.ConclusionOur results confirmed the previous studies showing that treatment of the cardiomyocytes with DOX increased the expression of Cx43. However, our investigations produced a novel finding that knocking down AMPKα1 also increased the Cx43 but did not enhance the effect of DOX on Cx43 expression, suggesting a role for AMPK in the pathway that regulates the Cx43 to mediate the DOX‐induced cardiotoxicity. Our investigations also showed an isoform‐specific effect of AMPK on Cx43 expression since only α1 but not α2 KD increased Cx43 levels. Whether the increase in Cx43 is due to increased gene expression or inhibited protein degradation remains to be determined. Further investigation is also warranted to elucidate the roles of other AMPK subunits or isoforms in regulating the Connexin 43 expression in the context of doxorubicin cardiotoxicity.