Regulation of cardiomyocyte DNA damage and cell death by the type 2A protein phosphatase regulatory protein alpha4

The type 2A protein phosphatase regulatory protein alpha4 (alpha 4) constitutes an anti-apoptotic protein in non-cardiac tissue, however it's anti-apoptotic properties in the heart are poorly defined. To this end, we knocked down alpha 4 protein expression (alpha 4 KD) using siRNA in cultured H9c2 cardiomyocytes and confirmed the lack of DNA damage/cell death by TUNEL staining and MTT assay. However, alpha 4 KD did increase the phosphorylation of p53 and ATM/ATR substrates, decreased the expression of poly ADP-ribose polymerase and associated fragments. Expression of anti-apoptotic proteins Bcl-2 and Bcl-xL was reduced, whereas expression of pro-apoptotic BAX protein did not change. Alpha4 KD reduced basal H2AX Ser139 phosphorylation, whereas adenoviral-mediated re-expression of alpha 4 protein following alpha 4 KD, restored basal H2AX phosphorylation at Ser139. The sensitivity of H9c2 cardiomyocytes to doxorubicin-induced DNA damage and cytotoxicity was augmented by alpha 4 KD. Adenoviral-mediated overexpression of alpha 4 protein in ARVM increased PP2AC expression and augmented H2AX Ser139 phosphorylation in response to doxorubicin. Furthermore, pressure overload-induced heart failure was associated with reduced alpha 4 protein expression, increased ATM/ATR protein kinase activity, increased H2AX expression and Ser139 phosphorylation. Hence, this study describes the significance of altered alpha 4 protein expression in the regulation of DNA damage, cardiomyocyte cell death and heart failure.