Regulation of the PP2AC, PP4C, PP6C and alpha4 signalling axis in the myocardium : roles in calcium homeostasis and hypertrophy

Cardiac physiology and hypertrophy are regulated by the phosphorylation status of most proteins, which is controlled by the opposing reactions of protein kinases and phosphatases (PP). The type 2A protein phosphatase family is comprised of PP2A, PP4 and PP6, due to the high amino acid homology of their catalytic subunits (PP2ACα/β, PP4C and PP6C). The activity and expression of this family are partly regulated by alpha4, a common regulatory protein that is essential in type 2A phosphatase holoenzyme biogenesis. In the heart, more than 98% of protein dephosphorylation is mediated by serine/ threonine protein phosphatases, of which type 2A protein phosphatases along with protein phosphatase 1, contr ibute approximately 90%. Currently, the role(s) of type 2A protein phosphatases and their regulation by alpha4 in the heart is poorly defined and requires detailed investigation. In this study, quantitative PCR analysi s demonstrated that PP2ACβ mRNA was most abundant in H9c2 cardiomyocytes and neonatal rat ventricular myocytes (NRVM) whilst, in adult rat ventricular myocytes (ARVM), PP2ACα mRNA was the most abundantly transcribed. Surprisingly, immunoblotting analysis, using catalytic subunit-specific antibodies, identified the expression of all type 2A protein phosphatase catalytic subunits in H9c2 cardiomyocytes and NRVM, however, ARVM only expressed PP2AC and PP6C protein. PP4C protein expression was only detectable in ARVM following proteasomal inhibition with compound MG132. Using siRNA to selectively knockdown type 2A protein phosphatase catalytic subunits, it was revealed that PP2ACα alone dephosphorylates CaV1.2-Ser1928. The data also suggested that PP2ACα, PP2ACβ and PP4C dephosphorylate phospholemman at both Ser63 and Ser68 in cardiomyocytes. siRNA-mediated knockdown of alpha4 protein expression rapidly reduced the expression of all type 2A catalytic subunits. Interestingly, expression of both PP2AC and alpha4 protein expression was elevated in pressure overload-induced left ventricular (LV) hypertrophy. Even though PP6C expression was unchanged, expression of PP6C regulatory subunits (i) SIT4-associated protein 1 (SAP1) and (ii) ankyrin repeat domain (ANKRD) 28 and 44 proteins were upregulated, whereas SAP2 expression was downregulated in hypertrophied LV tissue. Co-immunoprecipitation experiments revealed that the cellular association between alpha4 protein and PP2AC or PP6C subunits was either unchanged or reduced in hypertrophied LV tissue, respectively. Exposure of cardiomyocytes to hydrogen peroxide increased levels of H2AX phosphorylation (γH2AX), indicating hydrogen peroxide-induced DNA damage, which was unaffected by the knockdown of PP6C, however, levels of both total H2AX and γH2AX were diminished by the knockdown of alpha4 protein. The novel findings in this study collectively, demonstrate the differences in th e expression, stability, substrate specificity and altered alpha4-mediated regulation of the type 2A protein phosphatases in normal and hypertrophied myocardium and provide new insights into the molecular mechanisms involved in cardiac calcium homeostasis and DNA repair and thereby help to identify potential targets for the development of new and improved therapies against cardiac pathological hypertrophy.