Peroxynitrite increases protein phosphatase activity and promotes the interaction of phospholamban with protein phosphatase 2a in the myocardium

Abstract

High levels of peroxynitrite have been shown to decrease cardiomyocyte contraction through a reduction in phospholamban (PLB) phosphorylation. However, previous reports did not examine the direct effect of peroxynitrite on protein phosphatase activity in the myocardium or the role of specific phosphatases. Here we test the effect of the peroxynitrite donor SIN-1 on protein phosphatase activity in whole heart homogenates, as well as the interaction of PLB with protein phosphatase 1 (PP1) and 2a (PP2a). SIN-1 (200 μmol/L) induced a significant increase in protein phosphatase activity, which was alleviated with the specific PP1/PP2a inhibitor okadaic acid. Conversely, lower concentrations of SIN-1 and the nitric oxide donor spermine NONOate (300 μmol/L) were both without effect on phosphatase activity. We next examined the effect of SIN-1 on the interaction of PLB with PP1 and PP2a using co-immunoprecipitation, since okadaic acid inhibited the effects of SIN-1 in our current and previous studies. SIN-1 significantly increased the interaction of PLB with PP2a, but had no effect on the interaction between PLB and PP1. Urate, a peroxynitrite scavenger, inhibited the effects of SIN-1 on phosphatase activity and the interaction of PLB with PP2a, thus implicating peroxynitrite as the causal species. The results of this study provide further insight into the mechanism through which high levels of peroxynitrite serve to decrease PLB phosphorylation and myocardial contraction. Therefore, peroxynitrite signaling could play a key role in the contractile dysfunction manifested in heart failure where peroxynitrite production and protein phosphatase activity are increased and PLB phosphorylation is decreased.