Our preliminary data indicate ischemic hearts from rats have a transient decrease in protein phosphatase 2a (PP2a) activity, and an increase in phosphorylation of phospholamban, ERK and AKT. The decrease in PP2a activity occurs concomitant with demethylation of the catalytic subunit of PP2a (PP2aC). Additional decreases in PP2a activity occur with post-ischemic oxidation of PP2aC. Ventricular myocytes undergoing intracellular Ca2+ overload or simulated ischemia show similar reductions in methylation and activity of PP2aC. Further, cellular studies indicate the various targeting subunits of PP2a, such as PP2aB561, PP2aB551, PP2aB72 and Alpha-4, differentially locate the PP2a complex to specific myocardial substrates. Thus, these data and that of others led us to hypothesize that myocardial ischemia induces a change in methylation, oxidation, and/or phosphorylation of PP2aC to alter substrate targeting by the PP2aBs. This causes a decrease in localized PP2a activity at select myocardial targets. We believe this is of benefit to the heart. Support of a beneficial effect comes from our studies in which we show that inducing demethylation of PP2aC prior to ischemia leads to an improvement in post-ischemic function in isolated hearts. Further, when we used siRNA to decrease PP2aB561 expression this led to an increase in phosphorylation of phospholamban and a resistance to Ca2+ overload-induced hypercontracture in myocytes. Thus, we hypothesize ischemia-induced, select reductions in localized PP2a activity and resulting protein hyperphosphorylation is cardioprotective. We have 2 Aims to address our hypothesis.
In Aim 1 we will characterize post-translational modifications of PP2aC and PP2a targets over the course of ischemia-reperfusion (I/R), identify the degree to which these changes and specific PP2a subunit localizations improve function and viability, and test a clinically available therapy and a novel peptide for their ability to modify PP2a and improve post-ischemic recovery in isolated hearts.
In Aim 2 we will determine if demethylation or oxidation of PP2aC, and/or attenuating PP2aB561, PP2aB551, PP2aB72, or Alpha-4 - dependent translocations of PP2aC reduce PP2a activity at specific targets to delay onset of osmotic fragility, reduce Ca2+ overload and loss of contractility, or attenuate myofilament proteolysis induced by simulated I/R in ventricular myocytes. To complete these Aims we will use agents that selectively induce demethylation and/or oxidation of PP2aC, and siRNA to reduce expression of specific PP2a related subunits. The significance of the work is that it will determine the role of post-translational modifications of PP2aC and select PP2aB subunits have in modifying target protein phosphorylations and attendant functional changes to improve myocardial health. Thus, this work is foundational to a reasoned design of therapies which would selectively manipulate PP2a to improve clinical outcomes.
The proposed work is relevant to public health in that it addresses questions such as: Does a current clinical therapy, with an unrecognized """"""""side-effect"""""""" of modifying protein phosphatase 2a (PP2a), or our newly developed inhibitor of PP2a improve post-ischemic recovery of the heart? By experimentally mimicking the inherent PP2a-dependent protective mechanisms used by the heart, can we improve the short and long term outcomes from ischemic events in the heart? Which of the unique PP2aB targeting subunits is key to improving post-ischemic recovery in the heart, and by what mechanisms does this occur?
