Protein kinase C (PKC) plays an important role in modulating cardiac contractile function. Experiments in the previous funding cycle showed activation of PKC phosphorylates the thin filament protein, cardiac troponin I (cTnl) in intact myocytes, and this phosphorylation event accelerated relaxation. PKC phosphorylates 3 clusters of residues on purified cTnl (e.g. Ser23/24, Ser43/45, Thr144). Our long term goal is to determine the contribution of these cTnl phosphorylation sites to the PKC-mediated relaxation response in intact myocytes. In our earlier work, Thr144 accelerated relaxation in response to acute PKC activation. More prolonged PKC activation phosphorylated Ser23/24, which also accelerated relaxation. However, the role of Ser43/45 phosphorylation in myocyte relaxation is controversial, and its contribution to relaxation relative to the other Tnl phosphorylation sites remains unclear. The hypothesis tested in the first aim of this proposal is that there is a dose-dependent divergent influence of Ser43/45 phosphorylation on myocyte relaxation. Low level Ser43/45 phosphorylation is expected to accelerate relaxation, while more extensive phosphorylation is predicted to slow relaxation.
Aim 2 is designed to examine the relative contribution and/or functional hierarchy of the 3 phosphorylation clusters on relaxation. Viral-based gene transfer will be used in both aims to achieve a range of cTnl replacement with modified cTnl containing substitutions in specific phosphorylation site(s). These substitutions include non-phosphorylatable Ala and negatively charged Asp to mimic phosphorylation. Rapid, specific and efficient myofilament replacement with modified Tnl is achieved in fully differentiated adult rat myocytes using this powerful approach. These studies will provide new insights into the individual and integrated role of the cTnl phosphorylation clusters in the relaxation response to activated PKC.
The final aim focuses on the state of PKC-dependent cTnl phosphorylation and its influence on function during the development of heart failure. The key hypothesis tested is that PKCmediated cTnl phosphorylation is decreased and contributes to delayed relaxation during heart failure. This hypothesis will be tested in a pressure-overload rat model, and in explanted human myocardium from failing hearts. The ability to restore relaxation by gene transfer of Asp-substituted cTnl will be examined in failing myocytes. These studies could aid in the development of therapies to improve relaxation during heart failure.
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