Influence of Troponin I Phosphorylation by PKC on Contractile Function

描述（由申请人提供）：蛋白激酶C（PKC）在调节心脏收缩功能中起重要作用。上一个资助周期的实验表明，PKC的激活使完整肌细胞中的细丝蛋白、心肌肌钙蛋白I（cTnI）磷酸化，并且这种磷酸化事件加速了松弛。PKC磷酸化纯化的cTnI上的3簇残基（例如，Ser 23/24、Ser 43/45、Thr 144）。我们的长期目标是确定这些cTnI磷酸化位点对完整肌细胞中PKC介导的舒张反应的贡献。在我们早期的工作中，Thr 144加速了对急性PKC激活的响应。更长时间的PKC激活磷酸化Ser 23/24，这也加速了松弛。然而，Ser 43/45磷酸化在肌细胞松弛中的作用是有争议的，并且其相对于其他Tnl磷酸化位点对松弛的贡献仍然不清楚。在该提议的第一个目的中测试的假设是，Ser 43/45磷酸化对肌细胞松弛具有剂量依赖性的发散影响。低水平的Ser 43/45磷酸化预期会加速松弛，而更广泛的磷酸化预期会减缓松弛。目的2旨在研究3个磷酸化簇对松弛的相对贡献和/或功能层次。基于病毒的基因转移将用于这两个目的，以实现用在特定磷酸化位点中含有取代的修饰的cTnI替代一系列cTnI。这些取代包括不可磷酸化的Ala和带负电荷的Asp以模拟磷酸化。使用这种强大的方法，在完全分化的成年大鼠肌细胞中实现了用修饰的TnI进行的快速、特异性和有效的肌丝替换。这些研究将为cTnI磷酸化簇在活化PKC的松弛反应中的个体和综合作用提供新的见解。最终的目标集中在PKC依赖性cTnl磷酸化的状态及其对心力衰竭发展过程中功能的影响。检验的关键假设是PKC介导的cTnl磷酸化降低，并导致心力衰竭期间舒张延迟。这一假设将在压力超负荷大鼠模型中进行测试，并在来自衰竭心脏的心肌中进行测试。将在衰竭的肌细胞中检查通过基因转移Asp-substituted cTnl恢复松弛的能力。这些研究可以帮助开发治疗方法，以改善心力衰竭期间的放松。

DESCRIPTION (provided by applicant): 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.