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。先前资助周期的实验表明，PKC的激活会使完整肌细胞中的细丝蛋白、心肌肌钙蛋白I （cTnl）磷酸化，这种磷酸化事件会加速松弛。PKC磷酸化纯化cTnl上的3个残基簇（例如Ser23/24， Ser43/45, Thr144）。我们的长期目标是确定这些cTnl磷酸化位点对完整肌细胞中pkc介导的松弛反应的贡献。在我们早期的工作中，Thr144加速了对PKC急性激活的松弛反应。更长时间的PKC激活使Ser23/24磷酸化，也加速了松弛。然而，Ser43/45磷酸化在肌细胞松弛中的作用是有争议的，其相对于其他Tnl磷酸化位点的松弛作用仍不清楚。在本提案的第一个目的中测试的假设是，Ser43/45磷酸化对肌细胞松弛有剂量依赖性的发散影响。低水平的Ser43/45磷酸化预计会加速弛豫，而更广泛的磷酸化预计会减缓弛豫。目的2旨在检查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种目的，以实现在特定磷酸化位点上用含有替代的修饰的cTnl替代一系列cTnl。这些取代包括不可磷酸化的Ala和带负电荷的Asp来模拟磷酸化。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修饰Tnl快速、特异性和高效的肌丝替代。这些研究将为cTnl磷酸化簇在激活PKC的松弛反应中的个体和综合作用提供新的见解。最后的目的是关注pkc依赖的cTnl磷酸化状态及其对心力衰竭发展过程中的功能的影响。检验的关键假设是pkc介导的cTnl磷酸化降低并有助于心力衰竭时的延迟松弛。这一假设将在压力过载大鼠模型和从衰竭心脏移植的人类心肌中得到验证。通过基因转移asp取代的cTnl恢复松弛的能力将在衰竭的肌细胞中进行检查。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.