Influence of Troponin I Phosphorylation by PKC on Contractile Function

描述(申请人提供)：蛋白激酶C(PKC)在调节心脏收缩功能中起着重要作用。在前一个资金周期中的实验表明，在完整的心肌细胞中，PKC的激活磷酸化了细丝蛋白，即心肌肌钙蛋白I(CTnl)，而这种磷酸化事件加速了松弛。PKC在纯化的cTn1上磷酸化3个残基簇(例如Ser23/24、Ser43/45、Thr144)。我们的长期目标是确定这些cTn1磷酸化位点对完整心肌细胞中PKC介导的松弛反应的贡献。在我们早期的工作中，Thr144加速了对急性PKC激活的松弛。更长时间的PKC激活使Ser23/24磷酸化，这也加速了松弛。然而，Ser43/45磷酸化在心肌细胞松弛中的作用是有争议的，它相对于其他TNL磷酸化位点在松弛中的作用尚不清楚。在这项提议的第一个目的中检验的假设是，Ser43/45磷酸化对心肌细胞松弛存在剂量依赖性的不同影响。低水平的Ser43/45磷酸化预计会加速松弛，而更广泛的磷酸化预计会减缓松弛。AIM 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标，以实现一系列的cTn1替换与修饰的cTn1，其中包含特定磷酸化位点的替换(S)。这些取代包括非磷酸化的丙氨酸和带负电荷的天冬氨酸来模拟磷酸化。利用这一强大的方法，在完全分化的成年大鼠心肌细胞中，实现了用改良的TNL快速、特异和有效地替换肌丝。这些研究将为cTn1磷酸化簇在对激活的PKC的松弛反应中的单独和综合作用提供新的见解。最终目的是研究在心力衰竭发展过程中PKC依赖的cTn1磷酸化状态及其对功能的影响。验证的关键假设是，PKC介导的cTn1磷酸化减少，并在心力衰竭过程中导致延迟松弛。这一假设将在压力超负荷的大鼠模型和从衰竭的心脏移植的人类心肌中得到验证。通过基因转移天冬氨酸取代的cTn1恢复松弛的能力将在衰竭的心肌细胞中进行检测。这些研究可能有助于心力衰竭期间改善放松的治疗方法的开发。

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.