Post-translational modification of the thin filament leads to progressive pump dysfunction

摘要 心脏病仍然是美国的主要死亡原因。病人和动物 心力衰竭模型持续出现蛋白激酶C活性升高及其下游 肌丝分子开关蛋白肌钙蛋白I(CTnI)在Ser43/45处的磷酸化 (S43/45)。先前，我们发现收缩功能障碍与cTnI的慢性磷酸化有关。 聚集，并损害收缩和松弛在分离的心肌细胞。工作假说 指导这一应用的是cTnI p-S43/45是用于稳态的短期调制的主制动器 功能，但慢性它会导致心脏功能障碍，并作为肌节的非传统作用 压力信号。这种类型的肌节应激与线粒体沟通以激活活性氧。 在显著的收缩功能障碍之前，物种产生和启动线粒体重塑。这 沟通为心脏功能不全的螺旋式下降奠定了基础 导致心力衰竭的重构的目标是证明cTnIS43/45作为一种快速的 活体主刹车，在慢性条件下会引起剂量依赖性的收缩功能障碍，还 通过导致早期线粒体ROS的产生，改变能量和 改建。在这种方法中，带有一系列模拟磷酸化cTnIS43/45D或a的转基因小鼠 产生了新磷酸零cTnIS43/45N以实现剂量依赖的替代 内源性cTnI。在目标1中，体内和细胞的心脏结构和收缩功能是一体化的 通过对肌丝和钙信号的分析，深入了解这一簇所起的作用。这 方法将表明，剂量依赖的cTnIS43/45D替换在体内产生心脏和 心肌细胞功能障碍，导致后期重塑、进行性功能受损和心力衰竭。 这一目标的研究也将证明，在重大重塑之前，这一集群对 对β-肾上腺素能受体刺激的正性变力和趋性反应及体内替代 与cTnIS43/45N在功能上是保守的替代。初步研究表明氧化应激 成年cTnIS43/45D小鼠在检测到 严重的功能障碍。因此，目标2研究了cTnIS43/45D扮演非规范的 刺激下游线粒体活性氧(ROS)产生的作用，其次是 能量和重塑改变是引发进行性心脏损害的早期途径 性能。每个目标的研究还包括概念验证实验，以表明早期目标 CTnI和/或下游线粒体的作用可预防和/或减弱功能障碍的螺旋式下降 和重塑到心力衰竭。

ABSTRACT Heart disease remains the leading cause of death in the United States. Patients and animal models of heart failure consistently develop elevated protein kinase C activity and downstream phosphorylation of the myofilament molecular switch protein, cardiac troponin I (cTnI) at Ser43/45 (S43/45). Previously, we showed contractile dysfunction is linked to chronic phosphorylation of this cTnI cluster and it impairs both contraction and relaxation in isolated myocytes. The working hypothesis guiding this application is that cTnI p-S43/45 is a master brake for short-term modulation of steady state function, but chronically it causes cardiac dysfunction, and serves a non-traditional role as a sarcomere stress signal. This type of sarcomere stress communicates with mitochondria to activate reactive oxygen species production and initiate mitochondrial remodeling prior to significant contractile dysfunction. This communication lays the foundation for the progressive downward spiral of cardiac dysfunction and remodeling that leads to heart failure The objectives are to demonstrate cTnIS43/45 acts as a rapid in vivo master brake, causes dose-dependent contractile dysfunction under chronic conditions and also communicates sarcomere stress by causing early mitochondrial ROS production, altered energetics and remodeling. For the approach, transgenic mice with a range of phospho-mimetic cTnIS43/45D or a novel phospho-null cTnIS43/45N were generated to achieve dose-dependent replacement of endogenous cTnI. In Aim 1, in vivo and cellular cardiac structure and contractile function are integrated with analysis of myofilament and Ca2+ signaling to gain insight into the role played by this cluster. This approach will show that dose-dependent cTnIS43/45D replacement produces in vivo cardiac and myocyte dysfunction that leads to later remodeling, progressively impaired function and heart failure. Studies in this aim also will prove that prior the significant remodeling, this cluster acts as a brake on the positive inotropic and lusitropic response to β-adrenergic receptor stimulation, and in vivo replacement with cTnIS43/45N is a functionally conservative substitution. Preliminary studies show oxidative stress and downstream mitochondrial alterations develop in adult cTnIS43/45D mice prior to detection of significant dysfunction. Thus, Aim 2 examines the novel idea that cTnIS43/45D plays a non-canonical role to stimulate downstream mitochondrial reactive oxygen species (ROS) production, followed by altered energetics and remodeling as an early route for triggering progressively impaired cardiac performance. Studies in each aim also include proof-of-concept experiments to show that early targeting of cTnI and/or downstream mitochondria prevents and/or attenuates the downward spiral of dysfunction and remodeling to heart failure.