Molecular Mechanisms of Myofilament Dysfunction in Heart Failure

充血性心力衰竭(CHF)与心脏细胞功能异常有关。CHF中这种受抑制的功能背后的分子机制尚不清楚。在以前的工作中，我们已经证明，在CHF中，肌丝功能在最大力量产生能力、钙反应和跨桥循环动力学方面受到抑制。实验机械/生化数据表明，Dys调节的肌丝收缩蛋白磷酸化导致CHF的肌丝功能障碍，可能是通过改变肌球蛋白轻链(MLC)、肌球蛋白结合蛋白C(MyoBPC)的磷酸化，以及 肌钙蛋白-L(TnL)。然而，确切的结构-功能关系尚未确定。在这项持续支持的建议中，我们将在压力过载继发的豚鼠中采用公认的充血性心力衰竭模型。豚鼠模型可以在与人类的肌丝和EC偶联参数非常相似的模型中研究控制、代偿性肥厚和充血性心力衰竭。生化蛋白质组学分析将被用来确定CHF的靶向通路和蛋白质，并将使用 各种生物物理技术，从完整的电刺激分离的肌肉到单一的肌原纤维(目标1)。收缩蛋白的翻译后修饰和信号通路将通过腺病毒基因传递、激酶/磷酸酶治疗和通透性分离心肌中的重组蛋白收缩蛋白交换来操纵(目标1)。正如我们最近证明的，心脏的局部肌丝功能并不统一，这种分布在心力衰竭时显著改变。 在特定目标2中提出的实验将确定这些现象背后的信号通路和收缩蛋白翻译后修饰。最后，AIM 3中提出的实验将确定驱动钙瞬变和机械动态收缩蛋白力产生之间的动态和时间耦合；这些实验将在单个心肌纤维中进行。总体而言，我们的目标是确定CHF收缩蛋白功能障碍的机制。我们的研究将有助于开发新的治疗策略来对抗OHF患者。

Congestive heart failure (CHF) is associated with an abnormality in cardiac cell function. The molecular mechanisms that underlie this depressed function in CHF are unknown. In previous work we have shown that myofilament function is depressed in CHF in terms of depressed maximum force generating capacity, calcium responsiveness, and cross-bridge cycle kinetics. Experimental mechanical/biochemical data suggest that dys-regulated myofilament contractile protein phosphorylation causes myofilament dysfunction in CHF, possibly via altered phosphorylation of myosin light chain (MLC), myosin binding protein C (MyoBPC), and Troponin-l (Tnl). However, the precise structure-function relationship has not been determined. In this proposal for continued support we will employ a well-established model of CHF in the guinea-pig secondary to pressure overload. The guinea-pig model allows study of control, compensatory hypertrophy, and CHF in a model that closely resembles the myofilament and EC-coupling parameters as found in the human. Biochemical proteomics analysis will be used to identify pathways and proteins that are targeted in CHF and the impact of identified post-translational modifications on contractile function will be determined using a variety of biophysical techniques ranging from intact electrically stimulated isolated muscle to single myofibrils (aim 1). Contractile protein post-translational modifications and signal pathways will be manipulated via adenoviral gene delivery, kinase/phosphatase treatment and recombinant protein contractile protein exchange in permeabilized isolated myocardium (aim 1). As we recently demonstrated, regional myofilament function is not uniform in the heart and this distribution is significantly altered in heart failure. Experiments proposed in specific aim 2 will determine the signal pathways and contractile protein posttranslational modifications that underlie these phenomena. Finally, experiments proposed in aim3 will determine the dynamic and temporal coupling between the driving Ca2+ transient and the mechanical dynamic contractile protein force production; these experiments will be performed in single cardiac myofibrils. Overall, our aim is to determine the mechanisms that underlie contractile protein dysfunction in CHF. Our research will aid in the development of new therapeutic strargies to combat OHF in patients.