Troponin Modulation in Heart Failure

描述（由申请人提供）：本文提出的实验继续我们对促进肥大和改变心肌肌丝结构和功能的信号通路的多重活性的研究。 我们的假设是，与肥大和衰竭相关的肌丝蛋白磷酸化的改变是限制人类收缩和舒张储备的重要因素。 在新的实验线，我们专注于新的信号通过效应的Rho亚家族的小G蛋白，包括Rho依赖性激酶（ROK）和p21激活的激酶（Pak 1）。 我们的目标是：目标一：目的：测定正常人（C）终末期心力衰竭（HF）和支持性HF（LVAD）患者肌钙蛋白（cTnT）复合物与含有未磷酸化或假磷酸化cTnT和cTnI的重组复合物交换前后肌丝中Ca 2+和力/ATP酶活性之间的稳态关系。 在pH和肌节长度变化的情况下进行测量。 目标二：通过比较肌丝蛋白（cTnI、cTnT、C蛋白、肌球蛋白轻链2）的磷酸化，确定C、H和LVAD心脏中cTn活性改变的机制。 目标3：确定RhoA和ROK活性改变对大鼠和小鼠心肌细胞功能（（Ca 2+）i和缩短）、肌丝张力成本和对Ca 2+的反应以及肌丝蛋白磷酸化位点和相对水平的急性影响。 目标4：确定Pak 1激活诱导肌丝蛋白去磷酸化和细胞功能改变的机制，以及该机制是否是抗肾上腺素能作用的重要机制。 我们的方法，这些目标，使用良好的表征样品的人心肌，转基因模型，以及腺病毒介导的转移的cDNA，以特异性激活的韩国和Pak 1途径。 方法包括测量完整肌细胞的缩短和Ca 2+，以及对照大鼠和小鼠的单个肌细胞和纤维束中的力、缩短和ATP酶速率，以及功能上重要的蛋白激酶A（PKA）和PKC肌丝位点缺陷的转基因模型和受磷蛋白。 使用磷酸特异性抗体、分析电泳和质谱分析翻译后修饰。 这些实验的数据为心力衰竭的机制和潜在的治疗方法提供了新的见解。

DESCRIPTION (provided by applicant): Experiments proposed here continue our investigation of the multiplex activities of signaling pathways that promote hypertrophy and alter the structure and function of cardiac myofilaments. Our hypothesis is that alterations in myofilament protein phosphorylation associated with hypertrophy and failure represent a significant factor limiting both contraction and relaxation reserve in humans. In new lines of experiments, we focus on novel signaling through effectors of the Rho subfamily of small G proteins including Rho dependent kinase (ROK) and p21-activated kinase (Pak 1). Our aims are: Aim #1 : To determine the steady state relation between Ca2+ and force/ATPase activity in myofilaments from normal (C) end-stage human failed hearts (HF), and supported HF (LVAD) before and after exchange of the troponin (cTn) complex with recombinant complex containing unphosphorylated or pseudo-phosphorylated cTnT and cTnl. Measurements are made with variations in pH and sarcomere length. Aim #2: To determine the mechanism for the altered cTn activity in C, H, and LVAD hearts by comparing phosphorylation of myofilament proteins (cTnl, cTnT, C-protein, myosin light chain 2). Aim #3: To determine acute effects of altered RhoA and ROK activity on rat and mouse cardiomyocyte function ((Ca2+)i and shortening), on myofilament tension cost and response to Ca2+, and on sites and relative levels of myofilament protein phosphorylation. Aim #4: To determine the mechanism by which activation of Pak 1 induces dephosphorylation of myofilament proteins and altered cellular function, and whether this mechanism is a significant mechanism in anti-adrenergic effects. We approach these aims using well-characterized samples of human myocardium, transgenic models, as well as adenoviral mediated transfer of cDNA to specifically activate the ROK and Pak 1 pathways. Methods include measurements of shortening and Ca2+ in intact myocytes and force, shortening, and ATPase rate in single myocytes and fiber bundles from control rats and mice and transgenic models deficient in functionally significant protein kinase A (PKA) and PKC myofilament sites, and phospholamban. Post-translational modifications are analyzed using phosphospecific antibodies, analytical electrophoresis, and mass spectrometry. Data from these experiments provide novel insights into the mechanisms of heart failure and potential therapies.