Troponin Modulation in Heart Failure

描述(申请人提供)：这里提出的实验继续我们对促进心肌肥大和改变心肌肌丝结构和功能的信号通路的复合活动的研究。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松弛储备的重要因素。在新的实验系列中，我们将重点放在通过小G蛋白Rho亚家族效应器的新信号，包括Rho依赖蛋白(ROK)和p21激活蛋白(PAK1)。我们的目标是：目的1：研究肌钙蛋白(CTn)与未磷酸化或假磷酸化的cTnT和cTn1重组复合体交换前后，正常(C)终末期人心衰(HF)和支持心衰(LVAD)肌丝中钙离子与FORCE/ATPase活性的稳态关系。测量是通过改变pH值和肌节长度来进行的。目的#2：通过比较肌丝蛋白(cTn1、cTnT、C蛋白、肌球蛋白轻链2)的磷酸化程度，探讨C、H和LVAD心脏cTn活性改变的机制。目的#3：测定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)的急性影响，对肌丝张力成本和对Ca~(2+)的反应，以及对肌丝蛋白磷酸化的部位和相对水平的影响。目的#4：探讨PAK1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。为了达到这些目的，我们使用了具有良好特性的人类心肌样本、转基因模型以及腺病毒介导的cDNA转移来特异性地激活ROK和PAK 1通路。方法包括测量正常大鼠和小鼠完整心肌细胞的缩短和钙离子，以及单个心肌细胞和纤维束的力量、缩短和ATPase比率，以及功能显著蛋白激酶A(PKA)和PKC肌丝位点缺失的转基因模型和磷蛋白。翻译后修饰使用磷酸特异性抗体、分析电泳法和质谱仪进行分析。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.