Troponin Modulation in Heart Failure

心力衰竭中的肌钙蛋白调节

• 批准号: 6826154
• 负责人: R John Solaro
• 金额: $ 34.88万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-01-15 至 2009-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6826154
• 关键词: adenosinetriphosphatase; calcium flux; clinical research; cytoskeletal proteins; enzyme activity; genetically modified animals; guanine nucleotide binding protein; heart failure; heart function; human subject; laboratory mouse; laboratory rat; microfilaments; molecular site; myocardium; myofibrils; myosins; pathologic process; patient oriented research; phosphorylation; posttranslational modifications; protein kinase; protein protein interaction; protein structure function; recombinant proteins; troponin; ventricular hypertrophy

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.

描述（由申请人提供）：此处提出的实验继续我们研究促进肥大并改变心脏肌膜的结构和功能的信号通路的多重活性。 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代表了人类的收缩和松弛储备的重要因素。 在新的实验线中，我们通过小型G蛋白的Rho亚家族的效应子（包括Rho依赖性激酶（ROK）（ROK）（ROK）和P21激活的激酶（PAK 1））重点关注新的信号传导（PAK 1）。 我们的目的是：目标1：确定正常（C）终末期人类失败的心脏（HF）的肌丝中Ca2+和力/ATPase活性之间的稳态关系，并在肌钙蛋白（CTN）（CTN）与重组复合物（CTN）复合物（COMBOLINANS复合物）复合物中含有无磷酸化或Pseudopsect and psseudolated Orpseudos-psseudy-phosphosphorysctn and conts and the Forsed HF（LVAD）之间的稳态关系。 测量是通过pH和肌节长度变化进行的。 AIM＃2：通过比较肌丝蛋白的磷酸化（CTNL，CTNT，CTNT，C蛋白，肌球蛋白轻链2），确定C，H和LVAD心脏中CTN活性改变的机制。 AIM＃3：确定RhoA和ROK活性改变对大鼠和小鼠心肌细胞功能（（Ca2+）I以及缩短）的急性影响，对Ca2+的肌膜张力成本和反应，以及肌膜蛋白蛋白磷酸化的位点和相对水平。 目标＃4：确定PAK 1激活诱导肌丝蛋白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。 我们使用人类心肌，转基因模型以及腺病毒介导的cDNA转移以特异性激活ROK和PAK 1途径的腺病毒介导的转移来实现这些目标。 方法包括在单个肌细胞中的完整肌细胞和力，缩短和ATPase速率中的缩短和Ca2+的测量，以及来自对照大鼠，小鼠，小鼠和转基因模型的纤维束，以及功能意义的蛋白质激酶A（PKA）（PKA）和PKC肌纤维丝部位和磷酸脂肪剂。 使用磷酸特异性抗体，分析电泳和质谱法分析翻译后修饰。 这些实验的数据为心力衰竭和潜在疗法的机制提供了新的见解。