Blunting of the Myofilament Beta-Adrenergic Response in HCM: Structural-Dynamic Mechanisms

项目摘要： 肥厚性心肌病（HCM）是一种复杂的遗传性心脏疾病，影响约1/300 - 1/500 世界各地的个人。肥厚型心肌病患者常见的临床表现是左心室功能损害， 舒张功能障碍（diastolic dysfunction）β-肾上腺素能刺激是舒张性能的关键调节剂。 在β-肾上腺素能刺激过程中，蛋白激酶A（PKA）介导多种肌节蛋白的磷酸化。 靶点，包括肌钙蛋白I（cTnI）丝氨酸23/24（Ser 23/24）。这种磷酸化事件导致 在肌丝水平的松弛（或失活）速率的显著增加。以前的工作表明， 这一观察结果是由于钙从心脏细丝解离速率的增加。此外，本发明还 一些细细丝HCM突变已显示出对磷酸化 ATP酶测定和力-pCa测量中的Ser 23/24。虽然几个小组的广泛工作 研究了钙解离速率增加的结构基础，所有以前的研究，最好的 我们的知识，缺乏关键的细丝结合伙伴肌动蛋白和原肌球蛋白，关键组成部分， 松弛的变构调节。在这个建议中，我们将进行TR-FRET实验，以评估两者 cTnI的N-末端和cTnI的C-末端之间的分子内和分子间相互作用以及 在存在和不存在Ser 23/24磷酸化的情况下，cTnI的N末端和cTnC的位点II。的 实验设计将提供我们将在我们的原子细丝模型中使用的距离。然后我们将 使用停流荧光各向异性，以探测C-末端的动态行为的转变， 当Ser 23/24被磷酸化时，钙从心脏细丝解离，cTnI。我们假设 Ser 23/24的磷酸化将改变这些转换发生的速率， 可能被HCM致病突变改变。探讨观察到的舒张功能的程度 损伤（以及可能的终末HCM表型的严重程度）可能是突变特异性的，我们建议 研究cTnI残基R145处3个独立已知cTnI突变的分子效应。这 突变热点包括HCM连锁突变R145 G、R145 Q和限制性心肌病（RCM）。 突变R145 W。我们将从TR-FRET实验中获得的结构数据与钙离子解离的变化相结合 研究结构变化如何影响功能，以及舒张功能障碍是否在 Ser 23/24磷酸化的存在。我们将采用元自洽模拟来获得自由能 变化，并确定从这些突变和磷酸化发生的相互作用中的特定变化， 以及在两种钙状态（开和关）下。本研究中提出的体外计算机耦合方法 应用程序将提供Ser 23/24上发生的结构变化的原子水平分辨率 在WT状态和已知cTnI连接的HCM/RCM突变的背景下， 确定有针对性的疾病机制的目标。

Project Summary: Hypertrophic cardiomyopathy (HCM) is a complex genetic cardiac disorder that affects ~1/300 – 1/500 individuals worldwide. A common clinical manifestation of patients with HCM is an impairment in left ventricular relaxation (diastolic dysfunction). Beta-adrenergic stimulation is a key regulator of diastolic performance. During beta- adrenergic stimulation protein kinase A (PKA) mediates phosphorylation of a variety of sarcomeric targets, including cardiac troponin I (cTnI) at serine 23/24 (Ser23/24). This phosphorylation event results in a significant increase in relaxation (or de-activation) rates at the myofilament level. Previous work has shown that this observation is due to increases in calcium dissociation rate from the cardiac thin filament. Additionally, some thin filament HCM mutations have been shown to exhibit an impaired response to phosphorylation of Ser23/24 in ATPase assays and force-pCa measurements. While extensive work by several groups has investigated the structural basis for this increase in calcium dissociation rate, all previous studies, to the best of our knowledge, lack the key thin filament binding partners actin and tropomyosin, crucial components for allosteric regulation of relaxation. In this proposal we will perform TR-FRET experiments to assess both intramolecular and intermolecular interactions between the N-terminus of cTnI and C-terminus of cTnI and the N-terminus of cTnI and Site II of cTnC in the presence and absence of phosphorylation at Ser23/24. The experimental design will provide distances that we will employ in our atomistic thin filament model. We will then use stopped flow fluorescence anisotropy in order to probe transitions in dynamic behavior in the C-terminus of cTnI when Ser23/24 is phosphorylated as calcium dissociates from the cardiac thin filament. We hypothesize that phosphorylation of Ser23/24 will alter the rate at which these transitions occur and that these mechanisms may be altered by HCM causative mutations. To explore the possibility that the degree of observed diastolic impairment (and potentially the severity of the end HCM phenotype) may be mutation-specific, we propose to investigate the molecular effects of 3 independent known cTnI mutations at residue R145 in cTnI. This mutational hotspot includes HCM- linked mutations R145G, R145Q and the restrictive cardiomyopathy (RCM) mutation R145W. We will couple structural data from TR-FRET experiments to changes in calcium dissociation rate to investigate how structural changes impact function and if the diastolic dysfunction is additive in the presence of Ser23/24 phosphorylation. We will employ metadynamics simulations to obtain free energy changes and identify specific changes in interactions that occur from these mutations and phosphorylation as well as in the two calcium states (on and off). The in-vitro—in-silico coupled approaches proposed in this application will provide atomic level resolution of the structural changes that occur upon Ser23/24 phosphorylation in the WT state and in the context of known cTnI-linked HCM/RCM mutations, with a long-term goal of identifying targetable disease mechanisms.