BACKGROUND Impaired left ventricular relaxation, high filling pressures, and dysregulation of Ca2+ homeostasis are common findings contributing to diastolic dysfunction in hypertrophic cardiomyopathy (HCM). Studies have shown that impaired relaxation is an early observation in the sarcomere-gene-positive preclinical HCM cohort, which suggests the potential involvement of myofilament regulators in relaxation. A molecular-level understanding of mechanism(s) at the level of the myofilament is lacking. We hypothesized that mutation-specific, allosterically mediated, changes to the cTnC (cardiac troponin C)-cTnI (cardiac troponin I) interface can account for the development of early-onset diastolic dysfunction via decreased PKA accessibility to cTnI. METHODS HCM mutations R92L-cTnT (cardiac troponin T; Arg92Leu) and Δ160E-cTnT (Glu160 deletion) were studied in vivo, in vitro, and in silico via 2-dimensional echocardiography, Western blotting, ex vivo hemodynamics, stopped-flow kinetics, time-resolved fluorescence resonance energy transfer, and molecular dynamics simulations. RESULTS The HCM-causative mutations R92L-cTnT and Δ160E-cTnT result in different time-of-onset diastolic dysfunction. R92L-cTnT demonstrated early-onset diastolic dysfunction accompanied by a localized decrease in phosphorylation of cTnI. Constitutive phosphorylation of cTnI (cTnI-D23D24) was sufficient to recover diastolic function to non-Tg levels only for R92L-cTnT. Mutation-specific changes in Ca2+ dissociation rates associated with R92L-cTnT reconstituted with cTnI-D23D24 led us to investigate potential involvement of structural changes in the cTnC-cTnI interface as an explanation for these observations. We probed the interface via time-resolved fluorescence resonance energy transfer revealing a repositioning of the N-terminus of cTnI, closer to cTnC, and concomitant decreases in distance distributions at sites flanking the PKA consensus sequence. Implementing time-resolved fluorescence resonance energy transfer distances as constraints into our atomistic model identified additional electrostatic interactions at the consensus sequence. CONCLUSIONS These data show that the early diastolic dysfunction observed in a subset of HCM is attributable to allosterically mediated structural changes at the cTnC-cTnI interface that impair accessibility of PKA, thereby blunting β-adrenergic responsiveness and identifying a potential molecular target for therapeutic intervention.

中文翻译：

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Arg92Leu-cTnT 改变 cTnC-cTnI 界面，破坏 PKA 介导的松弛。


背景 左心室舒张受损、充盈压高和 Ca2+ 稳态失调是导致肥厚型心肌病 （HCM） 舒张功能障碍的常见发现。研究表明，松弛受损是肌节基因阳性临床前 HCM 队列的早期观察结果，这表明肌丝调节剂可能参与松弛。缺乏对肌丝水平机制的分子水平理解。我们假设 cTnC （心肌肌钙蛋白 C）-cTnI （心肌肌钙蛋白 I） 界面的突变特异性、变构介导的变化可以解释通过降低 PKA 对 cTnI 的可及性而发生早发性舒张功能障碍的发展。方法 HCM 突变 R92L-cTnT （心肌肌钙蛋白 T;Arg92Leu） 和 Δ160E-cTnT （Glu160 缺失） 通过 2 维超声心动图、蛋白质印迹、离体血流动力学、停流动力学、时间分辨荧光共振能量转移和分子动力学模拟在体内、体外和计算机中进行了研究。结果 HCM 致病突变 R92L-cTnT 和 Δ160E-cTnT 导致不同时间的舒张功能障碍。R92L-cTnT 表现出早发性舒张功能障碍，伴有 cTnI 磷酸化的局部降低。cTnI （cTnI-D23D24） 的组成型磷酸化足以将舒张功能恢复到仅 R92L-cTnT 的非 Tg 水平。与 r92L-cTnT 重构相关的 Ca2+ 解离速率的突变特异性变化与 cTnI-D23D24 重构相关，这促使我们研究了 cTnC-cTnI 界面结构变化的潜在参与，以解释这些观察结果。 我们通过时间分辨荧光共振能量转移探测界面，揭示了 cTnI 的 N 末端重新定位，更接近 cTnC，并且伴随着 PKA 共有序列两侧位点的距离分布减少。将时间分辨荧光共振能量转移距离作为约束实施到我们的原子模型中，确定了共有序列中额外的静电相互作用。结论 这些数据表明，在 HCM 的一个子集中观察到的早期舒张功能障碍归因于变构介导的 cTnC-cTnI 界面结构变化，这些变化损害了 PKA 的可及性，从而减弱了 β-肾上腺素能反应性并确定治疗干预的潜在分子靶点。