Restrictive Cardiomyopathy Troponin I R145W Mutation Does Not Perturb Myofilament Length-dependent Activation in Human Cardiac Sarcomeres
Restrictive Cardiomyopathy Troponin I R145W Mutation Does Not Perturb Myofilament Length-dependent Activation in Human Cardiac Sarcomeres
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DOI：
10.1074/jbc.m116.746172
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发表时间：
2016-10-07
影响因子：
4.8
通讯作者：
de Tombe, Pieter P.
中科院分区：
文献类型：
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作者：
Dvornikov, Alexey V.;Smolin, Nikolai;de Tombe, Pieter P.
心肌肌钙蛋白 I (cTnI) R145W 突变与限制性心肌病 (RCM) 相关。最近的证据表明，这种突变在最大蛋白激酶 A (PKA) 刺激的条件下会诱导扰动肌丝长度依赖性激活 (LDA)。然而，一些引起心脏病的突变与 PKA 介导的磷酸化反应迟钝有关。这是否包括 LDA 尚不清楚。将分离的带皮人心肌中的内源肌钙蛋白交换为含有cTnI R145W、PKA/PKC拟磷酸电荷突变（S23D/S24D和T143E）或其各种组合的重组肌钙蛋白。测量肌丝 Ca2+ 力敏感性、张力消耗、LDA 和单个肌原纤维激活/松弛参数。我们的结果表明，R145W 和 T143E 均解耦了 S23D/S24D 拟磷剂对肌丝功能（包括 LDA）的影响。分子动力学模拟显示，在存在 cTnI RCM 突变或 cTnI PKC 磷酸模拟物的情况下，cTnC 的螺旋 C（残基 56、59 和 63）与 cTnI（残基 145）之间的相互作用显着减少。这些结果表明，RCM 相关的 cTnI R145W 突变诱导永久性结构状态，该状态与 PKC 介导的 cTnI Thr-143 磷酸化诱导的结构状态相似，但更广泛。我们认为，这种结构构象变化会导致肌丝 Ca2+ 敏感性增加，此外，还会解除由 PKA 在 Ser-23/Ser-24 靶位点介导的 cTnI 磷酸化的影响。然而，R145W RCM 突变本身不会影响 LDA。这些扰动的生物物理和生化肌丝特性可能会显着导致舒张期心泵功能障碍，这种情况在患有与 cTnI R145W 突变相关的限制性心肌病的患者中可见。
The cardiac troponin I (cTnI) R145W mutation is associated with restrictive cardiomyopathy (RCM). Recent evidence suggests that this mutation induces perturbed myofilament length-dependent activation (LDA) under conditions of maximal protein kinase A (PKA) stimulation. Some cardiac disease-causing mutations, however, have been associated with a blunted response to PKA-mediated phosphorylation; whether this includes LDA is unknown. Endogenous troponin was exchanged in isolated skinned human myocardium for recombinant troponin containing either cTnI R145W, PKA/PKC phosphomimetic charge mutations (S23D/S24D and T143E), or various combinations thereof. Myofilament Ca2+ sensitivity of force, tension cost, LDA, and single myofibril activation/relaxation parameters were measured. Our results show that both R145W and T143E uncouple the impact of S23D/S24D phosphomimetic on myofilament function, including LDA. Molecular dynamics simulations revealed a marked reduction in interactions between helix C of cTnC (residues 56, 59, and 63), and cTnI (residue 145) in the presence of either cTnI RCM mutation or cTnI PKC phosphomimetic. These results suggest that the RCM-associated cTnI R145W mutation induces a permanent structural state that is similar to, but more extensive than, that induced by PKC-mediated phosphorylation of cTnI Thr-143. We suggest that this structural conformational change induces an increase in myofilament Ca2+ sensitivity and, moreover, uncoupling from the impact of phosphorylation of cTnI mediated by PKA at the Ser-23/Ser-24 target sites. The R145W RCM mutation by itself, however, does not impact LDA. These perturbed biophysical and biochemical myofilament properties are likely to significantly contribute to the diastolic cardiac pump dysfunction that is seen in patients suffering from a restrictive cardiomyopathy that is associated with the cTnI R145W mutation.