In vitro rescue study of a malignant familial hypertrophic cardiomyopathy phenotype by pseudo-phosphorylation of myosin regulatory light chain
In vitro rescue study of a malignant familial hypertrophic cardiomyopathy phenotype by pseudo-phosphorylation of myosin regulatory light chain
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DOI：
10.1016/j.abb.2013.12.011
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发表时间：
2014-06-15
影响因子：
3.9
通讯作者：
Szczesna-Cordary, Danuta
中科院分区：
文献类型：
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作者：
Muthu, Priya;Liang, Jingsheng;Szczesna-Cordary, Danuta
心肌肌球蛋白调节轻链（RLC）的假磷酸化从未被研究作为缓解由肌球蛋白RLC中的致病突变引起的心肌病表型的拯救方法。本研究的重点是天冬氨酸缬氨酸取代（D166 V）的肌球蛋白RLC显示与恶性表型的家族性肥厚型心肌病（FHC）。该突变也被证明会导致心脏中表达D166 V的转基因小鼠出现严重的功能异常。为了探索这种新的拯救策略，使用D166 V的假磷酸化来确定D166 V诱导的有害表型是否可以恢复到野生型（WT）RLC的水平。将RLC磷酸化位点的S15 D取代插入重组WT和D166 V突变体中以模拟组成性磷酸化的RLC蛋白。非磷酸化（S15 A）构建体用作对照。采用多方面的方法来确定假磷酸化对肌球蛋白产生力和运动的能力的影响。使用突变重建猪心肌制剂，我们显示了S15 D诱导的救援D166 V-肌球蛋白肌动蛋白的酶和结合特性。在S15 D-D166 V与D166 V重构肌球蛋白的体外运动性测定中注意到力产生能力的显著增加。在用磷酸化模拟重组RLC重建的猪乳头肌条中，观察到类似的假磷酸化诱导效应对D166 V引起的异常Ca 2+力敏感性的影响。这项研究的结果表明，一种新的体外拯救策略，可用于在体内改善恶性心肌病表型。我们第一次证明了假RLC磷酸化可以逆转大多数突变诱导的表型，突出了RLC磷酸化在对抗心脏疾病中的重要性。(C)2013 Elsevier Inc. All rights reserved.
Pseudo-phosphorylation of cardiac myosin regulatory light chain (RLC) has never been examined as a rescue method to alleviate a cardiomyopathy phenotype brought about by a disease causing mutation in the myosin RLC. This study focuses on the aspartic acid to valine substitution (D166V) in the myosin RLC shown to be associated with a malignant phenotype of familial hypertrophic cardiomyopathy (FHC). The mutation has also been demonstrated to cause severe functional abnormalities in transgenic mice expressing D166V in the heart. To explore this novel rescue strategy, pseudo-phosphorylation of D166V was used to determine whether the D166V-induced detrimental phenotype could be brought back to the level of wild-type (WT) RLC. The S15D substitution at the phosphorylation site of RLC was inserted into the recombinant WT and D166V mutant to mimic constitutively phosphorylated RLC proteins. Non-phosphorylatable (S15A) constructs were used as controls. A multi-faceted approach was taken to determine the effect of pseudo-phosphorylation on the ability of myosin to generate force and motion. Using mutant reconstituted porcine cardiac muscle preparations, we showed an S15D-induced rescue of both the enzymatic and binding properties of D166V-myosin to actin. A significant increase in force production capacity was noted in the in vitro motility assays for S15D-D166V vs. D166V reconstituted myosin. A similar pseudo-phosphorylation induced effect was observed on the D166V-elicited abnormal Ca2+ sensitivity of force in porcine papillary muscle strips reconstituted with phosphomimic recombinant RLCs. Results from this study demonstrate a novel in vitro rescue strategy that could be utilized in vivo to ameliorate a malignant cardiomyopathic phenotype. We show for the first time that pseudo-RLC phosphorylation can reverse the majority of the mutation-induced phenotypes highlighting the importance of RLC phosphorylation in combating cardiac disease. (C) 2013 Elsevier Inc. All rights reserved.