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Abstract：
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 Ca(2+) 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.
摘要：
从未研究过心脏肌球蛋白调节轻链（RLC）的伪磷酸化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挽救方法。这项研究的重点是肌球蛋白RLC中的天冬氨酸对缬氨酸的取代（D166V），这与家族性肥厚型心肌病（FHC）的恶性表型有关。该突变也已被证明在心脏中表达D166V的转基因小鼠中引起严重的功能异常。为了探索这种新颖的救援策略，D166V的假磷酸化用于确定D166V诱导的有害表型是否可以恢复到野生型（WT）RLC的水平。将RLC磷酸化位点处的S15D取代插入重组WT和D166V突变体中以模拟组成型磷酸化的RLC蛋白。非可磷酸化(S15A)构建体用作对照。采取了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。使用突变重组猪心肌制剂，我们显示了S15D诱导的D166V-肌球蛋白对肌动蛋白的酶促和结合特性的挽救。在S15D-D166V的体外运动试验中，注意到力生产能力显着增加。D166V重组肌球蛋白。在用磷性重组RLCs重建的猪乳头状肌条中，D166V引起的异常Ca（2）力敏感性观察到类似的假磷酸化诱导作用。这项研究的结果证明了一种新颖的体外抢救策略，可在体内用于改善恶性心肌病表型。我们首次表明，伪RLC磷酸化可以逆转大多数突变诱导的表型，突出了RLC磷酸化在对抗心脏病中的重要性。
