Functional characterization of the human α-cardiac actin mutations Y166C and M305L involved in hypertrophic cardiomyopathy
Functional characterization of the human α-cardiac actin mutations Y166C and M305L involved in hypertrophic cardiomyopathy
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DOI：
10.1007/s00018-012-1030-5
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发表时间：
2012-10-01
影响因子：
8
通讯作者：
Mannherz, Hans Georg
中科院分区：
文献类型：
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作者：
Mueller, Mirco;Mazur, Antonina Joanna;Mannherz, Hans Georg
遗传性心肌病是由肌节基因产物（包括 α-心肌肌动蛋白 (ACTC1)）的点突变引起的。我们检查了肥厚型心肌病 (HCM) 中发现的 α-心脏肌动蛋白突变 Y166C 和 M305L 的生化和细胞生物学特性。未标记的野生型 (WT) 心脏肌动蛋白以及 Y166C 和 M305L 突变体由杆状病毒/Sf9 细胞系统表达，并通过固定化凝溶胶蛋白 G4-6 进行亲和纯化。它们的正确折叠通过许多测定得到验证。在原肌球蛋白、凝溶胶蛋白和 Arp2/3 复合物存在的情况下，突变肌动蛋白还表现出扰乱的内在 ATP 酶活性和改变的聚合行为。两种突变体仅刺激心脏 β-肌球蛋白 ATP 酶达到 WT 心脏 F-肌动蛋白的 50%。 WT 的共聚物和突变肌动蛋白数量的增加导致肌球蛋白 ATP 酶的刺激减少。已建立的细胞系的转染显示，EGFP 和血凝素 (HA) 标记的 WT 以及两种突变肌动蛋白并入细胞质应激纤维中。 HA 标记的 WT 和 Y166C 肌动蛋白的腺病毒载体已成功用于感染成年和新生大鼠心肌细胞 (NRC)。表达的 HA 标记肌动蛋白被整合到 NRC 细丝的负端，证明了与内源性肌动蛋白形成混合细丝的能力。在 NRC 中，与感染 WT 肌动蛋白的 NRC 相比，Y166C 突变体在 72 小时后导致肌节长度缩短。因此，我们的数据表明突变肌动蛋白可以整合到心肌细胞细丝中，并且通过其减少的肌球蛋白相互作用模式可能是 HCM 启动的基础。
Inherited cardiomyopathies are caused by point mutations in sarcomeric gene products, including alpha-cardiac muscle actin (ACTC1). We examined the biochemical and cell biological properties of the alpha-cardiac actin mutations Y166C and M305L identified in hypertrophic cardiomyopathy (HCM). Untagged wild-type (WT) cardiac actin, and the Y166C and M305L mutants were expressed by the baculovirus/Sf9-cell system and affinity purified by immobilized gelsolin G4-6. Their correct folding was verified by a number of assays. The mutant actins also displayed a disturbed intrinsic ATPase activity and an altered polymerization behavior in the presence of tropomyosin, gelsolin, and Arp2/3 complex. Both mutants stimulated the cardiac beta-myosin ATPase to only 50 % of WT cardiac F-actin. Copolymers of WT and increasing amounts of the mutant actins led to a reduced stimulation of the myosin ATPase. Transfection of established cell lines revealed incorporation of EGFP- and hemagglutinin (HA)-tagged WT and both mutant actins into cytoplasmic stress fibers. Adenoviral vectors of HA-tagged WT and Y166C actin were successfully used to infect adult and neonatal rat cardiomyocytes (NRCs). The expressed HA-tagged actins were incorporated into the minus-ends of NRC thin filaments, demonstrating the ability to form hybrid thin filaments with endogenous actin. In NRCs, the Y166C mutant led after 72 h to a shortening of the sarcomere length when compared to NRCs infected with WT actin. Thus our data demonstrate that a mutant actin can be integrated into cardiomyocyte thin filaments and by its reduced mode of myosin interaction might be the basis for the initiation of HCM.