Pathogenic properties of the N-terminal region of cardiac myosin binding protein-C in vitro.
Pathogenic properties of the N-terminal region of cardiac myosin binding protein-C in vitro.
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DOI：
10.1007/s10974-012-9292-y
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发表时间：
2012-05
影响因子：
2.7
通讯作者：
Sadayappan, Sakthivel
中科院分区：
文献类型：
--
作者：
Govindan, Suresh;Sarkey, Jason;Ji, Xiang;Sundaresan, Nagalingam R.;Gupta, Mahesh P.;de Tombe, Pieter P.;Sadayappan, Sakthivel
心肌肌球蛋白结合蛋白-C(cMyBP-C)在肌节的结构和稳定性以及调节心肌收缩方面起着重要作用。已证实150 kDa全长cMyBP-C在缺血再灌注损伤过程中发生蛋白水解性切割，产生一个N端40 kDa片段(质量29 kDa)，主要与缺血后收缩功能障碍有关。到目前为止，这种截短的cMyBP-C蛋白的致病特性还没有被阐明。在本研究中，我们假设，与110 kDa的C末端片段和FL cMyBP-C相比，这40 kDa片段的存在对心肌细胞是有毒的。为了验证这一假设，我们用表达cMyBP-C的FL、110和40 kDa片段的腺病毒感染新生大鼠和成年兔的心肌细胞，并测定了细胞毒性、钙瞬变、收缩和蛋白-蛋白相互作用。在此，我们发现新生大鼠心肌细胞表达40 kDa片段显著增加LDH释放和caspase3活性，显著降低细胞存活率，并损害对钙的处理。表达40 kDa片段的成年心肌细胞表现出类似的钙处理损伤，同时肌节长度缩短、松弛速度和收缩速度显著降低。重组蛋白的下拉分析表明，该40 kDa片段与肌节肌动蛋白显著结合，与C0-C2结构域相似。此外，我们在40 kDa片段中发现了几个可能影响肌动球蛋白功能的乙酰化位点。总之，我们的数据表明，cMyBP-C的40 kDa裂解片段对心肌细胞是有毒的，并通过抑制肌球蛋白功能而显著损害心肌细胞的收缩能力和钙处理能力。通过阐明内源性表达的cMyBP-C氨基末端片段对肌节功能的有害影响，这些数据有助于理解心肌损伤后收缩功能障碍。
Cardiac myosin binding protein-C (cMyBP-C) plays a role in sarcomeric structure and stability, as well as modulating heart muscle contraction. The 150 kDa full-length (FL) cMyBP-C has been shown to undergo proteolytic cleavage during ischemia–reperfusion injury, producing an N-terminal 40 kDa fragment (mass 29 kDa) that is predominantly associated with post-ischemic contractile dysfunction. Thus far, the pathogenic properties of such truncated cMyBP-C proteins have not been elucidated. In the present study, we hypothesized that the presence of these 40 kDa fragments is toxic to cardiomyocytes, compared to the 110 kDa C-terminal fragment and FL cMyBP-C. To test this hypothesis, we infected neonatal rat ventricular cardiomyocytes and adult rabbit ventricular cardiomyocytes with adenoviruses expressing the FL, 110 and 40 kDa fragments of cMyBP-C, and measured cytotoxicity, Ca2+ transients, contractility, and protein–protein interactions. Here we show that expression of 40 kDa fragments in neonatal rat ventricular cardiomyocytes significantly increases LDH release and caspase 3 activity, significantly reduces cell viability, and impairs Ca2+ handling. Adult cardiomyocytes expressing 40 kDa fragments exhibited similar impairment of Ca2+ handling along with a significant reduction of sarcomere length shortening, relaxation velocity, and contraction velocity. Pull-down assays using recombinant proteins showed that the 40 kDa fragment binds significantly to sarcomeric actin, comparable to C0–C2 domains. In addition, we discovered several acetylation sites within the 40 kDa fragment that could potentially affect actomyosin function. Altogether, our data demonstrate that the 40 kDa cleavage fragments of cMyBP-C are toxic to cardiomyocytes and significantly impair contractility and Ca2+ handling via inhibition of actomyosin function. By elucidating the deleterious effects of endogenously expressed cMyBP-C N-terminal fragments on sarcomere function, these data contribute to the understanding of contractile dysfunction following myocardial injury.
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