Thin filament incorporation of an engineered cardiac troponin C variant (L48Q) enhances contractility in intact cardiomyocytes from healthy and infarcted hearts.
Thin filament incorporation of an engineered cardiac troponin C variant (L48Q) enhances contractility in intact cardiomyocytes from healthy and infarcted hearts.
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工程心肌肌钙蛋白 C 变体 (L48Q) 的细丝掺入可增强健康和梗塞心脏的完整心肌细胞的收缩性。
DOI：
10.1016/j.yjmcc.2014.03.015
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发表时间：
2014-07
影响因子：
5
通讯作者：
Regnier, Michael
中科院分区：
文献类型：
--
作者：
Feest, Erik R.;Korte, F. Steven;Tu, An-yue;Dai, Jin;Razumova, Maria V.;Murry, Charles E.;Regnier, Michael
关键词：
目前许多治疗收缩性心力衰竭的药物治疗都是以细胞内[Ca~(2+)]([Ca~(2+)]i)代谢或心肌肌钙蛋白C(CTNC)为靶点的，并且可能会有显著的副作用，包括心律失常或对舒张期功能的不利影响。在这项研究中，我们测试了直接增加cTNC的钙结合特性以增强正常和心肌梗死(MI)心脏完整心肌细胞不依赖[Ca2+]i收缩的可行性。具体地说，通过腺病毒介导的心肌肌钙蛋白C(CTNC)变异体的过度表达，增强了心脏细丝的激活，该变异体旨在增加单一氨基酸取代(L48Q)所产生的钙结合亲和力。在有皮肤的心脏小梁和肌原纤维中，我们和其他人已经表明，用L48Q cTNC替代天然cTNC可以增加力的钙敏感性和力的最大发展速度。在这里，我们通过腺病毒转导将L48Q cTNC导入完整心肌细胞的肌丝中，从而为突变型或野生型(WT)cTNC蛋白提供c DNA。使用视频显微镜监测细胞收缩、松弛和细胞内钙瞬变(Fura-2)，我们报告了掺入L48Q cTNC显著增加了健康和心肌梗死心肌细胞的收缩能力，而不会对钙瞬变特性或松弛产生不利影响。L48Q cTNC表达导致的收缩能力的改善可能是由于细胞内钙瞬变幅度不受影响而导致收缩效率提高的结果。通过对转导的心肌细胞的肌原纤维进行Western印迹分析，证实了L48Q cTNC在肌丝中的表达和掺入，表明L48Q cTNC取代了18±2%的天然cTNC。这些实验证明了直接靶向心脏细丝蛋白以增强心肌梗死后受损的心肌细胞收缩能力的可行性。
Many current pharmaceutical therapies for systolic heart failure target intracellular [Ca2+] ([Ca2+]i) metabolism, or cardiac troponin C (cTnC) on thin filaments, and can have significant side-effects, including arrhythmias or adverse effects on diastolic function. In this study, we tested the feasibility of directly increasing the Ca2+ binding properties of cTnC to enhance contraction independent of [Ca2+]i in intact cardiomyocytes from healthy and myocardial infarcted (MI) hearts. Specifically, cardiac thin filament activation was enhanced through adenovirus-mediated over-expression of a cardiac troponin C (cTnC) variant designed to have increased Ca2+ binding affinity conferred by single amino acid substitution (L48Q). In skinned cardiac trabeculae and myofibrils we and others have shown that substitution of L48Q cTnC for native cTnC increases Ca2+ sensitivity of force and the maximal rate of force development. Here we introduced L48Q cTnC into myofilaments of intact cardiomyocytes via adeno-viral transduction to deliver cDNA for the mutant or wild type (WT) cTnC protein. Using video-microscopy to monitor cell contraction, relaxation, and intracellular Ca2+ transients (Fura-2), we report that incorporation of L48Q cTnC significantly increased contractility of cardiomyocytes from healthy and MI hearts without adversely affecting Ca2+ transient properties or relaxation. The improvements in contractility from L48Q cTnC expression are likely the result of enhanced contractile efficiency, as intracellular Ca2+ transient amplitudes were not affected. Expression and incorporation of L48Q cTnC into myofilaments was confirmed by Western blot analysis of myofibrils from transduced cardiomyocytes, which indicated replacement of 18±2% of native cTnC with L48Q cTnC. These experiments demonstrate the feasibility of directly targeting cardiac thin filament proteins to enhance cardiomyocyte contractility that is impaired following MI.
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影响因子：
5
作者：
Kreutziger, Kareen L.;Piroddi, Nicoletta;Regnier, Michael
通讯作者：
Regnier, Michael
DOI：
10.1152/ajpregu.00630.2006
发表时间：
2007-03-01
影响因子：
2.8
作者：
de Tombe, Pieter P.;Belus, Alexandra;Poggesi, Corrado
通讯作者：
Poggesi, Corrado
影响因子：
2.9
作者：
Dong, Wen-Ji;Jayasundar, Jayant James;Cheung, Herbert C.
通讯作者：
Cheung, Herbert C.
DOI：
10.1073/pnas.0812536106
发表时间：
2009-08-18
影响因子：
11.1
作者：
Cazorla, Olivier;Lucas, Alexandre;Lezoualc'h, Frank
通讯作者：
Lezoualc'h, Frank
DOI：
10.1152/ajpheart.00520.2005
发表时间：
2005-11-01
影响因子：
4.8
作者：
Arteaga, GM;Warren, CM;Solaro, RJ
通讯作者：
Solaro, RJ