Heart muscle contractility and performance are controlled by posttranslational modifications of sarcomeric proteins. Although myosin regulatory light chain (RLC) phosphorylation has been extensively studied both in vitro and in vivo, the precise role of cardiac myosin light chain kinase (cMLCK), the primary kinase acting upon RLC, in the regulation of cardiomyocyte contractility remains poorly understood. In the current study, using recombinantly expressed and purified proteins, various analytical methods, in vitro and in situ kinase assays, and mechanical measurements in isolated ventricular trabeculae, we demonstrate that human cMLCK is not a dedicated kinase for RLC, but can phosphorylate other sarcomeric proteins with well-characterized regulatory functions. We show that cMLCK specifically mono-phosphorylates Ser-23 of human cardiac troponin I (cTnI) both in isolation and in the trimeric troponin complex in vitro and in situ in the native environment of the muscle myofilament lattice. Moreover, we observed that human cMLCK phosphorylates rodent cTnI to a much smaller extent both in vitro and in situ, suggesting a species-specific adaptation of cMLCK. Although cMLCK treatment of ventricular trabeculae exchanged with either rat or human troponin increased their cross-bridge kinetics, the increase in sensitivity of myofilaments to calcium was significantly blunted by human TnI, suggesting that human cTnI phosphorylation by cMLCK modifies the functional consequences of RLC phosphorylation. We propose that cMLCK-mediated phosphorylation of TnI is functionally significant and represents a critical signaling pathway that coordinates the regulatory states of thick and thin filaments in both physiological and potentially pathophysiological conditions of the heart.

中文翻译：

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心肌肌球蛋白调节轻链激酶通过磷酸化肌球蛋白调节轻链和肌钙蛋白 I 来调节心脏收缩性。

心肌收缩力和性能由肌节蛋白的翻译后修饰控制。尽管肌球蛋白调节轻链 (RLC) 磷酸化已在体外和体内进行了广泛研究，但心脏肌球蛋白轻链激酶 (cMLCK)（作用于 RLC 的主要激酶）在调节心肌细胞收缩性中的确切作用仍然知之甚少。在目前的研究中，使用重组表达和纯化的蛋白质、各种分析方法、体外和原位激酶测定以及分离的心室小梁的机械测量，我们证明人类 cMLCK 不是 RLC 的专用激酶，但可以磷酸化其他肌节具有良好表征的调节功能的蛋白质。我们表明，cMLCK 在体外和在肌肉肌丝晶格的天然环境中，在体外和原位的三聚体肌钙蛋白复合物中，都能特异性地单磷酸化人心肌肌钙蛋白 I (cTnI) 的 Ser-23。此外，我们观察到人类 cMLCK 在体外和原位都以更小的程度磷酸化啮齿动物 cTnI，这表明 cMLCK 具有物种特异性适应。尽管 cMLCK 处理与大鼠或人肌钙蛋白交换的心室小梁增加了它们的交叉桥动力学，但肌丝对钙的敏感性增加被人 TnI 显着减弱，这表明 cMLCK 对人 cTnI 磷酸化改变了 RLC 磷酸化的功能后果。