Lee Kong Chian School of Medicine, Nanyang Technological University, Experimental Medicine Building, 59 Nanyang Drive, Singapore 636921, Singapore.
Laboratory of Precision Disease Therapeutics, Genome Institute of Singapore, 60 Biopolis Street, Singapore 138672, Singapore.
Int J Mol Sci. 2021 Dec 22;23(1):88. doi: 10.3390/ijms23010088.
Heart failure (HF) as a result of myocardial infarction (MI) is a major cause of fatality worldwide. However, the cause of cardiac dysfunction succeeding MI has not been elucidated at a sarcomeric level. Thus, studying the alterations within the sarcomere is necessary to gain insights on the fundamental mechansims leading to HF and potentially uncover appropriate therapeutic targets. Since existing research portrays regulatory light chains (RLC) to be mediators of cardiac muscle contraction in both human and animal models, its role was further explored In this study, a detailed characterisation of the physiological changes (i.e., isometric force, calcium sensitivity and sarcomeric protein phosphorylation) was assessed in an MI mouse model, between 2D (2 days) and 28D post-MI, and the changes were related to the phosphorylation status of RLCs. MI mouse models were created via complete ligation of left anterior descending (LAD) coronary artery. Left ventricular (LV) papillary muscles were isolated and permeabilised for isometric force and Ca sensitivity measurement, while the LV myocardium was used to assay sarcomeric proteins' (RLC, troponin I (TnI) and myosin binding protein-C (MyBP-C)) phosphorylation levels and enzyme (myosin light chain kinase (MLCK), zipper interacting protein kinase (ZIPK) and myosin phosphatase target subunit 2 (MYPT2)) expression levels. Finally, the potential for improving the contractility of diseased cardiac papillary fibres via the enhancement of RLC phosphorylation levels was investigated by employing RLC exchange methods, in vitro. RLC phosphorylation and isometric force potentiation were enhanced in the compensatory phase and decreased in the decompensatory phase of HF failure progression, respectively. There was no significant time-lag between the changes in RLC phosphorylation and isometric force during HF progression, suggesting that changes in RLC phosphorylation immediately affect force generation. Additionally, the in vitro increase in RLC phosphorylation levels in 14D post-MI muscle segments (decompensatory stage) enhanced its force of isometric contraction, substantiating its potential in HF treatment. Longitudinal observation unveils potential mechanisms involving MyBP-C and key enzymes regulating RLC phosphorylation, such as MLCK and MYPT2 (subunit of MLCP), during HF progression. This study primarily demonstrates that RLC phosphorylation is a key sarcomeric protein modification modulating cardiac function. This substantiates the possibility of using RLCs and their associated enzymes to treat HF.
心肌梗死后心力衰竭（HF）是全球范围内导致死亡的主要原因。然而，导致 MI 后心脏功能障碍的原因在肌节水平上尚未阐明。因此，研究肌节内的变化对于了解导致 HF 的基本机制并可能发现适当的治疗靶点是必要的。由于现有研究表明调节轻链（RLC）在人类和动物模型中都是心肌收缩的调节剂，因此进一步探索了其在该研究中的作用。本研究详细描述了 MI 小鼠模型中 2D（2 天）和 28D 后心肌梗死后的生理变化（即等长力、钙敏感性和肌节蛋白磷酸化），并将变化与 RLC 的磷酸化状态相关联。通过完全结扎左前降支（LAD）冠状动脉来创建 MI 小鼠模型。分离和透化左心室（LV）乳头肌以进行等长力和 Ca 敏感性测量，而 LV 心肌用于测定肌节蛋白（RLC、肌钙蛋白 I（TnI）和肌球蛋白结合蛋白-C（MyBP-C））的磷酸化水平和酶（肌球蛋白轻链激酶（MLCK）、拉链相互作用蛋白激酶（ZIPK）和肌球蛋白磷酸酶靶亚基 2（MYPT2））的表达水平。最后，通过体外 RLC 交换方法，研究了通过增强 RLC 磷酸化水平来改善患病心脏乳头纤维收缩力的潜力。RLC 磷酸化和等长力增强在 HF 衰竭进展的代偿期增强，在失代偿期减弱。在 HF 进展过程中，RLC 磷酸化和等长力的变化之间没有明显的时间滞后，这表明 RLC 磷酸化的变化立即影响力的产生。此外，在 14D 后 MI 肌肉段（失代偿期）体外增加 RLC 磷酸化水平增强了其等长收缩力，证实了其在 HF 治疗中的潜力。纵向观察揭示了在 HF 进展过程中涉及 MyBP-C 和调节 RLC 磷酸化的关键酶（如 MLCK 和 MYPT2（MLCP 的亚基））的潜在机制。本研究主要表明 RLC 磷酸化是调节心脏功能的关键肌节蛋白修饰。这证实了使用 RLC 及其相关酶治疗 HF 的可能性。
