PDF(Pubmed)
Abstract：
Cardiac-specific myosin light chain kinase (cMLCK), encoded by MYLK3, regulates cardiac contractility through phosphorylation of ventricular myosin regulatory light chain. However, the pathophysiological and therapeutic implications of cMLCK in human heart failure remain unclear. We aimed to investigate whether cMLCK dysregulation causes cardiac dysfunction and whether the restoration of cMLCK could be a novel myotropic therapy for systolic heart failure.
We generated the knock-in mice (Mylk3+/fs and Mylk3fs/fs) with a familial dilated cardiomyopathy-associated MYLK3 frameshift mutation (MYLK3+/fs) that had been identified previously by us (c.1951-1G>T; p.P639Vfs*15) and the human induced pluripotent stem cell-derived cardiomyocytes from the carrier of the mutation. We also developed a new small-molecule activator of cMLCK (LEUO-1154).
Both mice (Mylk3+/fs and Mylk3fs/fs) showed reduced cMLCK expression due to nonsense-mediated messenger RNA decay, reduced MLC2v (ventricular myosin regulatory light chain) phosphorylation in the myocardium, and systolic dysfunction in a cMLCK dose-dependent manner. Consistent with this result, myocardium from the mutant mice showed an increased ratio of cardiac superrelaxation/disordered relaxation states that may contribute to impaired cardiac contractility. The phenotypes observed in the knock-in mice were rescued by cMLCK replenishment through the AAV9_MYLK3 vector. Human induced pluripotent stem cell-derived cardiomyocytes with MYLK3+/fs mutation reduced cMLCK expression by 50% and contractile dysfunction, accompanied by an increased superrelaxation/disordered relaxation ratio. CRISPR-mediated gene correction, or cMLCK replenishment by AAV9_MYLK3 vector, successfully recovered cMLCK expression, the superrelaxation/disordered relaxation ratio, and contractile dysfunction. LEUO-1154 increased human cMLCK activity ≈2-fold in the Vmax for ventricular myosin regulatory light chain phosphorylation without affecting the Km. LEUO-1154 treatment of human induced pluripotent stem cell-derived cardiomyocytes with MYLK3+/fs mutation restored the ventricular myosin regulatory light chain phosphorylation level and superrelaxation/disordered relaxation ratio and improved cardiac contractility without affecting calcium transients, indicating that the cMLCK activator acts as a myotrope. Finally, human myocardium from advanced heart failure with a wide variety of causes had a significantly lower MYLK3/PPP1R12B messenger RNA expression ratio than control hearts, suggesting an altered balance between myosin regulatory light chain kinase and phosphatase in the failing myocardium, irrespective of the causes.
cMLCK dysregulation contributes to the development of cardiac systolic dysfunction in humans. Our strategy to restore cMLCK activity could form the basis of a novel myotropic therapy for advanced systolic heart failure.
We generated the knock-in mice (Mylk3+/fs and Mylk3fs/fs) with a familial dilated cardiomyopathy-associated MYLK3 frameshift mutation (MYLK3+/fs) that had been identified previously by us (c.1951-1G>T; p.P639Vfs*15) and the human induced pluripotent stem cell-derived cardiomyocytes from the carrier of the mutation. We also developed a new small-molecule activator of cMLCK (LEUO-1154).
Both mice (Mylk3+/fs and Mylk3fs/fs) showed reduced cMLCK expression due to nonsense-mediated messenger RNA decay, reduced MLC2v (ventricular myosin regulatory light chain) phosphorylation in the myocardium, and systolic dysfunction in a cMLCK dose-dependent manner. Consistent with this result, myocardium from the mutant mice showed an increased ratio of cardiac superrelaxation/disordered relaxation states that may contribute to impaired cardiac contractility. The phenotypes observed in the knock-in mice were rescued by cMLCK replenishment through the AAV9_MYLK3 vector. Human induced pluripotent stem cell-derived cardiomyocytes with MYLK3+/fs mutation reduced cMLCK expression by 50% and contractile dysfunction, accompanied by an increased superrelaxation/disordered relaxation ratio. CRISPR-mediated gene correction, or cMLCK replenishment by AAV9_MYLK3 vector, successfully recovered cMLCK expression, the superrelaxation/disordered relaxation ratio, and contractile dysfunction. LEUO-1154 increased human cMLCK activity ≈2-fold in the Vmax for ventricular myosin regulatory light chain phosphorylation without affecting the Km. LEUO-1154 treatment of human induced pluripotent stem cell-derived cardiomyocytes with MYLK3+/fs mutation restored the ventricular myosin regulatory light chain phosphorylation level and superrelaxation/disordered relaxation ratio and improved cardiac contractility without affecting calcium transients, indicating that the cMLCK activator acts as a myotrope. Finally, human myocardium from advanced heart failure with a wide variety of causes had a significantly lower MYLK3/PPP1R12B messenger RNA expression ratio than control hearts, suggesting an altered balance between myosin regulatory light chain kinase and phosphatase in the failing myocardium, irrespective of the causes.
cMLCK dysregulation contributes to the development of cardiac systolic dysfunction in humans. Our strategy to restore cMLCK activity could form the basis of a novel myotropic therapy for advanced systolic heart failure.
摘要：
背景：心脏特异性肌球蛋白轻链激酶（cMLCK），由MYLK3编码，通过心室肌球蛋白调节轻链的磷酸化调节心脏收缩力。然而,cMLCK在人类心力衰竭中的病理生理和治疗意义尚不清楚.我们旨在研究cMLCK失调是否会导致心脏功能障碍，以及cMLCK的恢复是否可能是收缩性心力衰竭的新型肌力疗法。
方法：我们产生了具有家族性扩张型心肌病相关MYLK3移码突变（MYLK3+/fs）的敲入小鼠（Mylk3+/fs和Mylk3fs/fs），该突变已被我们先前鉴定（c.1951-1G>T;p.P639Vfs*15）和人类诱导的多能干细胞衍生突变。我们还开发了一种新的cMLCK小分子激活剂(LEUO-1154)。
结果：两只小鼠（Mylk3+/fs和Mylk3fs/fs）由于无义介导的信使RNA衰变而显示cMLCK表达降低，减少心肌中的MLC2v（心室肌球蛋白调节轻链）磷酸化，和cMLCK剂量依赖性方式的收缩功能障碍。与这个结果一致，来自突变小鼠的心肌显示心脏超松弛/无序松弛状态的比率增加，这可能导致心脏收缩力受损。通过通过AAV9_MYLK3载体补充cMLCK来挽救在敲入小鼠中观察到的表型。MYLK3+/fs诱导的多能干细胞衍生的心肌细胞cMLCK表达减少50%，收缩功能障碍，伴随着超松弛/无序松弛比的增加。CRISPR介导的基因校正，或通过AAV9_MYLK3载体补充cMLCK，成功恢复cMLCK表达，超弛豫/无序弛豫比，和收缩功能障碍。LEUO-1154在心室肌球蛋白调节轻链磷酸化的Vmax中增加人cMLCK活性约2倍，而不影响Km。LEUO-1154治疗人MYLK3+/fs诱导的多能干细胞衍生的心肌细胞恢复了心室肌球蛋白调节轻链磷酸化水平和超松弛/无序松弛率，并改善了心脏收缩力，而不影响钙瞬变，表明cMLCK激活剂充当肌胶。最后,多种原因导致的晚期心力衰竭的人心肌MYLK3/PPP1R12B信使RNA表达率明显低于对照心脏，提示肌球蛋白调节轻链激酶和磷酸酶之间的平衡在衰竭的心肌，不管原因。
结论：cMLCK失调有助于人类心脏收缩功能障碍的发展。我们恢复cMLCK活性的策略可以为晚期收缩性心力衰竭的新型肌力疗法奠定基础。
方法：我们产生了具有家族性扩张型心肌病相关MYLK3移码突变（MYLK3+/fs）的敲入小鼠（Mylk3+/fs和Mylk3fs/fs），该突变已被我们先前鉴定（c.1951-1G>T;p.P639Vfs*15）和人类诱导的多能干细胞衍生突变。我们还开发了一种新的cMLCK小分子激活剂(LEUO-1154)。
结果：两只小鼠（Mylk3+/fs和Mylk3fs/fs）由于无义介导的信使RNA衰变而显示cMLCK表达降低，减少心肌中的MLC2v（心室肌球蛋白调节轻链）磷酸化，和cMLCK剂量依赖性方式的收缩功能障碍。与这个结果一致，来自突变小鼠的心肌显示心脏超松弛/无序松弛状态的比率增加，这可能导致心脏收缩力受损。通过通过AAV9_MYLK3载体补充cMLCK来挽救在敲入小鼠中观察到的表型。MYLK3+/fs诱导的多能干细胞衍生的心肌细胞cMLCK表达减少50%，收缩功能障碍，伴随着超松弛/无序松弛比的增加。CRISPR介导的基因校正，或通过AAV9_MYLK3载体补充cMLCK，成功恢复cMLCK表达，超弛豫/无序弛豫比，和收缩功能障碍。LEUO-1154在心室肌球蛋白调节轻链磷酸化的Vmax中增加人cMLCK活性约2倍，而不影响Km。LEUO-1154治疗人MYLK3+/fs诱导的多能干细胞衍生的心肌细胞恢复了心室肌球蛋白调节轻链磷酸化水平和超松弛/无序松弛率，并改善了心脏收缩力，而不影响钙瞬变，表明cMLCK激活剂充当肌胶。最后,多种原因导致的晚期心力衰竭的人心肌MYLK3/PPP1R12B信使RNA表达率明显低于对照心脏，提示肌球蛋白调节轻链激酶和磷酸酶之间的平衡在衰竭的心肌，不管原因。
结论：cMLCK失调有助于人类心脏收缩功能障碍的发展。我们恢复cMLCK活性的策略可以为晚期收缩性心力衰竭的新型肌力疗法奠定基础。
