Single-molecule imaging reveals how mavacamten and PKA modulate ATP turnover in skeletal muscle myofibrils.
Single-molecule imaging reveals how mavacamten and PKA modulate ATP turnover in skeletal muscle myofibrils.
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DOI：
10.1085/jgp.202213087
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发表时间：
2023-01-02
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肌肉收缩的控制仍然不清楚。在这里，我们成像荧光标记的ATP与单分子精度，以揭示在不同的肌节位置的个别肌球蛋白的活动。我们展示了PKA和FDA批准的药物mavacamten治疗如何影响松弛的肌原纤维。肌肉收缩在两个层次上受到控制：细的和粗的肌丝。后一种控制水平涉及肌球蛋白头部的三种状态：活跃、无序松弛（DRX）和超松弛（SRX），其分布控制着可与肌动蛋白相互作用的肌球蛋白的数量。如何控制这些仍然是不确定的。使用荧光标记的ATP，我们能够在空间上分配肌节内的单个肌球蛋白的活性。我们观察到，SRX在C区的所有股骨头中占53%，而在P区和D区分别占35%和44%。最近FDA批准的肥厚型心肌病药物mavacamten（mava）显著降低了DRX，有利于C区和D区的SRX，分别为60%和63%。由于粗丝调节部分由肌球蛋白结合蛋白-C（MyBP-C）调节，我们还研究了PKA磷酸化。这与mava的效果相反，特别是在C区，它将SRX降低到34%，有利于DRX。这些结果直接表明，过量浓度的mava确实会增加SRX，但在肌节中的作用有限，表明mava对骨骼肌的作用较小。此外，我们表明PKA直接影响骨骼肌的收缩机制，导致被压抑的头部的解放。由于这种作用集中在C区，这表明它可能是通过MyBP-C磷酸化，尽管我们的数据表明PKA治疗无法获得的肌球蛋白的进一步储备。
Control of muscle contraction is still not understood. Here, we image fluorescently tagged ATP with single-molecule precision to reveal the activity of individual myosins in different sarcomeric locations. We show how treatment with PKA and the FDA-approved drug mavacamten affects relaxed myofibrils. Muscle contraction is controlled at two levels: the thin and the thick filaments. The latter level of control involves three states of myosin heads: active, disordered relaxed (DRX), and super-relaxed (SRX), the distribution of which controls the number of myosins available to interact with actin. How these are controlled is still uncertain. Using fluorescently labeled ATP, we were able to spatially assign the activity of individual myosins within the sarcomere. We observed that SRX comprises 53% of all heads in the C-zone compared with 35% and 44% in the P- and D-zones, respectively. The recently FDA-approved hypertrophic cardiomyopathy drug, mavacamten (mava), significantly decreased DRX, favoring SRX in both the C- and D-zones at 60% and 63%, respectively. Since thick filament regulation is in part regulated by the myosin-binding protein-C (MyBP-C), we also studied PKA phosphorylation. This had the opposite effect as mava, specifically in the C-zone where it decreased SRX to 34%, favoring DRX. These results directly show that excess concentrations of mava do increase SRX, but the effect is limited across the sarcomere, suggesting mava is less effective on skeletal muscle. In addition, we show that PKA directly affects the contractile machinery of skeletal muscle leading to the liberation of repressed heads. Since the effect is focused on the C-zone, this suggests it is likely through MyBP-C phosphorylation, although our data suggest that a further reserve of myosins remain that are not accessible to PKA treatment.