An industrial collaboration to develop new microscopy tools to image ATP usage in muscle

当粗丝中的肌凝蛋白II利用ATP为含有肌动蛋白的细丝的相对运动提供动力时，心肌收缩发生。考虑到大量的肌凝蛋白马达，如果所有的马达都是活跃的，心脏将迅速排出ATP。因此，为了节省能量，肌凝蛋白在与肌动蛋白分离时可以进入“超放松（SRX）”状态，肌动蛋白具有抑制atp酶。当需要时，例如在运动期间，这些SRX肌球蛋白可以被重新激活。肥厚性心肌病（HCM）是最常见的遗传性心脏病，被认为是由心肌收缩过度引起的，而心肌收缩过度可能是由SRX基因数量不足引起的。我们将研究SRX在放松和主动条件下是如何调节的。我们的方法是研究肌凝蛋白和肌动蛋白在其原生肌原纤维环境中的作用。这种高度有序的结构能够在钙和ATP存在的情况下收缩。在这个项目中，我们将构建一个装置，使用玻璃微针抓住肌原纤维的两端，这将使收缩力得以测量。独特的是，我们将把这与ATP在猪/人心肌肌原纤维和干细胞来源的心肌细胞中被翻转的单分子成像结合起来。这是以前从未实现过的，为理解肌肉如何受到钙、磷酸化、力、药物干预和疾病相关突变的影响开辟了巨大的可能性。在本项目结束时，我们将全面了解肌球蛋白SRX状态在正常和病变组织中的作用和调控。

Cardiac muscle contraction occurs when myosin II in the thick filament uses ATP to power the relative movement of the actincontaining thin filament. Given the vast numbers of myosin motors, if all were active, the heart would be rapidly drained of ATP. Therefore, to save energy, myosin can enter the 'super relaxed (SRX)' state when detached from actin, which has an inhibited ATPase. When needed, e.g., during exercise, these SRX myosins can be reactivated. Hypertrophic cardiomyopathy (HCM), the most common genetic heart disease, is thought to be due to hypercontractility, which may be caused by underpopulation of SRX. We will investigate how SRX is regulated in both relaxed and active conditions. Our approach is to study myosin and actin in their native myofibril environment. This highly ordered structure is capable of contracting in the presence of calcium and ATP. In this project, we will construct a device that uses glass microneedles to grab either end of a myofibril, this will enable the contractile force to be measured. Uniquely, we will combine this with single molecule imaging of ATP being turned over in the myofibrils ofporcine/human cardiac muscle, and stem-cell derived cardiomyocytes. This has not been achieved before and opens up a huge possibility for understanding how muscle is affected by calcium, phosphorylation, force, pharmacological interventions, and disease-associated mutations. By the end of this project, we will provide a comprehensive understanding of the role and regulation of myosin's SRX states in normal and diseased tissue.