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

当粗丝中的肌球蛋白II使用三磷酸腺苷为含放线的细丝的相对运动提供动力时，心肌就会收缩。考虑到大量的肌球蛋白马达，如果所有马达都是活跃的，心脏将迅速耗尽三磷酸腺苷。因此，为了节省能量，肌球蛋白在与肌动蛋白分离时可以进入超松弛(SRX)状态，肌动蛋白具有抑制的ATPase。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。肥厚型心肌病(HCM)是最常见的遗传性心脏病，被认为是由于SRX基因数量不足引起的心肌收缩功能亢进。我们将调查在宽松和活跃的条件下SRX是如何受到监管的。我们的方法是研究肌球蛋白和肌动蛋白在它们天然的肌原纤维环境中。这种高度有序的结构能够在钙和三磷酸腺苷存在的情况下收缩。在这个项目中，我们将建造一种装置，使用玻璃微针抓住肌原纤维的两端，这将使收缩力量能够被测量。独一无二的是，我们将把它与猪/人心肌和干细胞来源的心肌细胞的肌原纤维中的ATP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打开了巨大的可能性。到本项目结束时，我们将全面了解肌球蛋白SRX状态在正常组织和疾病组织中的作用和调节。

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.