Decipher Mechano-Chemo-Transduction Pathway and Function in Cardiomyocytes

心脏将血液泵入血液循环，以抵抗全身血管阻力；心脏也可以感觉到 机械负荷改变和调节收缩能力以维持心输出量。心脏的负荷适应性 是弗兰克-斯塔林和安雷普在108年前描述的。目前的理解是Anrep 心肌细胞收缩时上调钙瞬变以增强收缩能力的作用 后负荷在病理性压力超负荷中占主导地位。然而，机械力-化学转导 (MCT)将机械负荷转换为生化信号以调节兴奋的机制-钙 心肌细胞中的信号-收缩(E-C)偶联仍未解决。新出现的证据表明， 负荷适应性存在于单个心肌细胞内。我们最近发现，收缩的心肌细胞 粘弹性水凝胶对后负荷代偿反应中钙瞬变和收缩的调节作用 变化，显示收缩的自动调节。但潜在的MCT机制尚未解决。 一条重要的线索来自我们的数学模型预测，观察到的自动调节行为 可由细胞表面机械传感器产生，该传感器可感知心肌细胞在 在三维粘弹性环境中的收缩，如在原位。这笔赠款的目标有两个：首先，我们将破译 细胞-表面机械传感器、抗肌营养不良蛋白-糖蛋白复合体(DGC)及其下游的MCT途径； 接下来，我们将确定MCT在调节心肌细胞E-C偶联中的功能影响。 机械载荷。我们的多学科团队将结合生物工程、电生理学、钙成像和 肌肉力学要发展创新技术，实现三个具体目标。 创新：我们将开发具有分子系链和应力报告功能的新型细胞凝胶(Cell-in-Gel-TS) 这项技术被用来控制心跳收缩期间心肌细胞的机械负荷，据报道 应力水平，以及锚定/非锚定细胞表面机械传感器，以探测MCT途径。 目的-1：破译从DGC机械传感器到化学转导再到E-C偶联分子的MCT途径。 目的-2：确定机械负荷如何调节兴奋-钙信号-收缩偶联系统。 目的-3：验证DGC突变破坏MCT通路导致E-C偶联失调的假说。 成功的结果将(A)将E-C耦合范式从当前的前馈模式转变为新的 MCT反馈自动调节模型，(B)为理解心脏的 对健康和疾病中机械负荷变化的内在适应性反应，以及(C)开发新的细胞- In-Gel-TS平台在单细胞和分子水平上控制后负荷，可广泛用于研究 后负荷(替代无负荷设置)下的心肌细胞模拟三维心肌的病理生理负荷。 1

The heart pumps blood into circulation against systemic vascular resistance; the heart can also sense mechanical load changes and regulate contractility to maintain cardiac output. The heart’s load adaptivity had been described by Frank-Starling and Anrep over 108 years ago. The current understanding is that the Anrep effect upregulates Ca2+ transient to enhance contractility when the cardiomyocyte is contracting under afterload, which is predominant in pathological pressure overload. However, the mechano-chemo-transduction (MCT) mechanism that transduces mechanical load to biochemical signals to regulate excitation-Ca2+ signaling-contraction (E-C) coupling in cardiomyocytes remains unresolved. Emerging evidence show that the load adaptivity resides within single cardiomyocytes. We recently found that the cardiomyocytes contracting in viscoelastic hydrogels can regulate the Ca2+ transient and contractility in compensatory response to afterload changes, showing autoregulation of contraction. But the underlying MCT mechanisms are yet to be resolved. An important clue comes from our mathematical model prediction that the observed autoregulatory behavior can arise from cell-surface mechanosensors that sense the 3D mechanical stress on cardiomyocytes during contraction in a 3D viscoelastic environment as in situ. The goals of this grant are 2-fold: first we will decipher the cell-surface mechanosensor, dystrophin-glycoprotein complex (DGC) and the downstream MCT pathway; next, we will determine the functional impact of MCT on regulating E-C coupling in the cardiomyocyte under mechanical load. Our multi-disciplinary team will combine bioengineering, electrophysiology, Ca2+ imaging, and muscle mechanics to develop innovative technology and achieve three specific aims. INNOVATION: We will develop new Cell-in-Gel with molecular-tether and stress-reporter (Cell-in-Gel-TS) technology, which is used to control mechanical load on myocytes during beat-to-beat contraction, report the stress level, and tether/untether cell surface mechanosensors to probe MCT pathways. Aim-1: Decipher the MCT pathway from DGC mechanosensor to chemotransducer to E-C coupling molecules. Aim-2: Determine how mechanical load regulates the excitation-Ca2+ signaling-contraction coupling systems. Aim-3: Test hypothesis that DGC mutations disrupt the MCT pathway to cause dysregulation of E-C coupling. Successful outcomes will (A) shift the E-C coupling paradigm from the current feed-forward model to a new MCT feedback autoregulatory model, (B) open a unifying conceptual framework for understanding the heart’s intrinsic adaptive response to mechanical load changes in health and diseases, and (C) develop the new Cell- in-Gel-TS platform to control afterload at single-cell and molecular levels, which can be widely used to study myocytes under afterload (replacing load-free setting) to mimic pathophysiological loading in 3D myocardium. 1