Decipher Mechano-Chemo-Transduction Pathway and Function in Cardiomyocytes

心脏将血液泵入循环以对抗全身血管阻力;心脏还可以感知 机械负荷变化和调节收缩力以维持心输出量。心脏的负荷适应性 在108年前被弗兰克-斯塔林和安瑞普描述过。目前的理解是， 作用上调Ca 2+瞬变，以增强心肌细胞收缩时， 后负荷，这是占主导地位的病理压力超负荷。然而，机械-化学-转导 (MCT)将机械负荷转换为生化信号以调节兴奋性Ca 2+的机制 心肌细胞中的信号传导-收缩（E-C）偶联仍然没有解决。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 粘弹性水凝胶可调节Ca 2+瞬变和对后负荷的代偿性收缩反应 变化，显示收缩的自动调节。但MCT的潜在机制尚未得到解决。 一个重要的线索来自于我们的数学模型预测， 可以由细胞表面机械传感器产生，该传感器在心肌细胞的运动过程中感测心肌细胞上的3D机械应力。 在3D粘弹性环境中的收缩，如在原位。这项补助金的目标有两个方面：首先，我们将破译 细胞表面机械传感器、肌营养不良蛋白-糖蛋白复合物（DGC）和下游MCT通路; 接下来，我们将确定MCT对调节心肌细胞中E-C偶联的功能影响， 机械负荷我们的多学科团队将结合联合收割机生物工程，电生理学，钙离子成像， 肌肉力学旨在开发创新技术并实现三个具体目标。 创新：我们将开发新的Cell-in-Gel分子链和压力报告（Cell-in-Gel-TS） 技术，这是用来控制机械负荷的心肌细胞在心跳到心跳收缩，报告说， 应力水平，以及系留/解开细胞表面机械传感器以探测MCT途径。 目的一：解析MCT从DGC机械传感器到化学转换器再到E-C偶联分子的通路。 目的-2：确定机械负荷如何调节兴奋-Ca 2+信号-收缩耦合系统。 目的-3：检验假设DGC突变破坏MCT通路，导致E-C偶联失调。 成功的结果将（A）将E-C耦合范式从当前的前馈模型转变为新的前馈模型。 MCT反馈自动调节模型（B）为理解心脏的功能提供了一个统一的概念框架。 内在的适应性反应，以机械负荷的变化，在健康和疾病，和（三）发展新的细胞- 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 后负荷下的心肌细胞（替代无负荷设置），以模拟3D心肌中的病理生理负荷。 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