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