Significance: In every heartbeat, cardiac muscle cells generate contractile force to pump blood into circulation against a mechanical load. Cardiomyocytes also sense load changes and adjust the contractility to maintain cardiac output. Excessive overload in pathological conditions leads to heart diseases such as arrhythmias and heart failure. However, fundamental knowledge gaps still exist in the molecular and cellular mechanisms of mechano-transduction in cardiomyocytes, and therapeutic treatments for mechanical stress associated heart diseases (e.g., hypertension induced arrhythmias and heart failure, DCM, HFpEF) are severely limited to date. Innovations: Previous experiments using load-free cardiomyocytes largely missed mechanical load effects on regulating cardiomyocytes. We will develop an innovative Cell-in-Gel-TR technology to control mechanical load at the single-cell level. Our studies reveal that the mechanical load on the cell during contraction can feedback to regulate the 3 dynamic systems in excitation-Ca2+ signaling-contraction (E-C) coupling; closing these feedback loops enables the cardiomyocyte to autoregulate E-C coupling in response to load changes. This conceptual innovation will be explored in our R35 research to understand how mechanical load affects cardiomyocyte function and heart diseases. Research Plan: The central theme of my research is to elucidate how the 3 dynamic systems in E-C coupling feedforward and feedback to control the heart function as a dynamically regulated smart pump. In R35, we will expand and deepen our research beyond the 2 R01s to do multi-scale systematic studies of the mechano-transduction mechanisms and functional consequences. (1) Molecular level study to decipher mechano-chemo-electro-transduction (MCET) pathways, identify the key players, and determine molecular mechanisms. (2) Cell level study to investigate how mechanical load regulates the dynamic systems of excitation-Ca2+ signaling-contraction coupling. (3) Heart level study to probe how mechanical load regulates the intact heart function. (4) Study of heart diseases to understand why/how pathological overload leads to cardiac remodeling, arrhythmias, and heart failure. These 4 parts are designed to inform and enhance one another to provide a comprehensive view on how mechano-transduction pathways work at molecular level, integrate at the cell level, and manifest to the heart’s ability to autoregulate contractility in response to mechanical load changes in health and diseases. Capability and Adaptability: The strength of my research stems from interdisciplinary approach. The history of my research shows a strong track record in developing new technologies by combining rigorous methods in physics, chemistry, and biology. In R35, I will continue developing innovative solutions and to use cutting-edge technologies to achieve the transformative research goals. Expected Outcome and Impact: The research outcome will shift the paradigm of cardia...