ABSTRACT. Aim 1 of this project is built around years of collaborative work between Drs. Regnier and Murry studying human stem cell derived cardiomyocytes as a potential cell replacement strategy for cardiac repair following myocardial infarction (MI). We have shown that human stem cells can be differentiated into cardiomyocytes (CMs), produced at a scale and purity that permit testing in rodent models and non-human primates (NHP; Macaca nemestrina) and that these cells engraft and integrate with host tissue to improve left ventricular performance. The premise for the proposed experiments is based on two fundamental discoveries: 1) 2-deoxy ATP (dATP) is a potent natural nucleotide stimulant of contractility when used by cardiac myosin, and 2) hiPSC-CMs that overexpress the rate-limiting enzyme for dATP synthesis, ribonucleotide reductase (RNR), have increased contractility and also deliver dATP to the native myocardium heart via gap junctions. In our current award we made excellent progress in testing the hypothesis that engineered hiPSC-CMs with elevated RNR (hiPSC-CMRNR) improve outcomes in cell replacement therapy for MI (compared with control hiPSC-CMs). For this proposal, we have generated new hiPSC-CM lines with gene-edited RNR and different transcriptional promotors. These cells have greater RNR expression and produce multi-fold greater levels of cellular dATP. Thus, we will test the dose dependence of elevated dATP for hiPSC-CMRNR engrafted into infarcted rat hearts. The novel aspect of our approach is to go beyond replacement of lost tissue (with hiPSC-CMs) by using engineered hiPSC-CMRNR to produce and deliver a small molecule therapeutic (dATP) that improves native heart muscle contraction. This has the potential to substantially recover the post-MI depressed function of native myocardium. Aim 2 will explore the mechanistic basis of how small increases in myocardial dATP result in significant increases in contractile force and kinetics of activation and relaxation of muscle, and in the magnitude of LV pressure development (LVDP) and kinetics of pressure development (+dP/dt) and decline (-dP/dt) of the heart. Our recent reports and preliminary data strongly suggest at least three mechanisms are involved: 1) disruption of the super-relaxed state (SRX) from the myosin backbone to a disordered relaxed state (DRX), 2) movement of DRX myosin towards thin filaments via greater electrostatic interactions with actin, and 3) faster crossbridge cycling. We have published multiple studies on the chemo-mechanics of faster crossbridge cycling (3), so will focus primarily on mechanisms 1 and 2 here using multiple state of the art approaches. These include low angle x-ray diffraction analysis of isolated myosin, cardiac muscle, and whole heart (Langendorff) levels, stopped-flow ATPase, super-localization single molecule microscopy of thick filament zones, structure-based computational models of myosin ± actin and multi-scale models of the heart. This ...