ABSTRACT: The parent project is built on two fundamental discoveries, spanning 20 years of mechanistic and translational research: 1) 2-deoxy ATP (dATP) is a potent natural nucleotide stimulant of cardiac contractility (via increased myosin binding to actin and faster detachment following the power stroke), and 2) hiPSC-CMs overexpressing the rate-limiting enzyme for dATP synthesis, ribonucleotide reductase (RNR), exhibit both increased contractility and dATP delivery to the rest of the heart via gap junctions. Consequently, we are investigating the hypothesis that altering hiPSC-CMs to increase RNR (RNR-hiPSC-CMs) improves outcomes in cell replacement treatment for MI (as compared to control hiPSC-CMs), by increasing the contractility of both the graft and native myocardium. Our technique incorporates several highly innovative elements. 1) This is the first time that cellular nucleotide modification has been offered as a means of improving in vivo heart function. 2) The technique is not restricted to replacing lost tissue (with hiPSC-CMs) with a more functional graft but may also significantly benefit the native myocardium's post-MI depressed function. 3) The first application of modified hiPSC-CMs to deliver a small molecule treatment (dATP) that enhances cardiac muscle contraction. This essentially transforms hiPSC-CMs into a cardiac-specific medication delivery system. Aim 1 is to generate and characterize engineered mutations in RNR that enhance its stability and activity in cardiomyocytes, as well as their ability to titrate increasing quantities of dATP produced in hiPSC-CMs. Aim 2 investigates the ability of RNR variations identified in Aim 1 to improve cardiac function in a mouse model of myocardial infarction and heart failure using AAV vectors. Aim 3 will generate engineered hiPS cell lines that, upon differentiation, will act as dATP 'donor cells' for transplantation into acute MI and more problematic chronic MI arrhythmic rat models to test their capacity to increase function beyond that of non-designed hiPSC-CMs. We will assess the persistence of these effects and the cell lines' long-term survival and stability. We anticipate a significant contractile improvement in both the graft and native myocardium using RNR-hiPSC-CMs vs. hiPSC- CMs, which will be controlled by the transplanted cells' dATP producing capacity. These investigations will shed light on the potential for this combination cell- and small-molecule therapy to improve or perhaps restore pump performance in failing hearts. This addition, as the candidate's research project, will expand the scope of the study by pursuing two objectives. Aim 1 will examine the mechanical and structural mechanisms that allow cardiac muscle utilizing dATP to be less sensitive to contractile strength losses when the pH is decreased, as occurs during ischemia. Aim 2 will investigate whether cardiac function can be improved in a different cardiomyopathy model (dilated instead of MI), utilizin...