Understanding the roles of cardiac NAD+ pools and therapeutic effects of precursor supplements in heart failure We are exploring the hypothesis that nicotinamide adenine dinucleotide (NAD+) metabolism can be targeted to improve functional capacity in failing human hearts. NAD+ is a ubiquitous molecule that is required as a redox cofactor or substrate for hundreds of enzymes within the cell. It is derived from dietary tryptophan, niacin, nicotinamide, or synthetic intermediates, but the majority of synthesis in the heart is via nicotinamide. NAD+ concentration falls in failing human hearts and in some rodent models of heart failure. High doses of precursors including nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN) have therapeutic effects in rodent models. However, the doses used exceed what is tolerable in humans and the potential for effects at human-relevant doses remains uncertain. Our preliminary and published results suggest that high doses of NR and NMN may be required in rodent models because both molecules are extensively metabolized in the intestines and liver when delivered orally, with only a tiny fraction reaching the circulation intact. In contrast, intravenous delivery allows a much higher proportion of the dose to reach organs such as the heart. In addition to questions about dosing, the mechanism of protection has remained unclear. It is presumed to involve cardiac NAD+ levels, but whole-body supplementation studies leave open the possibility that other tissues mediate protection, for example through lowering blood pressure. We present a knockout mouse with cardiomyocyte-specific loss of NAD+ that impairs heart function and propose the generation of a new model to specifically test the role of mitochondrial NAD+ within the cardiomyocytes. This will be accomplished by targeting SLC25A51, which we recently identified as the mitochondrial NAD+ transporter. We propose three specific aims: Aim 1) Test whether heart-specific NAD+ depletion is sufficient to recapitulate the metabolic and electrical consequences of heart failure, Aim 2) Test whether alternate delivery routes can allow cardiac NAD+ to be rescued by low, human-relevant doses in mice, and Aim 3) Test whether altering mitochondrial NAD+ is sufficient to modulate heart function on its own or modifies susceptibility to induced heart failure. Our approach of using AAV to target SLC25A51 expression in the heart will be the first time that modulation of this protein to alter the mitochondrial NAD+ pool has been attempted in vivo. Together, these studies will reveal fundamental details of how NAD+ metabolism influences cardiac physiology, and will help guide efforts to develop novel therapeutic approaches for the treatment or prevention of heart failure in human patients.