Project Summary/Abstract Nicotinamide adenine dinucleotide (NAD+) is a redox cofactor for energy metabolism and a co-substrate of sirtuins for protein deacetylation. Cellular NAD+ levels decline with age in the heart and other organs. Previous studies have shown that cellular NAD+ repletion or boosting is protective against aging and age-related diseases. NAD+ is compartmentalized at subcellular levels and the proper distribution of NAD+ into mitochondria is crucial for NAD+-dependent metabolic and signaling function in mitochondria. SLC25A51 was recently identified as the primary mammalian mitochondrial NAD+ (mtNAD+) transporter that imports NAD+ from cytosol into mitochondria. Unlike the established roles of NAD+ synthesis and consumption, there is a critical knowledge gap in the role of mtNAD+ transport by SLC25A51 in aging and heart function. Despite the important roles of mtNAD+ in mitochondrial energy metabolism and protein deacetylation, how mtNAD+ levels change with age and impact cardiac aging remain unknown. Our preliminary study discovered that old murine hearts had reduced mtNAD+ levels and lower SLC25A51 expression compared to young hearts. The objective of this study is to determine the mechanistic role of SLC25A51 in cardiac aging using our newly developed genetic tools to overexpress or knockdown SLC25A51 in the heart. Multiple preclinical studies and clinical trials have examined or are investigating the therapeutic potential of various NAD+ boosting strategies in different diseases. Importantly, the existing NAD+ boosting strategies do not directly boost mtNAD+ levels, which may limit their efficacy. Our preliminary data showed that supplementation with an NAD+ precursor NMN significantly elevated total NAD+ levels in old murine hearts but did not effectively increase mtNAD+ levels. This highlights the potential of enhancing mtNAD+ transport as a novel approach to improve the benefits of NAD+ boosting strategies. We hypothesize that an age-related decline in SLC25A51 function reduces mtNAD+ levels, leading to compromised mitochondrial metabolism and cardiac dysfunction. We also propose that enhancing SLC25A51-mediated mtNAD+ transport will improve the protective effects of NAD+ boosting strategy in the old heart. In this study, we will 1) determine if SLC25A51 deficiency lowers mtNAD+ levels and accelerates cardiac aging; 2) determine if cardiac-specific SLC25A51 overexpression enhances mtNAD+ levels and cardiac function in old mice; and 3) test the hypothesis that levels of SLC25A51 expression regulate the efficacy of NAD+ precursor to improve cardiac function. The findings of this study will establish the roles of SLC25A51 in cardiac aging and open new avenues towards developing new interventions to enhance mtNAD+ metabolism or to maximize the efficacy of NAD+ boosting strategies.