Abstract 1808 Loss of Mitochondrial NAD+ Transporter SLC25A51 in Cardiomyocytes Impairs Complex I Respiration and Induces Cardiac Hypertrophy

Background: The heart serves as the central pump, supplying oxygen and nutrients to the rest of the body, and its proper functioning is essential for sustaining life. Cardiac function is intrinsically intertwined with the energy production orchestrated by mitochondria. Nicotinamide adenine dinucleotide (NAD+) is a coenzyme crucial for many cellular processes, including cellular signaling, DNA repair and many metabolic pathways such as glycolysis or tricarboxylic acid cycle (TCA). NAD+ also acts as a substrate for NAD+-dependent enzymes like sirtuins e.g. SIRT3, which control diverse mitochondrial functions from mitochondrial respiration to biogenesis by NAD+-dependent protein deacetylation. Among all subcellular compartments, mitochondria have the highest NAD+ concentrations and the mitochondrial NAD+ (mtNAD+) pool constitutes up to 70% of the total cellular NAD+ pool. Recent studies discovered SLC25A51 as the mammalian mtNAD+ transporter and a major source of mtNAD+ pool. Efficient transport of NAD+ into the mitochondria is essential for mitochondrial respiration and other NAD+-dependent reactions in mitochondria. However, the specific role of mtNAD+ transport and SLC25A51 in the heart remain unknown. Methods: CRISPR-Cas9 technology was used to generate mice with a loxP-flanked ("floxed") allele of Slc25a51. Floxed mice were bred with mice expressing Cre recombinase under Myh6 promoter to excise Slc25a51 and generate cardiac-specific SLC25A51 heterozygous (cHet-KO) and homozygous (cKO) knockout mice. mtNAD+ levels in mitochondria isolated from mouse hearts were measured by using NAD+ cycling assay. Mitochondria Complex-I and Complex-II-driven respiration was measured by Fluorescence Lifetime Micro Oxygen Monitoring System (Instech). Results: To unravel the significance of SLC25A51 in the heart tissue, we compared young cHet-KO and cKO knockout mice with their littermate controls (Ctrl) to determine the impact of SLC25A51 deficiency on cardiac and mitochondrial function. NAD cycling assay of cardiac mitochondrial extracts revealed that heterozygous and homozygous KO mice have dose-dependent decreases in mtNAD+ levels, with 30% and 75% reduction of mtNAD+ levels compared to Ctrl, respectively. Interestingly, cKO mice, but not cHet-KO mice exhibit mitochondrial protein hyperacetylation in the heart, while mitochondrial respiration in cHet-KO mice was not different compared to Ctrl. cKO mice exhibit impaired NADH-driven complex I respiration but not NADH-independent complex II respiration. This result suggests that mtNAD+ depletion compromises complex I of the electron transport chain in a dose-dependent manner. Intriguingly, cKO mice faced premature mortality at two months of age, accompanied by dramatic cardiac hypertrophy. These observations suggest that there may exist a threshold of mtNAD+ decline which the heart is able to tolerate and still effectively maintain its proper functionality. These findings not only emphasize the critical role of SLC25A51 in maintaining mitochondrial health but also shed light on its potential implications in cardiac hypertrophy and overall cardiac function. We acknowledge support from the PHF Seed Grant (YAC) and the Glenn/AFAR Junior Faculty Grant (YAC).