Abstract Body (Do not enter title and authors here): Introduction: Advanced age is a major risk factor for heart diseases. Exercise training has emerged as an effective intervention to improve functional outcomes in age-related cardiac dysfunction. N-acetyltransferase (NAT10), the only known N4-acetylcytidine (ac4C) ‘writer’ enzyme, has recently been implicated in heart failure. However, the role of NAT10 in cardiac aging and the cardiac benefits of exercise on age-related cardiac dysfunction are unclear.

Hypothesis: NAT10 is necessary and sufficient for cardiac benefits of exercise in aging.

Methods: Ten-week-old (young) or twenty-month-old (aged) mice received sedentary (Sed) or voluntary wheel running (Run) for eight weeks. AAV9 encoding cardiac troponin-T-driven NAT10 was tail vein-injected to overexpress NAT10 in cardiomyocytes (CMs).

Results: Compared with young mice, aged mice exhibited increased E/e’ (Young: -14.68±1.09 vs Aged: -20.16±1.61, n=8, p=0.03) and decreased global longitudinal strain (GLS, Young: -20.04±1.47 vs Aged: -15.88±0.84, n=8, p=0.04) with preserved ejection fraction. Aged hearts also displayed increased ferroptosis (reduced GPX4 and FSP1, p<0.05 vs Sed) and senescence (increased gene expression of p16, p21, and PAI, all p<0.05). In aged mice, running improved diastolic function (E/e’, Sed: -20.16±1.61 vs Run: -15.4±1.06, n=8, p=0.04; GLS, Sed: -15.88±0.84 vs Run: -20.66±1.02, n=8, p=0.02), reduced cardiac ferroptosis (increased GPX4 and FSP1, p<0.05 vs Sed), and decreased senescence gene expression. Cardiac NAT10 was downregulated in aged mice and was upregulated by exercise (all p<0.05). In aged mice, AAV9 injection for eight weeks led to ~6-fold increase in cardiac NAT10, resulting in reduced E/e’ (AAV9-NAT10: -22.67±1.03 vs Control: -26.33±1.12, n=7, p=0.03) and increased GLS (AAV9-NAT10: -19.23±0.93 vs Control: -15.26±1.01, n=7, p=0.04) as well as reduced senescence gene expression (reduced p16, p21, and PAI, p<0.05 vs AAV9-control). Mechanistically, NAT10 functioned by increasing ferritin ac4C deposition and promoting its translation, thereby inhibiting CM ferroptosis.

Conclusions: NAT10 regulates cardiac aging through ferritin, and it mediates cardiac benefits of exercise in aging. NAT10 is a promising therapeutic target for combating cardiac aging.