Abstract Body: Introduction: Exercise is an effective strategy for improving functional outcomes in patients with diabetic cardiomyopathy (DiaCM), yet the underlying molecular mechanisms are not fully understood. N-acetyltransferase (NAT10), the only known N4-acetylcytidine (ac4C) writer, has been linked to cardiac disease. However, the roles of NAT10 in the benefits of exercise in DiaCM are unknown.

Hypothesis: NAT10 is necessary for cardiac benefits of exercise in DiaCM.

Methods: NAT10 floxed mice were injected with AAV9 encoding cTnT-driven Cre recombinase to knockout (KO) NAT10 in cardiomyocytes (CMs). CM-specific NAT10 KO mice, NAT10 floxed mice, and their wild-type littermates were subjected to either chow diet or high-fat (HFD) diet feeding and injection of streptozotocin (STZ) to induce DiaCM, followed by an eight-week exercise training. Cardiac NAT10 expressions were assessed in patients with nonischemic primary dilated cardiomyopathies with diabetes (DM/DCM). Downstream effectors of NAT10 were identified through ac4C RNA immunoprecipitation-sequencing (RIP-seq).

Results: HFD+STZ mice displayed increased E/e’, decreased fractional shortening (FS), reduced cardiac NAT10 and ac4C levels (p<0.05 for all). Exercise improved E/e’ (Sedentary: -37.01 ± 1.31 vs Exercise: -31.01 ± 1.21, p=0.02, n=6) and FS (Sedentary: 26.31% ± 1.49 vs Exercise: 32.24% ± 1.48, p=0.04, n=6) and increased cardiac NAT10 protein expression by ~two-fold (p<0.01, n=6) and ac4C level. These effects of exercise were canceled by CM-specific NAT10 KO (E/e’: Control: -22.42 ± 2.78 vs KO: -39.09 ± 2.50; FS: Control: 32.86% ± 0.93 vs KO: 25.99% ± 1.19, p<0.01 for all, n=6). Pathway analysis from RIP-seq revealed a significant enrichment of the ferroptosis pathway. In HFD+STZ mice, ac4C levels of key ferroptosis regulators FTH1, SLC7A11, and GPX4 were increased by exercise while reduced by CM-specific NAT10 KO (p<0.01 for all, n=4). Cardiac NAT10 and ac4C levels, and protein expressions of ferroptosis markers SLC7A11 and GPX4 were reduced in DM/DCM patients (vs. nonfailing controls, p<0.01 for all; n=12 in DM/DCM, n=8 in nonfailing).

Conclusions: Exercise via NAT10 enhances ac4C deposition on ferroptosis regulators, reducing ferroptosis and improving cardiac function in DiaCM.