Abstract Body: Introduction: Heart failure with preserved ejection fraction (HFpEF) is the most common form of HF. Despite the best available therapies, prognosis remains poor for many patients with HFpEF. N6-methyladenosine (m6A) is the most common form of mRNA modification in eukaryotes and has been implicated in cardiac disease. However, whether m6A plays a role in HFpEF is unknown.

Methods: Cardiomyocyte (CM)-specific methyltransferase like 3 (METTL3) knockout (KO) was achieved by injecting AAV9 encoding cTnT-driven Cre into METTL3 floxed mice. CM-specific METTL3 overexpression (OE) was achieved by injecting AAV9 encoding cTnT-driven METTL3 into wild-type (WT) mice. HFpEF mouse models were induced by (1) high-fat diet (HFD) and Nω-nitrol-arginine methyl ester for 15 weeks (HFD+L-NAME); or (2) HFD and transverse aortic constriction-induced moderate pressure overload for 15 weeks (HFD+mTAC). Cardiac METTL3 expression was assessed in patients with HFpEF.

Results: METTL3 was the most downregulated m6A writer, at both the mRNA (by ~70%, p<0.001, n=12) and protein (by ~80%, p<0.001, n=12) levels, in hearts of HFpEF patients compared to matched controls. In HFD+L-NAME mice, cardiac METTL3 protein expression was reduced by ~70% (p<0.001, n=8), and associated with cardiac diastolic dysfunction, decreased contractile reserves, and increased fibrosis. These HFpEF phenotypes were further exacerbated in HFD+L-NAME mice with CM-specific METTL3 KO (E/e’: WT: -32.82±2.31 vs KO: -41.27±2.49, p=0.03, n=6). Conversely, CM-specific METTL3 OE improved cardiac diastolic function, increased contractile reserves, and reduced fibrosis in both HFD+L-NAME (E/e’: Control: -34.13±1.62 vs OE: -28.40±1.78, p=0.04, n=6) and HFD+mTAC mice (E/e’: Control: -33.80±1.92 vs OE: -28.34±0.94, p=0.02, n=6). Mechanistically, METTL3 functioned by increasing m6A deposition on Ankrd1 (a cardiac stress-response protein) and promoting its degradation, thereby reducing subsequent cardiac inflammation.

Conclusions: CM METTL3 deficiency is part of the causal pathobiology of HFpEF and its CM-specific induction is sufficient to improve cardiac diastolic function and cardiac remodeling in HFpEF. These findings identify METTL3 as a promising therapeutic target for HFpEF.