Heart failure with preserved ejection fraction (HFpEF) is associated with low cardiac nicotinamide adenine dinucleotide (NAD⁺), while oral supplementation of the NAD⁺ precursor, nicotinamide, improves preclinical HFpEF in rodents. However, NAD⁺ is a pleiotropic molecule and, thus, it remains elusive which cell-autonomous mechanisms are required for HFpEF therapy by NAD⁺. Because autophagy is an NAD⁺-dependent mechanism that is essential for maintaining cellular homeostasis, especially in long-lived cardiomyocytes, we set out to test whether cardiac autophagy is causally involved in NAD+ repletion therapy. 

Here, we show that moderate elevation of NAD⁺ using a clinically feasible dose of nicotinamide (0.3% w/v in the drinking water) effectively improved cardinal signs of HFpEF in the ZSF1 obese rats, a model of metabolic syndrome-induced HFpEF. Specifically, nicotinamide reduced cardiac hypertrophy, diastolic dysfunction, and pulmonary congestion. These cardioprotective effects correlated with increased expression of several mRNA species involved in the autophagic-lysosomal pathway in the heart. Consistently, nicotinamide stimulated the autophagy marker LC3B, leading to an increase in its electrophoretic mobility (LC3B-II) and reduced the autophagic substrate p62, indicating increased autophagic flux. To examine whether autophagy is causally required for the anti-HFpEF effects of nicotinamide, we used Atg5-deficient (Atg5^-/-) mice, which lack constitutive autophagy specifically in cardiomyocytes. Atg5^-/- mice and their control littermates (Atg5^+/+) were subjected to a ‘two-hit’ HFpEF model using a high-fat diet (HFD) and the nitric oxide synthase inhibitor N-nitro-l-arginine methyl ester (L-NAME). After 5 weeks of HFD+L-NAME administration, both Atg5^-/- and Atg5^+/+ mice developed diastolic dysfunction, left ventricular remodeling and hypertrophy, with preserved ejection fraction and no signs of dilation. Again, nicotinamide significantly attenuated diastolic dysfunction and ventricular remodeling in HFD+L-NAME-fed Atg5^+/+ mice, however; nicotinamide failed to exert similar cardioprotective effects in Atg5^-/- mice. Mechanistically, autophagy activation was not associated with changes in the acetylation levels of essential autophagy-related proteins, as evaluated by immunoblotting and confirmed in a comprehensive acetylome analysis. By contrast, the insulin/IGF-1 pathway, which inhibits autophagy, emerged amongst the top hits in an unbiased gene-set enrichment analysis (Benjamini-Hochberg-adjusted P=0.006; KEGG) of the cardiac transcriptome. Accordingly, nicotinamide reduced the expression of cardiac IGF-1 receptor, and diminished the activity of IGF-1 signaling, as indicated by reduced phosphorylation of the downstream effector serine/threonine-protein kinase Akt at Thr³⁰⁸ and Ser⁴⁷³. Importantly, co-administration of IGF-1 to nicotinamide-fed mice attenuated the pro-autophagic action of nicotinamide in vivo.

In sum, these results indicate that activation of cardiac autophagy is essential for NAD⁺ repletion therapy by nicotinamide in experimental HFpEF.