Urolithin A Restores Mitochondrial Function and Reverses Cardiac Remodeling in Heart Failure with Preserved Ejection Fraction

Abstract Background Heart failure with preserved ejection fraction (HFpEF) accounts for nearly half of all heart failure cases; however, mechanism-based therapies targeting cellular dysfunction remain absent. Emerging evidence suggests that mitochondrial dysfunction(definition) and impaired quality control, particularly defective mitophagy, are central to the pathogenesis of HFpEF. Urolithin A (UA), a gut microbiome-derived postbiotic metabolite, has shown promise as a mitophagy activator in preclinical models; however, its therapeutic efficacy in HFpEF remains unknown. Methods We used a clinically relevant two-hit HFpEF mouse model (high-fat diet (HFD) plus Nω-nitro-L-arginine methyl ester (L-NAME)) and co-administered UA during disease progression. The cardiac structure and function were assessed using echocardiography, histology, and transmission electron microscopy. Mitochondrial bioenergetics were evaluated using Seahorse-based respirometry. Mitophagy flux was monitored using mt-Keima assays in H9c2 cells and human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Mechanistic insights were obtained using immunoblotting, integrated shotgun metagenomic and lipidomic profiling, and single-nucleus RNA sequencing (snRNA-seq). Results UA treatment significantly attenuated cardiac remodeling and fibrosis in HFpEF mice while improving diastolic function parameters. Transmission electron microscopy revealed restoration of the mitochondrial ultrastructure, and mitochondrial stress tests demonstrated enhanced oxidative phosphorylation capacity and glycolytic reserves. Immunoblot assays revealed that UA treatment recovered PINK1/Parkin-mediated mitophagy markers (reduced LC3-II and p62/SQSTM1). Mt-Keima assays confirmed enhanced mitophagic flux in H9c2 cells under HFpEF-like stress conditions. Integrated metagenomics and lipidomics revealed significant reductions in cardiovascular risk-associated ceramides. snRNA-seq demonstrated that HFpEF-like stress downregulated contractile, calcium-handling, and mitochondrial mitophagy gene programs. UA treatment restored the mitochondrial quality control signatures and normalized profibrotic and conduction-type cell populations. Conclusions Urolithin A restored mitochondrial quality control through the activation of mitophagy and reversed cardiac remodeling in HFpEF. These findings establish UA as a mitochondria-targeted therapeutic candidate for HFpEF. Novelty and Significance What is known? ✓ Heart failure with preserved ejection fraction (HFpEF) is a heterogeneous syndrome driven by metabolic stress and myocardial remodeling, for which effective disease-modifying therapies are lacking. ✓ Mitochondrial dysfunction and impaired mitophagy have been implicated in HFpEF, but their roles in cardiomyocyte state remodeling and fibrosis remain incompletely defined. ✓ Urolithin A, a gut microbiome–derived metabolite, enhances mitophagy in aging and metabolic tissues, but its relevance to HFpEF has not been established. What new information does this article contribute? ✓ Urolithin A restores mitochondrial quality control and mitophagic flux in cardiomyocytes, improving diastolic function and attenuating cardiac remodeling in a two-hit HFpEF model. ✓ Integrated metagenomic and lipidomic analyses identify suppression of gut microbiome–associated ceramide biosynthesis as a systemic mechanism linked to Urolithin A–mediated cardioprotection. ✓ Single-nucleus RNA sequencing reveals that Urolithin A reverses maladaptive cardiomyocyte transcriptional state transitions toward fibrogenic and conduction-like programs.