Heart failure with preserved ejection fraction (HFpEF) remains a major unmet clinical challenge due to its poor prognosis and limited therapeutic options. C57BL/6J (B6J) mice are protected from the early development of HFpEF in the two-hit model compared to C57BL/6N (B6N) mice because of a naturally occurring loss-of-function variant in the mitochondrial enzyme Nicotinamide Nucleotide Transhydrogenase (Nnt). Under physiological conditions, Nnt maintains mitochondrial redox and energy balance by transferring hydrides from NADH to NADP⁺, producing NAD⁺ and NADPH to support redox homeostasis. Under pathological workload, however, this reaction reverses, leading to NAD⁺ and NADPH depletion, mitochondrial reactive oxygen species (ROS) accumulation, and maladaptive remodeling. Metabolomic profiling at an early disease-driving stage (three days after diet induction) revealed a higher susceptibility to oxidative stress in B6N hearts, characterized by depletion of glutathione (GSH) and NAD⁺. This redox imbalance coincided with reduced levels of cellular energy currencies (ATP, AMP, GTP, and GDP), indicating mitochondrial dysfunction and impaired energy turnover. Furthermore, activation of nucleotide salvage pathways, reflected by elevated free nucleotides, suggests nucleic acid degradation occurs to compensate for energy loss, while reduced nicotinamide riboside (NR) levels indicate exhausted cellular attempts to restore NAD⁺ pools through salvage metabolism. Complementary genetic models, including Nnt knockout in B6N mice and Nnt knock-in in B6J mice, demonstrate protection from and susceptibility to diastolic dysfunction, respectively, confirming the causal role of Nnt-driven oxidative stress in HFpEF. Notably, this protective effect was independent of adiposity or lean mass status, indicating that early mitochondrial redox disturbances and Nnt-mediated mechanisms act upstream of later systemic and inter-organ complications. To therapeutically target this pathway, cardiac-specific CRISPRi AAV9-mediated Nnt knockdown is being employed to reduce mitochondrial ROS generation, while structural Nnt mutant screening aims to selectively suppress pathological reverse Nnt activity while preserving its physiological forward function. This study identifies reverse Nnt activity as an early driver of mitochondrial oxidative stress and energetic dysfunction in HFpEF, revealing a previously underappreciated mechanism of disease initiation. These findings highlight Nnt as a promising therapeutic target and provide a foundation for precision-medicine approaches aimed at preserving mitochondrial function and preventing adverse cardiac remodeling in HFpEF.
