Abstract:
Heart failure with preserved ejection fraction (HFpEF) is characterized by poor clinical outcomes and resistance to most guideline-directed therapies effective in HFrEF. C57BL/6J (B6J) mice show protection against a two-hit HFpEF model compared to C57BL/6N (B6N) mice, owing to a naturally occurring loss-of-function variant in the mitochondrial protein Nicotinamide Nucleotide Transhydrogenase (Nnt). Nnt contributes to mitochondrial redox balance and energy metabolism by catalyzing hydride transfer from NADH to NADP⁺, generating NADPH and NAD⁺, and maintaining the cellular NADH/NADPH ratio. Under pathological workload, this reaction can reverse, leading to NAD⁺ and NADPH depletion, mitochondrial ROS accumulation, and maladaptive cardiac remodeling.
Our metabolomic profiling at early disease-driving stages (three days post-diet) revealed that B6N hearts exhibit higher susceptibility to oxidative stress than B6J hearts, indicated by depletion of glutathione (GSH) and NAD⁺ pools. Consistent with this redox imbalance, a rapid decline in cellular energy currencies (ATP, AMP, GTP, GDP) reflects mitochondrial dysfunction and impaired energy turnover in B6N cardiomyocytes. This is accompanied by activation of nucleotide salvage pathways, evidenced by increased free nucleotide levels suggestive of DNA and RNA degradation for energy recovery. Moreover, a marked reduction in nicotinamide riboside (NR) levels indicates a compensatory attempt to restore NAD⁺ through salvage mechanisms during metabolic stress.
In parallel, a Nnt knockout B6N model as well as a Nnt Knock-in B6J was used to assess protection and susceptibility to diastolic dysfunction, respectively, serving as proof-of-concept to pinpoint the pivotal role of Nnt-driven oxidative stress in driving the HFpEF phenotype. To counteract the pathogenic reverse activity of Nnt, a cardiac-specific CRISPRi AAV9-mediated Nnt knockdown strategy is employed in B6N mice to test rescue of HFpEF after an established diastolic dysfunction. Using real-time endogenous ROS monitoring, a series of Nnt structural mutants have been evaluated for their ability to blunt the reverse mode of enzymatic activity to mitigate HFpEF pathology.
This study, for the first time, identifies the primary source and mechanistic link between mitochondrial ROS accumulation and the molecular remodeling of the heart at the earliest stages of HFpEF. These findings establish the foundation for targeted gene therapies aimed at modulating Nnt function and redox balance as early interventions against HFpEF.
