Mitochondrial NNT Promotes Diastolic Dysfunction in Cardiometabolic HFpEF

BACKGROUND: Clinical management of heart failure with preserved ejection fraction (HFpEF) is hindered by a lack of disease-modifying therapies capable of altering its distinct pathophysiology. Despite the widespread implementation of a 2-hit model of cardiometabolic HFpEF to inform precision therapy, which utilizes HFD+L-NAME (ad libitum high-fat diet and 0.5% N[?]-nitro-L-arginine methyl ester), we observe that C57BL6/J mice exhibit less cardiac diastolic dysfunction in response to HFD+L-NAME. METHODS: Genetic strain-specific single-nucleus transcriptomic analysis identified disease-relevant genes that enrich oxidative metabolic pathways within cardiomyocytes. Because C57BL/6J mice are known to harbor a loss-of-function mutation affecting the inner mitochondrial membrane protein Nnt (nicotinamide nucleotide transhydrogenase), we established an isogenic model of Nnt loss-of-function to determine whether intact NNT is necessary for the pathological cardiac manifestations of HFD+L-NAME. Twelve-week-old mice cross-bred to isolate wild-type (Nnt+/+) or loss-of-function (Nnt-/-) Nnt in the C57BL/6N background were challenged with HFD+L-NAME for 9 weeks (N=6–10). RESULTS: Nnt+/+ mice exhibited impaired ventricular diastolic relaxation and pathological remodeling, as assessed via noninvasive echocardiographic quantification of early diastolic pulse-wave velocity (E) to mitral annular velocity (e') ratio (E/e') (42.8 versus 21.5, P=1.2×10-10), E/A (early-to-late mitral inflow velocity ratio) (2.3 versus 1.4, P=4.1×10-2), diastolic stiffness (0.09 versus 0.04 mm Hg/µL, P=5.1×10-3), and myocardial fibrosis (P=2.3×10-2). Liquid chromatography and mass spectroscopy exposed a 40.0% reduction in NAD+ (P=8.4×10-3) and a 38.8% reduction in the ratio of reduced-to-oxidized glutathione (GSH: GSSG, P=2.6×10-2) among Nnt+/+ mice after HFD+L-NAME feeding. Using single-nucleus ligand-receptor analysis, we implicate Fgf1 (fibroblast growth factor 1) as a putative NNT-dependent mediator of cardiomyocyte-to-fibroblast signaling in myocardial fibrosis. CONCLUSIONS: Together, these findings underscore the pivotal role of mitochondrial dysfunction in HFpEF pathogenesis, implicating both NNT and Fgf1 as novel therapeutic targets. Overall design: The initial pilot experiment consisted of twenty 12-week-old male mice from the C57BL/6N and C57BL/6J backgrounds that were purchased from 2 vendors (N=5 per diet-genotype)—Janvier and Charles River laboratory—and housed at the Klinisch Experimenteller Bereich mouse facility of Heidelberg University. All cohorts were group-housed (=4 mice/cage) on a 12:12-hour light-dark cycle from 06:00 to 18:00 at 25±1 °C and constant humidity with either ad libitum access to control laboratory diet (CON, no. 2916; Teklad) and water (CON) or HFD (58.0 kcal% fat, D12492) with water containing L-NAME (0.5 g/L, pH=7.4; Sigma-Aldrich) stored in light-sensitive bottles (HFD+L-NAME), as previously described.