Abstract Body (Do not enter title and authors here): Intro: The 2-hit HFD/L-NAME model is extensively used to develop new therapies for patients suffering from Heart failure with Preserved Ejection Fraction (HFpEF). It captures the features of one of the leading phenogroups of patients; the cardiometabolic and mild hypertension associated HFpEF.

Hypothesis: We assessed the effect of the duration of HFD+L-NAME on the progression of diastolic dysfunction. Then, we chose the optimal timing to enable time-effective proof-of-concept studies for drugs targeting this phenotype. Cardiac, metabolic and renal aspects were assessed in the chosen optimal model, in addition to the mitochondria state in cardiac tissue. Empagliflozin (EMPA), the clinical benchmark, was used for pharmacological validation.

Methods: C57BL6N mice were fed HFD (60% Kcal from fat)+water with L-NAME (0.5g/l) up to 20 weeks. Control mice (Ctrl) received normal chow+water. Cardiac function was evaluated at several timepoints to set the optimal timing. In the 2nd set of experiments, mice were randomized after 10 weeks of diet to receive vehicle or EMPA (10mg/kg) QD for 6 weeks followed by treadmill exercise tolerance test, echocardiography, invasive hemodynamic and quantification of metabolic and renal biomarkers. Cardiac tissue was used to assess cardiac fibrosis using sirius red and mitochondrial functional defects in this HFpEF model using Oroboros®

Results: HFD/L-NAME mice showed preserved systolic function and progressive diastolic dysfunction reaching a moderate phenotype characterized by inverted or pseudonormal profiles (E’/A’=0.8±0.02 E/E’=33.2±1.1 in HFD/L-NAME vs E’/A’=1.5±0.02 E/E’=26.9±2.1 in Ctrl). These mice showed left ventricular concentric hypertrophy, increased arterial and left ventricular end-diastolic pressure, cardiac fibrosis (p<0.05 vs. Ctrl, respectively), and altered exercise capacity (p<0.01 for running distance and time vs. Ctrl). A decrease in complex I activity was also observed. Insulin resistant state, increased blood glucose, cholesterol, ALT and FGF-21 were noted but not urea/creatinine ratio. EMPA alleviated cardiac dysfunction (E’/A’=1.3±0.03 E/E’=28.0±0.9) and had positive outcome on cardiac remodeling and metabolic biomarkers.

Conclusion: These data demonstrated that HFD/L-NAME captures several cardiac and metabolic aspects of patients suffering from moderate HFpEF associated with obesity/metabolic syndrome and mild hypertension. This model showed also cardiac fibrosis and impairment of metabolic flexibility.