Effects of empagliflozin on cardiomyocyte function and mitochondrial respiration in a rat model of HFpEF: a focus on sex-specific responses

Background: Sodium-glucose co-transporter 2 (SGLT-2) inhibitors, such as empagliflozin (EMPA), have shown cardiovascular benefits, particularly in heart failure with preserved ejection fraction (HFpEF) associated with obesity. However, the mechanisms driving these effects, including potential sex-specific responses, remain unclear.

Methods and Results: Male and female Wistar rats (6-7 weeks) were fed standard chow (CO) or a high-fat/fructose (HFD) diet combined with L-NAME (0.25 mg/ml for males, 0.15 mg/ml for females due to tolerance differences) via drinking water for 8 weeks to induce HFpEF. HFpEF rats were then randomly assigned to receive EMPA (10 mg/kg/day in drinking water) or regular tap water for an additional 8 weeks. HFpEF groups had ad libitum access to either HFD or a low-fat diet. Echocardiography (n=3–6 per group) was conducted at the end of treatment, and cardiomyocytes (n=3 per group, minimum of 33 cells) were isolated to assess sarcomere shortening and mitochondrial redox state (NAD(P)H/FAD ratios) using a customized manual IonOptix setup. Cardiomyocytes were paced at baseline (0.3 Hz) and under stress (3 Hz) with isoprenaline (30 nM) for 3 minutes. Additionally, mitochondria (n=4–8 per group) were isolated from the same and additional hearts for high-resolution respirometry using an Oroboros Oxygraph-2k.

In male HFpEF rats, echo revealed a significantly reduced E/A ratio, indicative of diastolic dysfunction, but with preserved ejection fraction (EF). CMs analysis showed shorter diastolic and systolic sarcomere lengths, along with marked oxidation of the mitochondrial redox state, reflecting elevated metabolic demand. Fractional shortening remained unchanged. EMPA treatment restored the E/A ratio and diastolic sarcomere length to control levels and partially recovered systolic sarcomere length during stress pacing, though not at baseline (0.3 Hz). EMPA also increased fractional shortening and enhanced the NAD(P)H/FAD ratio beyond control levels, suggesting an improved mitochondrial redox state. Mitochondrial respiration was markedly increased with EMPA treatment across pyruvate/malate, succinate, or fatty acid as substrates compared to HFpEF vehicle and even healthy controls. In female HFpEF rats, echocardiography similarly showed a reduced E/A ratio, consistent with diastolic impairment and preserved EF. Shortened diastolic and systolic sarcomere lengths were also observed, with no change in fractional shortening across groups. EMPA treatment slightly improved diastolic and systolic sarcomere lengths during stress pacing, though not to control levels, and partially restored the E/A ratio. EMPA also enhanced the NAD(P)H/FAD ratio, indicating an improved mitochondrial redox state. In terms of mitochondrial respiration, both HFpEF and HFpEF+EMPA groups exhibited significantly higher respiration rates with pyruvate/malate and succinate substrates compared to controls.

Conclusions: EMPA demonstrates beneficial effects on overall mitochondria function in HFpEF, with notable sex-specific responses. In males, EMPA restores mitochondrial function, contractility, and the E/A ratio, while in females, EMPA improves the mitochondrial redox state and partially alleviates diastolic dysfunction, including a modest improvement in the E/A ratio. These findings suggest that EMPA’s cardioprotective mechanisms in HFpEF may differ between sexes, underscoring the importance of sex-based analyses in cardiovascular treatment strategies.