Combined effects of semaglutide and empagliflozin on cardiomyocyte function and mitochondrial respiration in a rat model of HFpEF: a sex-specific analysis

Background: Sodium-glucose co-transporter 2 (SGLT-2) inhibitors like empagliflozin (EMPA), and Glucagon-like peptide-1 receptor (GLP-1R) agonists, such as semaglutide (SEMA), have shown individual cardiovascular benefits, particularly in heart failure with preserved ejection fraction (HFpEF). However, the combined effects of these agents, as well as potential sex-specific differences, remain largely unexplored. This study evaluates the impact of SEMA+EMPA on cardiomyocyte function and mitochondrial energetics in a diet-induced HFpEF model with L-NAME, a pan-nitric oxide synthase inhibitor that induces hypertension, thereby mimicking the HFpEF conditions observed in humans.

Methods and Results: Male and female Wistar rats (6-7 weeks old) were fed standard chow (CO) or a high-fat/fructose (HFD) diet with L-NAME (0.25 mg/ml for males, 0.15 mg/ml for females due to tolerance differences) for 8 weeks to induce HFpEF. HFpEF rats were then randomly assigned to receive a combination of EMPA (10 mg/kg/day in drinking water) and SEMA (120 μg/kg/day subcutaneously) for an additional 8 weeks or to remain on tap water with saline injections. HFpEF groups had ad libitum access to either HFD or a low-fat diet. Cardiomyocytes (³33 cells of n=3 animals per group), and mitochondria (n=6–8 per group) were isolated to assess sarcomere length, mitochondrial redox state (NAD(P)H/FAD ratio), and respiration using high-resolution respirometry (Oroboros Oxygraph-2k).

In male HFpEF cardiomyocytes, we observed significantly shorter diastolic and systolic sarcomere lengths, along with an oxidized mitochondrial redox state indicative of higher metabolic demand. Cardiomyocyte fractional shortening remained unchanged. SEMA+EMPA treatment restored both diastolic and systolic sarcomere lengths to control levels, significantly increased cardiomyocyte shortening capacity, and enhanced the NAD(P)H/FAD ratio, indicating an improved mitochondrial redox state. Mitochondrial respiration in HFpEF was lower than in controls but was fully restored by SEMA+EMPA across pyruvate/malate, succinate, and fatty acid substrates. In female HFpEF cardiomyocytes, we similarly observed reductions in both diastolic and systolic sarcomere lengths. With SEMA+EMPA treatment, only systolic sarcomere length was restored to control levels, while diastolic length remained shorter than controls. The NAD(P)H/FAD ratio improved substantially with treatment, reflecting a more reduced (or improved) mitochondrial state. In terms of mitochondrial respiration, both HFpEF and SEMA+EMPA-treated HFpEF groups exhibited significantly higher respiration rates with pyruvate/malate and succinate substrates compared to control.

Conclusions: Combined SEMA+EMPA treatment in the HFpEF model demonstrates clear sex-specific benefits. In males, treatment restores both sarcomere length and mitochondrial function, while in females it improves systolic function and mitochondrial redox balance, although diastolic sarcomere length remains partially impaired. Importantly, mitochondrial respiration in HFpEF females is substantially elevated at baseline compared to controls, and this elevation persists after treatment. These findings underscore the potential of combined SGLT2 and GLP-1 therapies in HFpEF, while highlighting the importance of sex-based analyses in cardiovascular treatment strategies.