Effects of semaglutide plus empagliflozin in a rat model of heart failure with preserved ejection fraction: Focus on excitation-contraction coupling and mitochondrial function

Background. SGLT2-inhibitors improve the outcome of patients with heart failure with preserved ejection fraction (HFpEF). The underlying mechanisms are, however, unresolved. Semaglutide (SEMA), a GLP-1 receptor agonist with highest body-weight reducing potential, has proven beneficial effects on cardiovascular outcomes, especially on HFpEF. Here, we evaluated the impact of SEMA plus EMPA on excitation-contraction coupling and mitochondrial energetics in a rat model with diet-induced obesity and HFpEF.

Methods and Results Male Wistar rats were fed standard chow (CO) or high fat/fructose (HFD) diet combined with L-Name (0.25mg/ml) via drinking water for 8-weeks to induce obesity and HFpEF. Afterwards, rats on HFD were administered EMPA via drinking water (10mg/kg/day) combined with SEMA (HF+E&S) (120μg/kg s.c.) or regular tap water and a comparable volume of saline s.c. (HF) for 8-weeks. During treatment, animals of the HF and HF+E&S group could choose between HFD and low fat diet. After 16 weeks, cardiac ventricular myocytes (n=3 per group, min. 33 cardiac myocytes) and mitochondria (n=6-8 per group) were isolated. Sarcomere length, cytosolic Ca²⁺ (Indo1, AM) and mitochondrial redox state (auto fluorescents NAD(P)H and FAD+), membrane potential (TMRM), and ROS (DCF) in myocytes were measured using an automatic Ionoptix fluorescence setup. Pacing at 0.3 Hz, followed by β-adrenergic stimulation and increasing stimulation rate at 3 Hz for 3 minutes, was used to subject cardiac myocytes to a physiological stress regimen.

The HF group showed a significantly shorter diastolic sarcomere length and increased diastolic & systolic [Ca²⁺] compared to CO. Fractional shortening was comparable, while Ca-transient amplitude was increased in HF. The mitochondrial redox status was more oxidized in the HF compared to the CO group, but mitochondrial membrane potential was more negative and more stable, furthermore ROS production was reduced. BNP blood levels were significantly increased in HF compared to CO. In HF+E&S, diastolic sarcomere length was rescued and fractional shortening increased compared to HF. Consistent with a higher Ca-transient amplitude compared to CO, diastolic and systolic [Ca²⁺] remained increased in HF+E&S. Mitochondrial redox status was rescued and, additionally, ROS production was lower in HF+E&S compared to CO. Interestingly, mitochondrial membrane potential was more stable in HF vs. HF+E&S, but more negative in HF+E&S than in CO. In isolated mitochondria, mitochondrial respiration, Ca²⁺-retention capacity using Calcium-Green, mitochondrial membrane potential using TMRM and H₂O₂ production using AmplexRed was measured. HF+E&S rescued mitochondrial respiration, with pyruvate/malate and fatty acids as substrates, to CO levels, but only partially with succinate as substrate. Mitochondrial membrane potential was more stable in HF vs. CO in the presence of pyruvate/malate. Ca²⁺-retention capacity was lowered in HF+E&S compared to HF and CO. H₂O₂ production was unchanged in the presence of HF+E&S, independent of the given substrate.

Conclusions The combined treatment of rats with HFD and L-Name is sufficient to generate calcium mishandling and mitochondrial dysfunction, typical for HFpEF. A therapy with SEMA+EMPA is able to rescue these typical HFpEF findings and reduce ROS burden. This underlines the efficiency of SEMA in combination with EMPA in the treatment of HFpEF, as shown for each of the drugs individually in clinical studies.