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

Background. In heart failure with preserved ejection fraction (HFpEF) and obesity, inhibitors of the sodium dependent glucose co-transporter 2 (SGLT-2) have been shown to be particularly beneficial. However, the mechanisms behind this are still unknown. Here, empagliflozin (EMPA) was evaluated in a rat model of HFpEF (diet-induced obesity plus L-NAME) aiming to determine the effects on excitation-contraction coupling and mitochondrial energetics in the heart.

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 (HF+EMPA) via drinking water (10mg/kg/day) or regular tap water for 8-weeks (HF), and could choose between HFD and low fat diet. There were no changes for the CO group. Following this, cardiac ventricular myocytes (n=3 per group, min. 33 cardiac myocytes) and mitochondria (n=4-8 per group) were isolated. Sarcomere length, cytosolic Ca²⁺ (Indo1, AM) and mitochondrial redox state (auto fluorescence 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.

Compared to CO, the HF group showed a significantly shorter diastolic sarcomere length and increased diastolic and systolic [Ca²⁺]. 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. Treatment with EMPA rescued diastolic sarcomere length and Ca-transient amplitude to CO levels, but fractional shortening was increased. Also mitochondrial redox status was rescued and ROS production was lower in HF+EMPA compared to CO. Interestingly, mitochondrial membrane potential was more negative and stable in HF vs HF+EMPA.

In isolated mitochondria, mitochondrial respiration, Ca2+-retention capacity using Calcium-Green, mitochondrial membrane potential using TMRM, and H₂O₂ production using AmplexRed was examined. EMPA rescued mitochondrial respiration, independent of the given substrate (pyruvate/malate, succinate, fatty acid-mix) to CO levels, while mitochondrial membrane potential was more stable in HF vs. CO in the presence of pyruvate/malate. Ca²⁺-retention capacity was lowered in HF compared to CO. H₂O₂ production was not different in EMPA treated rats compared to CO.

Conclusions

The combined treatment of rats with HFD and L-Name is sufficient to generate calcium mishandling and mitochondrial dysfunction, typically for HFpEF. Treatment with EMPA rescues these typical HFpEF findings, reduces ROS burden and improves mitochondrial dysfunction. This is in line with beneficial effects of empagliflozin in HFpEF patients shown in clinical studies.