Background: In patients with heart failure (HF), dysregulated Ca²⁺ and Na⁺ handling in cardiac myocytes underlie defects in systolic and diastolic function. Intracellular Na⁺ concentrations ([Na⁺]_i) are elevated in HF and deteriorate mitochondrial Ca²⁺ accumulation during b-adrenergic stimulation, since mitochondrial Ca²⁺decay is governed by a mitochondrial Na⁺/Ca²⁺ exchanger (NCLX). Since in mitochondria, Ca²⁺ is required to stimulate Krebs cycle dehydrogenases to adapt regeneration of NADH, NADPH and FAD during elevated cardiac workload, increased [Na⁺]_i provokes NAD(P)H oxidation, contributing to energetic deficit and increased emission of reactive oxygen species. Inhibitors of sodium/glucose co-transporter 2 (SGLT2) improve hospitalization for HF and cardiovascular mortality in patients with HF, and since these effects are independent of whether patients have diabetes, it has been proposed that off-target effects independent of SGLT2-inhibition may be relevant. In fact, previous studies in animal models and human myocytes reported that SGLT2-inhibitors block the cardiac Na⁺/H⁺-exchanger (NHE1), thereby lowering [Na⁺]_i in cardiac myocytes, while one other study could not observe this effect in rat cardiac myocytes. Therefore, this issue is highly controversial. The aim of this study was therefore to analyze the effect of in vivo treatment of mice with pressure overload-induced HF with the SGLT2-inhibitor Empagliflozin (EMPA) or the established NHE1-inhibitor cariporide (CAR) on [Na⁺]_i in cardiac myocytes. 

Methods: Ten weeks-old mice underwent transversal aortic constriction (TAC) or sham surgery and were treated with either EMPA (0.01mg/g), cariporide (CAR; 0.003 mg/g) or vehicle (Veh) in the drinking water, assuming a fluid intake of 0.15ml/g/d. After eight weeks of treatment, cardiac myocytes were isolated, electrically stimulated at 0.5, 2 and 4 Hz and [Na⁺]_i determined using the fluorescent Na⁺-indicator SBFI (10 µM) using an automatic fluorescence microscope (IonOptix/ CytoCyfer). While a number of additional experiments were performed in vitro and in vivo, we here report only the “top-line” results of cardiac myocyte [Na⁺]_i.

Results: Cardiac myocyte [Na⁺]_i was elevated in mice with TAC-induced HF compared to sham-operated controls (22.3 ±1.2 vs. 17.0 ±0.8 mM; TAC-Veh, n=28/ Sham-Veh, n=20; p=0.024). Treatment with the NHE1-inhibitor CAR reduced [Na⁺]_i in both TAC- and Sham-operated compared to vehicle-treated mice: TAC, 17.7 ±0.9 mM (CAR, n=59) vs. 22.3 ±1.2 mM (Veh, n=28), p<0.01; Sham, 9.6 ±0.5 mM (CAR, n=42) vs. 17.0 ±0.8 mM (Veh, n=20), p<0.0001. In contrast, EMPA did not lower [Na⁺]_i in TAC-treated animals: TAC, 19.3 ±1.1 mM (EMPA, n=57) vs. 22.3 ±1.2 mM (Veh, n=28), p=n.s.. In Sham-operated animals, EMPA even slightly increased [Na⁺]_i: 20.1 ±0.5 mM (EMPA, n=37) vs. 17.0 ±0.8 mM (Veh, n=20), p=n.s..

Conclusion: Eight weeks of in vivo treatment with the NHE1-inhibitor cariporide, but not the SGLT2-inibitor empagliflozin reduces [Na⁺]_i in cardiac myocytes of mice with or without pressure-overload induced HF. These data argue against a relevant off-target effect of empagliflozin on cardiac NHE1. Therefore, other effects than NHE1-inhibition likely underlie the beneficial effects of SGLT2-inhibitors in HF.