Empagliflozin reduces Ca/calmodulin-dependent kinase II activity in isolated ventricular cardiomyocytes

Abstract

Aims: The EMPA-REG OUTCOME study showed reduced mortality and hospitalization due to heart failure (HF) in diabetic patients treated with empagliflozin. Overexpression and Ca²⁺ -dependent activation of Ca²⁺ /calmodulin-dependent kinase II (CaMKII) are hallmarks of HF, leading to contractile dysfunction and arrhythmias. We tested whether empagliflozin reduces CaMKII- activity and improves Ca²⁺ -handling in human and murine ventricular myocytes.

Methods and results: Myocytes from wild-type mice, mice with transverse aortic constriction (TAC) as a model of HF, and human failing ventricular myocytes were exposed to empagliflozin (1 μmol/L) or vehicle. CaMKII activity was assessed by CaMKII-histone deacetylase pulldown assay. Ca²⁺ spark frequency (CaSpF) as a measure of sarcoplasmic reticulum (SR) Ca²⁺ leak was investigated by confocal microscopy. [Na⁺ ]_i was measured using Na⁺ /Ca²⁺ -exchanger (NCX) currents (whole-cell patch clamp). Compared with vehicle, 24 h empagliflozin exposure of murine myocytes reduced CaMKII activity (1.6 ± 0.7 vs. 4.2 ± 0.9, P < 0.05, n = 10 mice), and also CaMKII-dependent ryanodine receptor phosphorylation (0.8 ± 0.1 vs. 1.0 ± 0.1, P < 0.05, n = 11 mice), with similar results upon TAC. In murine myocytes, empagliflozin reduced CaSpF (TAC: 1.7 ± 0.3 vs. 2.5 ± 0.4 1/100 μm^-1 s^-1 , P < 0.05, n = 4 mice) but increased SR Ca²⁺ load and Ca²⁺ transient amplitude. Importantly, empagliflozin also significantly reduced CaSpF in human failing ventricular myocytes (1 ± 0.2 vs. 3.3 ± 0.9, P < 0.05, n = 4 patients), while Ca²⁺ transient amplitude was increased (F/F₀ : 0.53 ± 0.05 vs. 0.36 ± 0.02, P < 0.05, n = 3 patients). In contrast, 30 min exposure with empagliflozin did not affect CaMKII activity nor Ca²⁺ -handling but significantly reduced [Na⁺ ]_i .

Conclusions: We show for the first time that empagliflozin reduces CaMKII activity and CaMKII-dependent SR Ca²⁺ leak. Reduced Ca²⁺ leak and improved Ca²⁺ transients may contribute to the beneficial effects of empagliflozin in HF.

Keywords: Ca leak; CaMKII; Calcium; Empagliflozin; Heart failure.