Empagliflozin decreases myocardial cytoplasmic Na+ through inhibition of the cardiac Na+/H+ exchanger in rats and rabbits

Abstract

Aims/hypothesis: Empagliflozin (EMPA), an inhibitor of the renal sodium-glucose cotransporter (SGLT) 2, reduces the risk of cardiovascular death in patients with type 2 diabetes. The underlying mechanism of this effect is unknown. Elevated cardiac cytoplasmic Na⁺ ([Na⁺]_c) and Ca²⁺ ([Ca²⁺]_c) concentrations and decreased mitochondrial Ca²⁺ concentration ([Ca²⁺]_m) are drivers of heart failure and cardiac death. We therefore hypothesised that EMPA would directly modify [Na⁺]_c, [Ca²⁺]_c and [Ca²⁺]_m in cardiomyocytes.

Methods: [Na⁺]_c, [Ca²⁺]_c, [Ca ²⁺]_m and Na⁺/H⁺ exchanger (NHE) activity were measured fluorometrically in isolated ventricular myocytes from rabbits and rats.

Results: An increase in extracellular glucose, from 5.5 mmol/l to 11 mmol/l, resulted in increased [Na⁺]_c and [Ca²⁺]_c levels. EMPA treatment directly inhibited NHE flux, caused a reduction in [Na⁺]_c and [Ca²⁺]_c and increased [Ca²⁺]_m. After pretreatment with the NHE inhibitor, Cariporide, these effects of EMPA were strongly reduced. EMPA also affected [Na⁺]_c and NHE flux in the absence of extracellular glucose.

Conclusions/interpretation: The glucose lowering kidney-targeted agent, EMPA, demonstrates direct cardiac effects by lowering myocardial [Na⁺]_c and [Ca²⁺]_c and enhancing [Ca²⁺]_m, through impairment of myocardial NHE flux, independent of SGLT2 activity.

Keywords: Calcium; Cardiac death; Diabetes; Glucose; Heart failure; Sodium.

Fig. 1

EMPA effects on [Na⁺]_c, [Ca²⁺]_c and [Ca²⁺]_m. (a–c) EMPA (1 μmol/l) acutely lowers (a) [Na⁺]_c, and (b) diastolic [Ca²⁺]_c and (c) systolic [Ca²⁺]_c in rabbit cardiomyocytes. (d,e) Effects of EMPA (3 h incubation) on [Na⁺]_c at (d) 11 mmol/l and (e) 5.5 mmol/l glucose (Gluc), respectively. *p < 0.05 vs 0 μmol/l EMPA, ANOVA with Dunnett’s post hoc tests. (f) Effects of EMPA (3 h pre-incubation; [EMPA] 1) on diastolic (Dias) and systolic (Sys) [Ca²⁺]_c at 11 mmol/l glucose. *p < 0.05 vs 0 μmol/l EMPA ([EMPA] 0), unpaired t test. (g) [Ca²⁺]_m as determined by the change in fluorescence ratio, YFP/CFP, relative to 0 min (t = 0) during a 15 min incubation with 1 μmol/l EMPA (white bars) or vehicle (black bars). *p < 0.05 vs vehicle at a similar time point, two-way ANOVA for repeated measures followed by post hoc contrast with Bonferroni correction at one time point. For (a–c) n = 6–8 cells from 3 rabbits per investigation; for (d–f), n = 20–30 cells from 3 rabbits per investigation; for (g) n = 8–9 cardiomyocytes from 2–3 rats for each group (EMPA or vehicle)

Fig. 2

The effects of EMPA on the NHE in the presence and absence of glucose. (a) Cariporide exerted little effect on [Na⁺]_c when preceded by EMPA inhibition. (b) Similarly, EMPA was of little effect on [Na⁺]_c when preceded by Cariporide. (c) pH traces for rabbit cardiomyocytes exposed to an acidic load (NH₄ ⁺) and during recovery for control (black solid line), Cariporide-treated (grey line) or EMPA-treated (black dashed line) cells in the presence of 11 mmol/l glucose. *p < 0.05 vs control for pH measured at 1000 s; †p < 0.05 vs Cariporide for pH measured at 1000 s, ANOVA with post hoc testing with Bonferroni corrections. (d) EMPA (1 μmol/l) acutely lowers [Na⁺ ] _c even in the absence of extracellular glucose (n = 6/3 rabbits). (e) EMPA effects on pH recovery in the absence of extracellular glucose (n = 6/3 rabbits). Control (black solid line), EMPA-treuted (black dashed line). *p < 0.05 vs control for pH measured at 1000 s, unpaired t test. n = 6 cells from 3 rabbits for all except for (c), where n = 5–6 cells from 4 rabbits