Empagliflozin inhibits cardiac late sodium current via Ca/Calmodulin-dependent kinase II

Recently, it was published in Circulation that empagliflozin inhibits H₂O₂-induced cardiac late sodium current (late I_Na).¹ Using computational modeling and point mutagenic approaches, Philippaert et al. suggested a possible site of empagliflozin-binding within Na_V1.5 similar to that of local anesthetics, supportive of direct drug binding to Na_V1.5, although this remains to be determined conclusively and alternative mechanisms may exist.¹ We have previously shown that Ca/calmodulin-dependent kinase II (CaMKII) binds to Na_V1.5, stimulates late I_Na and affects its H₂O₂-dependent regulation.^2,3 We also demonstrated that empagliflozin inhibits CaMKII in failing human and murine cardiomyocytes.⁴

Here we show that inhibition of H₂O₂-induced late I_Na by empagliflozin cannot solely be mediated via direct drug binding but depends on CaMKII-dependent phosphorylation of Na_V1.5 at serine 571. We demonstrate that empagliflozin inhibits late I_Na in patients with aortic stenosis (AS) and phenotypic features of heart failure (HF) with preserved ejection fraction (HFpEF).

Raw data/analytic methods can be made available for purposes of reproducing results or replicating procedures. Human tissue/proprietary antibodies cannot be made available for legal constraints. Experiments conform to the Declaration of Helsinki. Human/murine studies were approved by institutional committee. Written informed consent was obtained from patients prior to tissue donation. LV samples were obtained from septal resections of 11 patients (8 male/ 3 female, aged 69.3±2.6) with AS undergoing valve replacement. Patients had a HFpEF-like phenotype with hypertrophy and preserved EF (59.4±1.7%). Murine models of CaMKIIδ knock-out (CaMKIIδ^−/−)³, inhibition of CaMKII-dependent Na_V1.5 phosphorylation at serine 571 (S571A), and with CaMKII phospho-mimetic Na_V1.5 S571E mutation were tested for involvement of CaMKII-Na_V1.5 phosphorylation. Isolated ventricular myocytes were incubated (30 min) with empagliflozin (1 μmol/l) or control (DMSO). Some cardiomyocytes were incubated with inhibitors of open-state Na channel inactivation (ATX-II or veratridine) or lidocaine (100 μmol/l, 30 min) for direct Na channel inhibition. H₂O₂ (100 μmol/l, 5 min) was used to induce reactive oxygen species, which stimulate late I_Na in HF via CaMKII³ (tested with CaMKII-inhibitor myristoylated-AiP; 2 μmol/l, 30 min). For some experiments, empagliflozin was washed-in to ATX-II or H₂O₂ pre-incubated myocytes.

Late I_Na was measured as described previously^2,3. Resting membrane potential was held at -120 mV and I_Na elicited by depolarizing to -20 mV for 1000 ms, quantified by integrating from 100 to 500 ms of the start of depolarization (normalized to membrane capacitance). Western blots used human ventricular tissue exposed to empagliflozin/vehicle (30 min)⁴. Data were analyzed using mixed-effects analysis with Holm-Sidak, linear mixed model with random factor ‘individual’ and Sidak correction, or paired t-test (GraphPad Prism 9).

We demonstrate that late I_Na can be reduced by empagliflozin in ventricular myocytes from patients with AS similar to CaMKII-inhibitor AiP (figure 1A). ATX-II-dependent (figure 1B) enhancement of late I_Na in murine wildtype cardiomyocytes was not affected by empagliflozin (not even at 10 and 100 μmol/l), nor after wash-in (at 1 μM) to ATX-II pre-incubated myocytes, which would be expected if empagliflozin were a direct Na_V1.5 inhibitor. Moreover, wash-in of empagliflozin (up to 10 μM) also did not inhibit late I_Na in myocytes preincubated with a moderate concentration of veratridine (16 nM, experimentally determined as EC50 by dose-response, data not shown). In sharp contrast, both veratridine and ATX-II-enhanced late I_Na were blocked by lidocaine (not shown). Empagliflozin robustly inhibited H₂O₂-induced late I_Na, (figure 1C), with maximal efficacy at 6 min but not already at 2 min after onset of exposure (late I_Na integral during wash-in: 0 min −50.8±4.3 A*F⁻¹*ms, 2 min: −39.9±4.6 A*F⁻¹*ms, p=0.0934 vs. 0 min; 4 min: −24.2±4.6 A*F⁻¹*ms, p=0.0007 vs. 0 min; 6 min: −17.2±4.0 A*F⁻¹*ms, p<0.0001 vs. 0 min). No additional effect of empagliflozin on late I_Na was observed upon AiP (not shown), or in myocytes lacking either CaMKIIδ (CaMKIIδ^−/−) or CaMKII-dependent Na_V1.5 phosphorylation at serine 571 (S571A, figure 1C). Accordingly, the enhanced late I_Na in mice with CaMKII phospho-mimetic Na_V1.5 S571E was neither blocked by empagliflozin nor AiP (figure 1D). In contrast, lidocaine inhibited late I_Na in S571E cells, underscoring that empagliflozin primarily acts via CaMKII-Na_V1.5 phosphorylation. Empagliflozin dose-response revealed an IC₅₀ for inhibition of H₂O₂-dependent late I_Na of 0.086 μmol/l in murine myocytes (not shown). Empagliflozin inhibited CaMKII-autophosphorylation and CaMKII-dependent phosphorylation of Na_V1.5 in AS and HF (figure 1E+F).

Figure 1:

A) Original recordings and mean data of empagliflozin- or AiP-mediated inhibition of late I_Na in human ventricular cardiomyocytes from patients with AS (n=patients). B) Original recordings and mean data of late I_Na in murine cardiomyocytes from WT or CaMKIIδ^−/− mice (n=cells/mice). The ATX-dependent enhancement of late I_Na could not be blocked by empagliflozin. C) In contrast, the H₂O₂-dependent stimulation of late I_Na was blocked by either CaMKII inhibition (AiP, CaMKII^−/−), transgenic inhibition of CaMKII-dependent Na_V1.5 phosphorylation (S571A) or in the presence of empagliflozin. D) In contrast to local anesthetic lidocaine, neither empagliflozin nor AiP could block enhanced late I_Na in mice with phosphomimetic substitution of glutamic acid for serine at 571 (S571E). E+F) Western blots of cardiomyocytes upon empagliflozin shows reduced CaMKII-autophosphorylation (T287) and reduced CaMKII-dependent Na_V1.5 phosphorylation.

For comparison of multiple groups, mixed-effects analysis+Holm-Sidak (1A) or linear mixed model+Sidak were performed. For comparison of two groups, paired t-test was done (1F).

In conclusion, inhibition of late I_Na by empagliflozin is at least in part due to inhibition of CaMKII-dependent regulation of Na_V1.5^2,4. If cardiac Na channels were solely directly inhibited, empagliflozin, like local anesthetics, should have blocked ATX-II/veratridine-stimulated late I_Na, but it did not. Nevertheless, the target of empagliflozin in the heart remains unclear⁵ and further research is needed to better understand direct vs. indirect effects on late I_Na. We demonstrate that empagliflozin also inhibits late I_Na in patients with AS and features of HFpEF, which may reduce the propensity for arrhythmias and contribute to the positive results of the EMPEROR-Preserved trial.