Cardiac Late Sodium Channel Current Is a Molecular Target for the Sodium/Glucose Cotransporter 2 Inhibitor Empagliflozin

Figures

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  Figure 1.. Empagliflozin inhibits the late component of the cardiac sodium channel current (late-INa) in heart failure and long QT syndrome 3. A, Representative whole-cell patch-clamp recording and grouped data showing the lack of effect of empagliflozin (10 µmol/L, empa) on peak, late, or total sodium currents from single cardiomyocytes isolated from sham-operated hearts. B, Representative whole-cell patch-clamp recording and grouped data showing the effects of empa (10 µmol/L) on sodium currents from single cardiomyocytes isolated from transverse aortic constriction–operated hearts in heart failure. Grouped data are from 5 to 15 cardiomyocytes per group. AUC indicates area under the curve. ***P<0.001, unpaired t test. C, Representative whole-cell patch-clamp recordings of recombinant Nav1.5 channel sodium currents from either wild-type (WT) or the long QT syndrome 3 (LQT3) R1623Q mutant. D, Representative whole-cell patch-clamp recordings showing the effects of empa (10 µmol/L) on sodium currents from the R1623Q mutant Nav1.5 channel. E and F, Grouped data showing the inhibitory effects of empa on peak- and late-INa, respectively, in the R1623Q mutant. G and H, Grouped data showing the…

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  Figure 2.. The SGLT2 inhibitors are potent inhibitors of the late component of the cardiac sodium channel current (late-INa). A and B, Representative whole-cell patch-clamp recordings of H2O2-induced late-INa and the inhibitory effects of 5 µmol/L tetrodotoxin (TTX, A) and 1 or 10 µmol/L empagliflozin (empa, B). C, Concentration-inhibition curves for empa on peak- and late-INa. D, Grouped data showing the frequency-dependent relationship of peak- vs late-INa inhibition by 1 µmol/L empa. E and F, Representative whole-cell patch-clamp recordings of H2O2-induced late-INa and the inhibitory effects of 1 or 10 µmol/L dapagliflozin (dapa, E) and canagliflozin (cana, F). G, Concentration–late-INa inhibition curves for empagliflozin, dapagliflozin, and canagliflozin. For all experiments, n=5 to 7 cells per group, *P<0.05 compared with the vehicle control, paired t test. Insets in B, E, and F are expanded sections illustrating the lack of effect of these drugs on peak-INa. AUC indicates area under the curve; IC50, half-maximal inhibitory concentration; and peak INa, maximum peak sodium current amplitude.

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  Figure 3.. Molecular modeling of empagliflozin (empa) docking to Nav1.5. A, Macroscopic structure of the human Nav1.5 homology model used in this study. B, Sites chosen for focused docking shown as colored spheres, and dotted circle denotes the central cavity region. C, Molecular Mechanics Generalized Born Solvation Area binding free energies of empa in these different sites presented in Kcal/mol. Error bars, SD. D, Empa structure (crimson) docked to the fDI-DIV and fDIII-DIV sites; Selectivity filter residues are shown in blue circles (D372, E898, K1419, and A1711). E and F, Two-dimensional interaction sketches illustrating the predicted interactions of empa with residues located in the fDI-DIV (E) and fDIII-DIV (F) sites. Residues highlighted in orange are predicted to have significant interactions with the empa molecule (Y1767 in fDI-DIV, and F1760 and W1345 in fDIII-DIV). G through J, Representative whole-cell patch-clamp recordings showing the effects of empa (10 µmol/L) on sodium currents from wild-type (WT; G) and Y1767L (H), F1760L (I), and W1345L (J) mutant Nav1.5 channels. Representative current traces in G through J were normalized to peak-INa. ***P<0.001, *P<0.05, paired t test.…

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  Figure 4.. Empagliflozin improves cardiomyocyte calcium handling induced by the late component of the cardiac sodium channel current (late-INa) activation. A through D, Representative traces showing that empagliflozin reversibly reduces the incidence of spontaneous calcium events induced by the late-INa activator veratridine. Representative 50-s sections of calcium transient recordings (1 Hz field stimulation) observed in the same myocyte under (A) control conditions, (B) during the application of 20 nmol/L veratridine, (C) with veratridine and 1 µmol/L empagliflozin, and (D) with veratridine alone after washout of empagliflozin. F through H, Representative traces of calcium transient recordings from myocytes (1 Hz field stimulation) showing that tetrodotoxin (TTX; 5 µmol/L), ranolazine (10 µmol/L) and lidocaine (50 µmol/L) also reduce the incidence of veratridine-induced spontaneous calcium events. E, Box plot of grouped data indicating the number of spontaneous calcium transients observed in 50-s segments in control conditions, during application of veratridine, or during application of veratridine and empagliflozin (n=28 myocytes from 7 heart isolations). I through K, Box plots of…

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  Figure 5.. Empagliflozin reduces NLRP3 inflammasome activation and improves postischemic contractile function. A, Representative Western blot illustrating that ischemia-reperfusion of isolated mouse hearts results in the activation of NLRP3 and TXNIP, an effect that is significantly reduced by empa or the late component of the cardiac sodium channel current inhibitor TTX. B and C, Grouped data from A (control: n=4; empa: n=5; TTX: n=6, 1-way ANOVA). D, LVDP at the baseline before (B20) and after (B40) drug treatment, at ischemia, and at 10, 20, 30 and 40 minutes reperfusion (R10, R20, R30, and R40). Two-way repeated-measures ANOVA during the reperfusion phase and 1-way ANOVA in the inset. B through D, Values represent mean±SEM. ***P<0.001, **P<0.01, *P<0.05. empa indicates empagliflozin; IR, ischemia-reperfusion; LVDP, left ventricular developed pressure; mw, molecular weight; NLRP3, nucleotide-binding domain-like receptor protein 3; TTX, tetrodotoxin; and TXNIP, thioredoxin-interacting protein.