The sodium/glucose cotransporter 2 inhibitor empagliflozin is a pharmacological chaperone of cardiac Nav1.5 channels

Abstract

Diminished peak sodium current (I_Na) is a causative factor for slowed ventricular conduction and cardiac arrhythmias in patients with Duchenne muscular dystrophy (DMD), a devastating muscle disease triggered by dystrophin deficiency. Recently, we showed that chronic administration of the sodium/glucose cotransporter 2 (SGLT2) inhibitor empagliflozin (EMPA) restores diminished peak I_Na in ventricular cardiomyocytes from the dystrophin-deficient mdx mouse model of DMD. Here, we aimed to elucidate the underlying mechanism. Whole cell patch clamp studies revealed that 24-h incubation of dystrophic (mdx) ventricular cardiomyocytes with EMPA significantly increases peak I_Na in a concentration-dependent manner (EC₅₀ = 94 nM). The enhancing effect on peak I_Na also occurred in dystrophic cardiac Purkinje fibers, as well as in dystrophic (DMD^mdx) rat cardiomyocytes, and was also exerted by other SGLT2 inhibitors. Immunofluorescence studies suggested that chronic EMPA treatment fully restores wild-type Na_v1.5 plasma membrane expression in mdx cardiomyocytes. Peak I_Na enhancement by EMPA depended on functional anterograde trafficking of Na_v1.5. The local anesthetic mexiletine, a well-known pharmacological chaperone of Na_v1.5, enhanced peak I_Na in a similar manner to EMPA. Furthermore, mutation of human Na_v1.5 at a site important for local anesthetic binding (Y1767A) completely abolished the ability of both EMPA and mexiletine to enhance peak I_Na. Finally, the importance of Y1767 for drug-induced modulation of peak I_Na was confirmed by molecular docking simulations. Our findings suggest that EMPA acts as a pharmacological chaperone of Na_v1.5 channels. Its chronic administration may reduce arrhythmia vulnerability in patients with DMD and other arrhythmogenic pathologies associated with diminished peak I_Na.NEW & NOTEWORTHY Dystrophin deficiency in cardiomyocytes leads to diminished peak Na currents. These can be fully rescued by long-term treatment with empagliflozin via pharmacochaperoning of Na_v1.5 channels.

Keywords: Duchenne muscular dystrophy; arrhythmias; cardiomyocyte sodium currents; empagliflozin; pharmacochaperone.

Figure 1. EMPA and other SGLT2 inhibitors enhance peak I_Na densities in dystrophin-deficient cardiomyocytes.

A–C: concentration-response relationship of peak I_Na enhancement by 24-h EMPA incubation in ventricular cardiomyocytes derived from mdx mice. A: typical original I_Na traces of an mdx car-diomyocyte after 24-h incubation under control conditions and after 24-h incubation with 0.1 μM or 1 μM EMPA, elicited by the pulse protocol displayed in the inset. B: from a series of such experiments [n = 12 cells (mdx control), 12 cells (mdx 0.1 μM EMPA), and 13 cells (mdx 1 μM EMPA); all cells originating from the same 3 mdx hearts], peak I_Na density-voltage relationships were derived. Data points are represented as means ± SE. Parameters for I_Na activa-tion derived from fits of the current-voltage relationships (function described in METHODS) are given in Table 1. C: peak I_Na densities at −41 mV were plotted against the EMPA concentration. Data were fit with a function given in the METHODS and yielded an EC₅₀ value of 94 nM (Hill Coeff., 1.3). D–F, effect of 24-h incubation with 1 μM EMPA on peak I_Na of cardiac Purkinje fibers derived from mdx-Cx40^{eGFP/ +} mice. D: typical original current traces of an mdx Purkinje fiber after incubation under control conditions or after EMPA treatment. E: respective peak I_Na density-voltage relationships [n = 23 cells (mdx control) and 27 cells (mdx EMPA); all cells originating from the same 4 mdx hearts]. F: dot plot comparing the maximum peak I_Na densities of untreated control and EMPA-treated mdx Purkinje fibers at −41 mV. A significant difference existed between control and EMPA-treated cells. G–I: effect of 24-h incubation with 1 μM EMPA on peak I_Na of ventricular cardiomyocytes derived from DMD^mdx rats. G: typical original current traces of a DMD^mdx rat cardiomyocyte after incubation under control conditions or after EMPA treatment. H: respective peak I_Na density-voltage relationships [n = 35 cells (DMD^mdx control) and 37 cells (DMD^mdx EMPA); all cells originating from the same 4 DMD^mdx hearts]. I: dot plot comparing the maximum peak I_Na densities of untreated control and EMPA-treated DMD^mdx cardiomyocytes at −46 mV. A significant difference existed between control and EMPA-treated cells. J–L: effect of 24-h incubation with other SGLT2 inhibitors on peak I_Na of ventricular cardiomyocytes derived from mdx mice. J: chemical structure comparison of EMPA (PubChem CID 11949646), dapagliflozin (DAPA, PubChem CID 9887712), and sotagliflozin (SOTA, PubChem CID 24831714). K: peak I_Na density-voltage relationships from untreated control mdx cardiomyocytes (n = 28 cells), mdx myocytes treated with 1 μM DAPA (n = 30 cells), and mdx myocytes treated with 1 μM SOTA (n = 29 cells, all cells originating from the same 4 mdx hearts). L: dot plot comparing the maximum peak I_Na densities of untreated control, DAPA-treated, and SOTA-treated mdx cardiomyocytes at −41 mV. A significant difference existed between control and DAPA-treated cells, as well as between control and SOTA-treated cells. DMD, Duchenne muscular dystrophy; EMPA, empagliflozin; I_Na, peak sodium current; SGLT2, sodium/glucose cotransporter 2.

Figure 2. Chronic effects of EMPA on peak I_Na in nondystrophic cells.

A–C: effect of 24-h incubation with 1 μM EMPA on peak I_Na of ventricular cardiomyocytes isolated from wild-type (wt) mice. A: typical original current traces of a wt ventricular cardiomyocyte after incubation under control conditions or after exposure to 1 μM EMPA for 24 h (for pulse protocol see Fig. 1A). B: respective peak I_Na density-voltage relationships [n = 24 cells (wt control) and 33 cells (wt EMPA); all cells originating from the same 4 wt hearts]. C: dot plot comparing the maximum peak I_Na densities of untreated control and EMPA-treated wt ventricular cardiomyocytes at −41 mV. No difference existed between control and EMPA-treated cells. D–F: effect of 24-h incubation with 10 μM EMPA (estimated free EMPA concentration: 1 μM) on peak I_Na of tsA201 cells expressing wt Na_v1.5. D: typical original current traces of a Na_v1.5-expressing tsA201 cell after incubation under control conditions or after EMPA treatment (for pulse protocol see inset). E: respective peak I_Na density-voltage relationships [n = 19 cells (control) and 20 cells (EMPA); all cells originating from the same 4 transfections]. F: dot plot comparing the maximum peak I_Na densities of untreated control and EMPA-treated Na_v1.5-expressing tsA201 cells at −46 mV. A significant difference existed between control and EMPA-treated cells. EMPA, empagliflozin; I_Na, peak sodium current.

Figure 3. Chronic exposure to EMPA fully rescues Na_v1.5 plasma membrane expression in cardiomyocytes from dystrophin-deficient mdx mice.

A: representative confocal immunofluorescence images of isolated wild-type (wt) and mdx ventricular cardiomyocytes stained using a Na_v1.5-specific rabbit primary antibody (ASC-005; Alomone Labs; dilution 1:100) and a donkey anti-rabbit secondary antibody conjugated to Alexa Fluor Plus 555 (Cat. No. A32794; Thermo Fisher Scientific; dilution 1:500). Staining of the cells was performed after a 24-h incubation under control conditions (wt control, mdx control), or after a 24-h incubation with 1 μM EMPA (mdx EMPA). B: exemplary region of interest for quantification of fluorescence, always drawn in the transmitted light image to guarantee an unbiased selection of the areas. The mean fluorescence intensity (in arbitrary units, AU) within the region of interest (one for each cardiomyocyte) was used as a marker for Na_v1.5 expression. C: mean fluorescence intensities for control cardiomyocytes from 3 wt hearts, and for control or EMPA-treated cardiomyocytes from 3 mdx hearts [n = 80 cells (wt control), n = 82 cells (mdx control), and 104 cells (mdx EMPA)]. A significant difference existed between wt control and mdx control cells, as well as between mdx control and mdx EMPA cells. Each dot represents a single cell. In addition, means ± SE are shown. EMPA, empagliflozin.

Figure 4. EMPA enhances peak I_Na densities in dystrophin-deficient mdx ventricular cardiomyocytes via facilitation of Na_v1.5 trafficking to the plasma membrane.

A–F: effects of protein synthesis inhibition and inhibition of anterograde trafficking on peak I_Na enhancement by 1 μM EMPA in mdx cardiomyocytes. A: peak I_Na density-voltage relationships from untreated control mdx cardiomyocytes (n = 28 cells), and mdx myocytes incubated for 4 h with 1 μM EMPA (n = 29 cells, all cells originating from the same 4 mdx hearts). Parameters for I_Na activation are given in Table 1. B: dot plot comparing the maximum peak I_Na densities of untreated control and 4-h EMPA-treated mdx cardiomyocytes at −41 mV. A significant difference existed between control and EMPA-treated cells. C: peak I_Na density-voltage relationships from mdx cardiomyocytes treated with 50 μg/mL of the protein synthesis inhibitor cycloheximide (CHX) for 4 h (n = 25 cells), and mdx myocytes incubated for 4 h with 50 μg/mL CHX and 1 μM EMPA (n = 29 cells, all cells originating from the same 4 mdx hearts). D: respective dot plot (at −41 mV) showing a significant difference between only CHX- and CHX-plus EMPA-treated cells. E: peak I_Na density-voltage relationships from mdx cardiomyocytes treated with 5 μg/mL of the anterograde trafficking inhibitor brefeldin A (BFA) for 24 h (n = 22 cells), and mdx myocytes incubated for 24 h with 5 μg/mL BFA and 1 μM EMPA (n = 22 cells, all cells originating from the same 3 mdx hearts). F: respective dot plot (at −41 mV), showing that the presence of BFA abolished peak I_Na enhancement by EMPA in mdx cardiomyocytes. G and H: effect of 24-h incubation with mexiletine (MEXI) on peak I_Na of ventricular cardiomyocytes derived from mdx mice. G: peak I_Na density-voltage relationships from untreated control mdx cardiomyocytes (n = 34 cells), mdx myocytes treated with 10 μM MEXI (n = 33 cells), and mdx myocytes treated with 10 μM MEXI and 1 μM EMPA (n = 33 cells, all cells originating from the same 5 mdx hearts). H: respective dot plot (at −41 mV), showing that MEXI treatment significantly enhanced the peak I_Na density in mdx myocytes. The additional presence of EMPA had no effect. CHX, cycloheximide; EMPA, empagliflozin; I_Na, peak sodium current.

Figure 5. Disruption of EMPA and mexiletine (MEXI) binding to Na_v1.5 by mutagenesis abolishes the drugs’ enhancing effect on peak I_Na.

A–F: effect of 24-h incubation with 10 μM EMPA (estimated free EMPA concentration: 1 μM) on peak I_Na of tsA201 cells expressing mutant Na_v1.5 channels. A: typical original current traces of a Na_v1.5-F1760A-expressing tsA201 cell after incubation under control conditions or after EMPA treatment. B: from a series of such experiments [n = 19 cells (control) and 19 cells (EMPA); all cells originating from the same 6 transfections], peak I_Na density-voltage relationships were derived. Parameters for I_Na activation are given in Table 1. The inset shows respective peak I_Na density-voltage relationships derived from tsA201 cells expressing wt Na_v1.5 (also see Fig. 1E). C: respective dot plot at −46 mV. D: typical original current traces of a Na_v1.5-Y1767A-expressing tsA201 cell after incubation under control conditions or after EMPA treatment. E: respective peak I_Na density-voltage relationships [n = 17 cells (control) and 17 cells (EMPA); all cells originating from the same 4 transfections]. F: dot plot at −46 mV. G–J: effect of 24-h incubation with MEXI on peak I_Na of tsA201 cells expressing mutant Na_v1.5 channels. G: peak I_Na density-voltage relationships of Na_v1.5-F1760A-expressing tsA201 cells after incubation under control conditions (n = 17 cells) or after 24-h incubation with 100 μM MEXI (n = 17 cells, all cells originating from the same 4 transfections). H: respective dot plot at −46 mV. I: peak I_Na density-voltage relationships of Na_v1.5-Y1767A-expressing tsA201 cells after incubation under control conditions (n = 17 cells) or after 24-h incubation with 100 μM MEXI (n = 17 cells, all cells originating from the same 4 transfections). J: respective dot plot at −46 mV. EMPA, empagliflozin; I_Na, peak sodium current.

Figure 6. Molecular modeling analysis.

A: location of binding residues in different Nav1.5 cryo-EM structures and the Alphafold3 model. The side-chains of F1760 and Y1767 are shown as sticks, with oxygen atoms colored in red. B: differences in the activation gate diameter [residue positions taken from Jiang et al. (36)] between opposing C-alpha atoms are shown as gray dotted lines, values in Å. Residue numbering corresponds to human Na_v1.5 numbers. C: spheres representation of the consensus binding mode of MEXI at the fenestration of DI-DIV shown in side view. D: close-up view of residues within 5 Å of the drugs, shown as sticks. Hydrogen bonds are shown as yellow dotted lines. E: spheres representation of the consensus binding mode of EMPA at the fenestration is shown in side-view. F: close-up view of residues within 5 Å of the drugs, shown as sticks. Hydrogen bonds are shown as yellow dotted lines. EMPA, empagliflozin.