Comparison of survival rates in DM-HFrEF model zebrafish treated with empagliflozin or sotagliflozin.

Survival analysis of DM-HFrEF zebrafish larvae after treatment with 0.2, 1, 5 and 25 μM empagliflozin or sotagliflozin. a, c Kaplan‒Meier survival curves (n = 120 larvae per group). b, d Ccross-sectional comparisons at the time points of 8 and 9 dpf and are presented as the mean ± standard deviation; each dot represents the value from one experiment (n = 3–6 experiments per group). The non-DM and DM groups were tested under the same conditions on the same day, and the graph is shown in Fig. 1 and Supplementary Fig. 2. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. the indicated group.

Comparison of locomotion in DM-HFrEF zebrafish treated with empagliflozin or sotagliflozin.

a Representative locomotion tracking images of zebrafish larvae for a 5-minute period. b, c Average movement distance and movement duration per 1 min (n = 12 larvae per group). Data are presented as the mean ± standard deviation, and each dot represents the value for one zebrafish larva. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. the indicated group.

Comparison of morphology in DM-HFrEF zebrafish treated with empagliflozin or sotagliflozin.

Representative morphological images. a Non-DM, b non-DM with HFrEF, c DM, d DM-HFrEF. e–l DM-HFrEF zebrafish following treatment with e 0.2 μM, f 1 μM, g 5 μM and h 25 μM empagliflozin or i 0.2 μM, j 1 μM, k 5 μM, and l 25 μM sotagliflozin. a The pericardium is indicated by a black dashed line, the swim bladder by a white dashed line, l the pericardial edema by a black arrow, and uninflated swim bladder by a white arrow.

Comparison of cardiac contraction in DM-HFrEF zebrafish treated with various concentrations of empagliflozin or sotagliflozin.

a Representative fluorescence microscopy images of TG (myl7:EGFP) zebrafish hearts at end-diastole and end-systole of ventricle. b Ventricular fractional shortening calculated based on fluorescent images (n = 8–25 larvae per group). c Representative blood flow graphs. d Standard deviation of the beat-to-beat interval analyzed based on blood flow (n = 14–25 larvae per group). b, d Data are presented as the mean ± standard deviation. ****p < 0.0001 vs. control group.

Comparison of structural binding and functional inhibitory effects of empagliflozin and sotagliflozin on NHE1.

a, b Molecular docking analysis of empagliflozin, sotagliflozin, cariporide and D-glucose binding to a structural model of zebrafish NHE1 in silico. Empagliflozin is shown in blue, sotagliflozin is shown in green, cariporide is in purple, and D-glucose is in gray. D-glucose was used as a negative control, and cariporide was used as a positive control. a Binding site and b affinities. c Schematic illustration of the drug affinity responsive target stability (DARTS) assay in vitro. d–f DARTS analysis by immunoblotting with NHE1 and GAPDH antibodies (n = 3 samples per group). g–j Measurement of intracellular Na⁺ and Ca²⁺ to analyze the functional inhibitory effects of empagliflozin and sotagliflozin on NHE1. g Changes in intracellular Na⁺ and h Ca²⁺ concentrations in cardiomyocyte under HG condition were evaluated after treatment with (n = 14–27 samples per group). i, j Changes in intracellular Na⁺ and Ca²⁺ concentrations in response to empagliflozin or sotagliflozin treatment in cells pretreated with cariporide (n = 11–20 samples per group). Data are presented as the mean ± standard deviation, and each dot represents the value of each sample. e, i *p < 0.05, ***p < 0.001 vs. the indicated group. g, h^####p < 0.0001 vs. the LG group, **p < 0.01, ****p < 0.0001 vs. the HG group.