Cardiac SGLT2 Expression and Cell-Type-Specific Responses to Empagliflozin in iPSC-Derived Models of Diabetic Cardiomyopathy

Figures

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  Figure 1. SGLT2 protein expression in human iPSC-derived cardiomyocytes and endothelial cells. (A) Representative Western blot showing SGLT2 expression in cardiomyocytes (CM) and endothelial cells (EC) under control and diabetogenic (Diab) conditions. Human embryonic kidney (HEK) cells were included as a positive control. (B,C) Densitometry of SGLT2 protein expression in cardiomyocytes (B) and endothelial cells (C) following 48 h under control or diabetogenic conditions, normalised to calnexin. Data are presented as mean ± SEM; n = 3–4 independent experiments (B) and n = 4–5 independent experiments (C). * p < 0.05 by unpaired t-test. Calnexin: ~90 kDa; SGLT2: ~46–75 kDa.

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  Figure 2. SGLT2 protein localisation in iPSC-derived cardiomyocytes, endothelial cells, and cardiac fibroblasts. (A–C) Representative immunofluorescence images of SGLT2 (red) localisation in cardiomyocytes co-stained with cTnT (green; (A)), endothelial cells co-stained with CD31 (green; (B)), and cardiac fibroblasts co-stained with vimentin (green; (C)), under control or diabetogenic (Diab) conditions for 48 h. Cell nuclei were counterstained with DAPI (blue). Scale bar = 20 µm.

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  Figure 3. Therapeutic effects of empagliflozin on cardiomyocytes, endothelial cells, and cardiac fibroblasts. (A,C) Representative images of cardiomyocytes (A) and endothelial cells (C) under control or diabetogenic (Diab) conditions, stained with propidium iodide (PI; purple) and Hoechst 33258 (blue). Scale bar = 40 µm. (B,D) Quantification of cell death in cardiomyocytes (B) and endothelial cells (D), expressed as the percentage of PI-positive/Hoechst-positive cells; n = 6–8 from three to four independent experiments. * p < 0.05 by unpaired t-test. (E,F) Cell metabolic activity assessed by the CellTiter-Blue in cardiomyocytes (E) and endothelial cells (F) under control, diabetogenic, or diabetogenic + empagliflozin (Empa; 0.1, 1, or 10 µM) conditions for 48 h; n = 15 from six independent experiments. * p < 0.05, *** p < 0.001, and **** p < 0.0001 vs. the diabetogenic group by one-way ANOVA with Dunnett’s post hoc test. (G) Representative immunofluorescence images of cardiac fibroblasts stained for αSMA (green) and DAPI (blue) under control, diabetogenic, and diabetogenic + empagliflozin (10 µM) conditions. (H) Quantification of cardiac fibroblast activation (percentage of αSMA-positive…

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  Figure 4. Therapeutic effects of empagliflozin on 3D cardiac microtissues. (A) Representative immunofluorescence images of cardiac microtissues under control or diabetogenic (Diab) conditions showing cardiomyocytes (cTnT, red), endothelial cells (CD31, green), and cardiac fibroblasts (vimentin, green). Cell nuclei were counterstained with DAPI (blue). Scale bar = 100 µm. (B) Metabolic activity assessed by CellTiter-Glo assay. Microtissues were cultured under control or diabetogenic conditions for 48 h, after which empagliflozin (Empa; 10 µM) or vehicle control was introduced as a rescue intervention for an additional 48 h. (C) Representative single-cycle contraction traces per group. (D–G) Quantification of contraction duration (D), relaxation time (E), time-to-peak (F), and beat rate variability (RMSSD/RRint; G). Data are presented as mean ± SEM; n = 10–16 from five independent experiments. * p < 0.05 and ** p < 0.01 by one-way ANOVA with Tukey’s post hoc test.

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  Figure 5. Proposed mechanism of action based on cell-type-specific responses to empagliflozin under diabetogenic conditions. Empagliflozin increased metabolic activity in endothelial cells and reduced the activation of αSMA-positive cardiac fibroblasts, while cardiomyocyte metabolic activity remained unchanged. Empagliflozin also preserved metabolic activity in cardiac microtissues, although this did not extend to the rescue of contractile dysfunction. As endothelial dysfunction and cardiac fibrosis are major contributors to diabetic cardiomyopathy, we propose empagliflozin-responsive non-myocytes as a potential route of cardioprotection in diabetic cardiomyopathy. Created in BioRender. Su, N. (2026) https://BioRender.com/2127anf accessed on 10 July 2026.