Empagliflozin suppressed cardiac fibrogenesis through sodium-hydrogen exchanger inhibition and modulation of the calcium homeostasis

Fig. 1. Cell migration, collagen production, myofibroblast differentiation, and proliferation capabilities of atrial fibroblasts treated with empagliflozin. A Photographs and averaged data revealed the migration assay results of atrial fibroblasts treated with empagliflozin (1 or 5 μmol/L). The left upper panels displayed the initial scratch (baseline) in different groups. Left lower panels displayed the images 6 h after the scratch was created (after migration) (n = 6 independent experiments, statistic test by one-way repeated measures ANOVA). B Photographs and averaged data revealed expression of pro-collagen type I, III, and α-smooth muscle actin (SMA) (n = 6 independent experiments, statistic test by one-way repeated measures ANOVA) in control and empagliflozin (1 or 5 μmol/L)-treated atrial fibroblasts. GAPDH was used as a loading control. C Empagliflozin treatment for 24 h had no significant effect on the proliferation rate of atrial fibroblasts (n = 6 independent experiments, statistic test by one-way repeated measures ANOVA). * p < 0.05, # p < 0.01, $ p < 0.005

Fig. 2. Intracellular Ca2+ signaling in empagliflozin-treated atrial fibroblasts. A Representative intracellular Ca2+ tracing from control (left upper panels), and empagliflozin (1 μmol/L, right upper panels)-treated atrial fibroblasts. Where indicated, thapsigargin was added to the calcium-free buffer to induce ER Ca2+ depletion. After the intracellular Ca2+ surge induced by thapsigargin (ER calcium) was returned to the steady state, the extracellular Ca2+ concentration was then increased to 2 mmol/L to measure Ca2+ entry. F340/F380 was expressed as the relative intracellular Ca2+. The left lower panel displays the change (∆ F340/F380) of Ca2+ measured by area under curve of Ca2+ tracing (AUC, statistic test by unpaired t-test), the change from baseline to peak calcium amplitude (statistic test by Mann–Whitney Rank Sum Test), and the decay time of Ca2+ entry (T50, calculated from the peak to the 50% of the decay or the end of the calcium image recording, statistic test by unpaired t-test) in control (n = 5) and empagliflozin-treated atrial fibroblasts (n = 5). B Averaged data of the levels of IP3 in the control cells and fibroblasts treated with empagliflozin (1 μmol/L) for 48 h (n = 6…

Fig. 3. Effects of empagliflozin on Na+/H+ exchanger (NHE) and downstream signaling. A Averaged data of the intracellular pH in the control cells and empagliflozin (1 μmol/L)-treated atrial fibroblasts cotreated with or without cariporide (10 μmol/L) for 48 h (n = 6 experiments, statistic test by one-way repeated measures ANOVA). B Photographs and averaged data of the expression of pro-collagen type I, type III, phosphorylated phospholipase C (pPLC), and NHE1 protein in control cells and empagliflozin (1 μmol/L)-treated atrial fibroblasts cotreated with or without cariporide (10 μmol/L) for 48 h (n = 6 experiments, statistic test by one-way repeated measures ANOVA). GAPDH was used as loading control. * p < 0.05, $ p < 0.005

Fig. 4. The effects of Na+/H+ exchanger inhibitor (cariporide) on empagliflozin decreased migration in atrial fibroblasts. Photographs and averaged data present the results of migration assay for empagliflozin (1 μmol/L)-treated atrial fibroblasts treated with or without cariporide (10 μmol/L). Upper panels display the initial scratch (baseline) in different groups. Lower panels display the images 6 h after the scratch was created (after migration) (n = 6 independent experiments, statistic test by one-way repeated measures ANOVA). * p < 0.05, # p < 0.01, $ p < 0.005

Fig. 5. Intracellular Ca2+ signaling in empagliflozin or cariporide-treated atrial fibroblasts. Representative intracellular Ca2+ tracing from cariporide alone (10 μmol/L, left upper panels), and cariporide (10 μmol/L) mixed with empagliflozin (1 μmol/L, right upper panels). Where indicated, thapsigargin was added to the calcium-free buffer to induce ER Ca2+ depletion. After the intracellular Ca2+ surge induced by thapsigargin (ER calcium) was returned to the steady state, the extracellular Ca2+ concentration was then increased to 2 mmol/L to measure Ca2+ entry. F340/F380 was expressed as the relative intracellular Ca2+. The left lower panel displays the change (∆ F340/F380) of Ca2+ measured by area under curve of Ca2+ tracing (AUC, statistic test by unpaired t-test), the change from baseline to peak calcium amplitude (statistic test by statistic test by unpaired t-test), and the decay time of Ca2+ entry (T50, calculated from the peak to the 50% of the decay or the end of the calcium image recording, statistic test by unpaired t-test) in cariporide alone (n = 5) and cariporide mixed with empagliflozin-treated atrial fibroblasts (n = 5)

Fig. 6. Effects of empagliflozin on rats with isoproterenol-induced heart failure (HF). A Schematic summarizing the treatment protocol for Wistar rats with isoproterenol (100 mg/kg, subcutaneous injection)-induced HF receiving vehicles, HF rats receiving empagliflozin (10 mg/kg/day orally for 28 consecutive days), and control rats. B averaged data present the results of serum levels of ß-hydroxybutyrate (statistic test by one − way ANOVA) in HF rats receiving vehicles (n = 5), HF rats receiving empagliflozin (n = 5), and control rats (n = 5). C Photographs reveal atrial fibrosis (stained with blue color) studied using Masson’s trichrome staining (statistic test by one − way ANOVA) in left atrium (LA) tissues from different groups. Control rats (n = 5) and HF rats receiving empagliflozin (n = 5) exhibited less severe LA fibrosis than vehicle-treated HF rats (n = 5). The fibrosis levels of LA tissues were expressed as the collagen volume fraction, that is, the ratio of the LA total collagen surface area stained blue to the LA total surface area. * p < 0.05, # p < 0.01, $ p < 0.005

Fig. 7. Effects of empagliflozin on heart structure, systolic function, and heart rate of rats with isoproterenol-induced heart failure (HF). Photographs and averaged data present the results of left ventricle fractional shortening (LVFS, statistic test by one − way ANOVA), LV posterior wall (LVPW, statistic test by one − way ANOVA), LV end-diastolic diameter (LVEDD, statistic test by ANOVA on ranks), LV end-systolic diameter (LVESD, statistic test by one − way ANOVA), and heart rate (HR, statistic test by one − way ANOVA) in HF rats receiving vehicles (n = 5), HF rats receiving empagliflozin (n = 5), and control rats (n = 5). * p < 0.05, $ p < 0.005

Fig. 8. The proposed molecular mechanism underlying the anti-fibrotic effects of empagliflozin on atrial fibroblasts. By inhibiting the Na+-H+ exchanger (NHE), empagliflozin decreases the expression of phosphorylated phospholipase C (PLC) and inositol trisphosphate (IP3) production thereby reducing ER Ca2+ release, extracellular Ca2+ entry and decreasing profibrotic cellular activities of atrial fibroblasts