Empagliflozin reduces the senescence of cardiac stromal cells and improves cardiac function in a murine model of diabetes

Abstract

The sodium-glucose cotransporter 2 (SGLT2) inhibitor empagliflozin reduces heart failure in diabetes, but underlying mechanisms remain elusive. We hypothesized that empagliflozin could counteract the senescence of cardiac stromal cells (CSC), the action of which limits cardiac damage and cardiac fibrosis in diabetic-like conditions in vitro and in vivo. CSC were isolated from murine heart biopsies (n = 5) through cardiosphere (CSp) formation and incubated for 3 or 48 hours with 5.5 mmol/L normal glucose (NG), high glucose (12-5 and 30.5 mmol/L, HG) or a hyperosmolar control of mannitol (HM) in the presence or absence of empagliflozin 100 nmol/L. The senescent CSC status was verified by β-gal staining and expression of the pro-survival marker Akt (pAkt) and the pro-inflammatory marker p38 (p-P38). The cardiac effects of empagliflozin were also studied in vivo by echocardiography and by histology in a murine model of streptozotocin (STZ)-induced diabetes. Compared to NG, incubations with HG and HM significantly reduced the number of CSps, increased the β-gal-positive CSC and P-p38, while decreasing pAkt, all reversed by empagliflozin (P < .01). Empagliflozin also reversed cardiac dysfunction, cardiac fibrosis and cell senescence in mice with (STZ)-induced diabetes (P < .01). Empagliflozin counteracts the pro-senescence effect of HG and of hyperosmolar stress on CSC, and improves cardiac function via decreasing cardiac fibrosis and senescence in diabetic mice, possibly through SGLT2 off-target effects. These effects may explain empagliflozin unexpected benefits on cardiac function in diabetic patients.

Keywords: cardiac function; cardiac stromal cells; diabetes; empagliflozin; glucose; senescence.

Conflict of interest statement

The authors declare no conflict of interest.

© 2020 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.

Figures

Figure 1

Effects of high glucose and high mannitol with or without empagliflozin on cardiosphere number and senescence of murine cardiac stromal cells. Panels A and B, Quantitation of murine cardiospheres in multiple microscope fields, treated with normal glucose (5.5 mmol/L) or high glucose (12.5 and 30 mmol/L) or high mannitol (7.0 and 25 mmol/L) for 3 h (panel A) and 48 h (Panel B), in the presence or absence of 100 nmol/L empagliflozin. The graph bars showing the total number of cardiospheres over 5 microscope fields per sample represent means ± standard deviations (n = 3 independent experiments). Panel C, Quantitation of senescent cells in multiple microscope fields from murine cardiac stromal cells (CSC) treated with low (5.5 mmol/L) or high glucose (30 mmol/L) or high mannitol (25 mmol/L) for 48 h, in the presence or absence of 100 nmol/L empagliflozin. The graph bars, showing the ratio between the number of senescence‐associated β‐galactosidase (SA‐βGal) positive cells and the total number of cells over 5 microscope fields per sample, represent means ± standard deviations (n = 3 independent experiments). Panel D, Representative SA‐βGal staining images of CSC treated with normal glucose (5.5 mmol/L) or high glucose (30 mmol/L) or high mannitol (25 mmol/L) for 48 h, in the presence or absence of 100 nmol/L empagliflozin, as indicated by the blue staining (arrows). All images are shown at 10× magnification. *P < .05 and **P < .01 vs. normal glucose; °P < .05 and °°P < .01 vs without empagliflozin. E, empagliflozin; Glu, glucose; Man, mannitol; SA‐βGal, senescence‐associated β‐galactosidase

Figure 2

Effects of high glucose and high mannitol with or without empagliflozin on the expression of pAkt, P‐P38, IR‐1α and IRS‐1 in murine cardiac stromal cells. Western analysis of the pro‐survival marker pAkt and AKT (A), pro‐inflammatory marker P‐P38 (B), IR‐1α (C), IRS‐1 and pIRS‐1 (D) in murine cardiac stromal cells treated with normal glucose (5.5 mmol/L) or high glucose (12.5 and 30 mmol/L) or high mannitol (7.0 and 25 mmol/L) for 48 h, in the presence or absence of 100 nmol/L empagliflozin, with β‐actin serving as a loading control. The graph bars represent means ± standard deviations (n = 3 independent experiments). **P < .01 vs normal glucose; °°P < .01 vs without empagliflozin. Glu, glucose; IR, insulin receptor; IRS, Insulin receptor substrate; Man, mannitol; pAkt, phosphorilated Akt; P‐p38, phosphorylated p38

Figure 3

Effects of streptozotocin and empagliflozin on cardiac function in mice. Panels A, Ejection Fraction (EF) and B, Fractional Shortening (FS) measured by echocardiography in the different treatment groups such as control (vehicle), streptozotocin‐treated mice and streptozotocin + empagliflozin‐treated mice. Panels C, Representative M‐mode and B‐mode images recorded by echocardiography in the different treatment groups. Data are expressed as means ± standard deviations (n = 5 mice per treatment group). **P < .01 vs vehicle‐treated mice; °°P < .01 vs without empagliflozin. E, empagliflozin; STZ, streptozotocin

Figure 4

Effects of streptozotocin and empagliflozin on cardiac fibrosis in mice. Panels A‐C, Picro‐sirius red staining showing the collagen content in left ventricle sections from control (vehicle), streptozotocin‐treated mice and streptozotocin + empagliflozin‐treated mice. All images are shown at 20× original magnification. Panel D, Collagen content quantified on picro‐sirius red staining using the ImageJ software. Panel E, Quantification of collagen type III expression by western analysis. Panel F, Representative western blot showing type III collagen expression in each treatment group. β‐actin levels were assessed as a loading control. Data are expressed as means ± standard deviation (n = 5 mice per treatment group). **P < .01 vs vehicle‐treated mice; °°P < .01 vs without empagliflozin. E, empagliflozin; STZ, streptozotocin

Figure 5

Effects of streptozotocin and empagliflozin on cardiac senescence‐associated β‐galactosidase activity in mice. Panels A‐C, Senescence β‐galactosidase (SA β‐Gal) staining (magnification 5× and 40×) and quantitative evaluations (Panel D) in cardiac tissue. Data are expressed as means ± standard deviations (n = 5 mice per treatment group). **P < .01 vs vehicle‐treated mice; °°P < .01 vs without empagliflozin. E, empagliflozin; STZ, streptozotocin

Figure 6

A scheme of the putative signalling pathway through which empagliflozin target cardiac stromal cell senescence induced by hyperglycaemia and hyperosmotic, based on findings from the in vitro part of the present study. In the plasma membrane, hyperglycaemia induces hyperosmotic stress, and together, these metabolic and biophysical stressors impair the ability of cardiac stromal cells to respond to insulin. This causes the perturbation of the pro‐survival PI3K/Akt and pro‐inflammatory p38 pathways with insulin preserving its signalling capacity through the mitogenic and pro‐inflammatory arm, leading to the development and progression of a pro‐senescent phenotype. Empagliflozin is able to revert CSC senescence by targeting the insulin signalling pathway