Cardioprotective Effects of Sodium Glucose Cotransporter 2 Inhibition in Angiotensin II-Dependent Hypertension Are Mediated by the Local Reduction of Sympathetic Activity and Inflammation

Abstract

The cardioprotective effects of sodium glucose cotrasponter 2 (SGLT2) inhibitors seem to be independent from the effects on glycemic control, through little-known mechanisms. In this study, we investigate whether the cardioprotective effects of empagliflozin, a SGLT2 inhibitor, may be associated with myocardial sympathetic activity and inflammatory cell infiltration in an experimental model of angiotensin II-dependent hypertension. Angiotensin II (Ang II), Ang II plus Empagliflozin, physiological saline, or physiological saline plus empagliflozin were administered to Sprague Dawley rats for two weeks. Blood pressure was measured by plethysmographic method. Myocardial hypertrophy and fibrosis were analysed by histomorphometry, and inflammatory cell infiltration and tyrosine hydroxylase expression, implemented as a marker of sympathetic activity, were evaluated by immunohistochemistry. Ang II increased blood pressure, myocardial hypertrophy, fibrosis, inflammatory infiltrates and tyrosine hydroxylase expression, as compared to the control group. Empagliflozin administration prevented the development of myocardial hypertrophy, fibrosis, inflammatory infiltrates and tyrosine hydroxylase overexpression in Ang II-treated rats, without affecting blood glucose and the Ang II-dependent increase in blood pressure. These data demonstrate that the cardioprotective effects of SGLT2 inhibition in Ang II-dependent hypertension may result from the myocardial reduction of sympathetic activity and inflammation and are independent of the modulation of blood pressure and blood glucose levels.

Keywords: SGLT2-inhibitors; angiotensin II; hypertension; inflammatory infiltrates; myocardial fibrosis; myocardial hypertrophy; rats; tyrosine hydroxylase.

Figure 1

Myocardial hypertrophy. Effect of Empagliflozin administration on myocardial hypertrophy in Ang II-treated rats. (a) Representative images of microscopic transverse sections ((a) left panel) and related microscopic fields ((a) right panel) that show left ventricular hypertrophy with related enlarged cardiomyocyte nuclei. Major and minor nuclear diameters were measured to evaluate the nuclear volume, as described in the Methods. Control (A), control + Empa (B), Ang II (C), Ang II + Empa-treated (D) rats (scale bar 100 µm). (b) Quantification of myocardial hypertrophy in the different groups of rats. Data are means ± SEM. *: p < 0.05. §: p <  0.01.

Figure 2

Myocardial interstitial fibrosis. Effects of Empagliflozin administration on myocardial interstitial fibrosis in Ang II-treated rats. (a) Representative photomicrographs of interstitial fibrosis (Sirius red stain; ((a) left panel)) in control (A), control + Empa (B), Ang II (C), Ang II + Empa-treated (D) rats (scale bar 200 µm). Myocardial interstitial fibrosis is caused by an increase in type I collagen fibers, which appear orange/yellow under polarized-light microscopy ((a) right panel). (b) Quantification of interstitial fibrosis in the different groups of rats. Data are means  ±  SEM. §: p  < 0.01. &: p <  0.0001.

Figure 3

Myocardial inflammatory cell infiltration. Effects of Empagliflozin administration on myocardial inflammatory cell infiltration in Ang II-treated rats. (a) Immunohistochemical identification of interstitial monocyte/macrophage infiltration (indicated by the arrows, brown reaction) in control (A), control + Empa (B), Ang II (C), Ang II + Empa-treated (D) rats (scale bar 200 µm). (b) Quantification of staining of myocardial monocyte/macrophage inflammatory cells in the different groups of rats. Data are means  ±  SEM. * p < 0.05. §: p  < 0.01. &: p < 0.0001.

Figure 4

Myocardial tyrosine hydroxylase expression. Effects of Empagliflozin administration on myocardial tyrosine hydroxylase expression in Ang II-treated rats. (a) Immunohistochemical identification of tyrosine hydroxylase expression at intraparenchymal nerve fiber level (indicated by the arrows, brown reaction) in control (A), control + Empa (B), Ang II (C), Ang II + Empa-treated (D) rats (scale bar 100 µm). The images in the white boxes have been enlarged ((a) right panel) (b) Quantification of staining of myocardial tyrosine hydroxylase expression in the different groups of rats. Data are means  ±  SEM. §: p < 0.01.