Empagliflozin ameliorates diabetic cardiomyopathy probably via activating AMPK/PGC-1α and inhibiting the RhoA/ROCK pathway

Figure 1 Effects of empagliflozin on cardiac function in db/db mice. A: M-mode imaging revealed reduced systolic and diastolic function in db/db mice; B: Quantitation of E/A ratio, left ventricular ejection fraction, and left ventricular fractional shortening. NC: Normal control mice; DB: Db/db mice; EM/DB: Db/db mice treated with empagliflozin; LVEF: Left ventricular ejection fraction; LVFS: Left ventricular fractional shortening. ^aP < 0.05 vs NC; ^bP < 0.05 vs DB.

Figure 2 Effects of empagliflozin on mitochondrial injury, apoptosis, and AMP-activated protein kinase, peroxisome proliferator-activated receptor-γ coactivator-1α, and the RhoA/ROCK pathway in db/db mice. A: Left ventricular sections stained with hematoxylin and eosin and by TdT-mediated dUTP-biotin nick end labeling to assess cardiomyocyte apoptosis: The nuclei of normal cardiomyocytes were blue while the nuclei of apoptosis-positive cardiomyocytes were brown. Transmission electron micrographs showed the effects of empagliflozin on the ultrastructure of the myocardium in db/db mice; B: Quantitation of apoptotic cells; C and D: The phosphorylation of AMP-activated protein kinase and myosin phosphatase target subunit 1, as well as the protein expression of peroxisome proliferator-activated receptor-γ coactivator-1α were measured by Western blot in three groups. Bars indicate the mean ± SD from three independent experiments (n = 3). β-tubulin was set as a control for normalization. NC: Normal control mice; DB: Db/db mice; EM/DB: Db/db mice treated with empagliflozin; HE: Hematoxylin and eosin; TEM: Transmission electron micrographs; p-AMPK: Phosphorylated AMP-activated protein kinase; p-MYPT1: Phosphorylated myosin phosphatase target subunit 1; TUNEL: TdT-mediated dUTP-biotin nick end labeling. ^aP < 0.05 vs NC; ^bP < 0.05 vs DB.

Figure 3 Effects of empagliflozin, fasudil, and overexpression of peroxisome proliferator-activated receptor-γ coactivator-1α on high glucose-induced cardiomyocyte apoptosis in vitro. A: Effects of different concentrations of empagliflozin on viability of cardiomyocytes exposed to high glucose (HG); B and C: Cardiomyocyte apoptosis measured by flow cytometry; D: TdT-mediated dUTP-biotin nick end labeling staining of cardiomyocytes in each group. Bars indicate the mean ± SD from three independent experiments (n = 3). NG: Normal glucose; HG: High glucose; EM: Empagliflozin; CC: Compound C; FA: Fasudil; HG + Ad–PGC: Cells were transfected with peroxisome proliferator-activated receptor-γ coactivator-1α-GFP-Ad; HG + Ad-GFP: Cells transfected with GFP-Ad. ^aP < 0.05 vs NG; ^bP < 0.05 vs HG; ^cP < 0.05 vs HG + EM.

Figure 4 Effects of empagliflozin, fasudil, and overexpression of peroxisome proliferator-activated receptor-γ coactivator-1α on apoptotic genes and related proteins in vitro. A and B: Bax and Bcl-2 mRNA expression was quantified using real-time quantitative PCR in cardiomyocytes; C: Protein expression of active caspase-3 measured by Western blot. Bars indicate the mean ± SD from three independent experiments (n = 3). β-tubulin was set as a control for normalization. NG: Normal glucose; HG: High glucose; EM: Empagliflozin; CC: Compound C; FA: Fasudil; HG + Ad–PGC: Cells were transfected with peroxisome proliferator-activated receptor-γ coactivator-1α-GFP-Ad; HG + Ad-GFP: Cells transfected with GFP-Ad. ^aP < 0.05 vs NG; ^bP < 0.05 vs HG; ^cP < 0.05 vs HG + EM.

Figure 5 Effects of empagliflozin, fasudil, and overexpression of peroxisome proliferator-activated receptor-γ coactivator-1α on high glucose-induced mitochondrial injury and oxidative stress in vitro. A: Levels of intracellular high glucose; B and C: Changes in mitochondrial membrane potential; D: Cellular ATP production; E: Cellular superoxide dismutase activity. Bars indicate the mean ± SD from three independent experiments (n = 3). ROS: Reactive oxygen species; MMP: Mitochondrial membrane potential; SOD: Superoxide dismutase; NG: Normal glucose; HG: High glucose; EM: Empagliflozin; CC: Compound C; FA: Fasudil; HG + Ad–PGC: Cells were transfected with peroxisome proliferator-activated receptor-γ coactivator-1α-GFP-Ad; HG + Ad-GFP: Cells transfected with GFP-Ad. ^aP < 0.05 vs NG; ^bP < 0.05 vs HG; ^cP < 0.05 vs HG + EM.

Figure 6 Relationship of empagliflozin with AMP-activated protein kinase, peroxisome proliferator-activated receptor-γ coactivator-1α, and the RhoA/ROCK pathway in cardiomyocytes in HG conditions in vitro. A-D: The phosphorylation of AMP-activated protein kinase and myosin phosphatase target subunit 1, as well as the protein expression of peroxisome proliferator-activated receptor-γ coactivator-1α were measured by Western blot in three groups; E: Sodium-glucose cotransporter (SGLT)1 and SGLT2 protein expression in cardiomyocytes. Bars indicate the mean ± SD from three independent experiments (n = 3). β-tubulin was set as a control for normalization. NG: Normal glucose; HG: High glucose; EM: Empagliflozin; CC: Compound C; FA: Fasudil; HG + Ad–PGC: Cells were transfected with PGC-1α-GFP-Ad. ^aP < 0.05 vs NG; ^bP < 0.05 vs HG; ^cP < 0.05 vs HG + EM.