Dapagliflozin attenuates hypoxia/reoxygenation-caused cardiac dysfunction and oxidative damage through modulation of AMPK

Kun-Ling Tsai, Pei-Ling Hsieh, Wan-Ching Chou, Hui-Ching Cheng, Yu-Ting Huang, Shih-Hung Chan, Kun-Ling Tsai, Pei-Ling Hsieh, Wan-Ching Chou, Hui-Ching Cheng, Yu-Ting Huang, Shih-Hung Chan

Abstract

Background: Emerging evidence demonstrated dapagliflozin (DAPA), a sodium-glucose cotransporter 2 inhibitor, prevented various cardiovascular events. However, the detailed mechanisms underlying its cardioprotective properties remained largely unknown.

Results: In the present study, we sought to investigate the effects of DAPA on the cardiac ischemia/reperfusion (I/R) injury. Results from in vitro experiments showed that DAPA induced the phosphorylation of AMPK, resulting in the downregulation of PKC in the cardiac myoblast H9c2 cells following hypoxia/reoxygenation (H/R) condition. We demonstrated that DAPA treatment diminished the H/R-elicited oxidative stress via the AMPK/ PKC/ NADPH oxidase pathway. In addition, DAPA prevented the H/R-induced abnormality of PGC-1α expression, mitochondrial membrane potential, and mitochondrial DNA copy number through AMPK/ PKC/ NADPH oxidase signaling. Besides, DAPA reversed the H/R-induced apoptosis. Furthermore, we demonstrated that DAPA improved the I/R-induced cardiac dysfunction by echocardiography and abrogated the I/R-elicited apoptosis in the myocardium of rats. Also, the administration of DAPA mitigated the production of myocardial infarction markers.

Conclusions: In conclusion, our data suggested that DAPA treatment holds the potential to ameliorate the I/R-elicited oxidative stress and the following cardiac apoptosis via modulation of AMPK, which attenuates the cardiac dysfunction caused by I/R injury.

Keywords: AMPK; Dapagliflozin; Ischemia/reperfusion injury.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Administration of dapagliflozin (DAPA) increases the phosphorylation of AMPK and suppresses the phosphorylation of PKC under hypoxia/reoxygenation condition. The expression levels of phosphorylated AMPK in H9c2 cells treated with DAPA were enhanced in dose-dependent (a, b) and time-dependent (c, d) manners. Representative Western blot images (e) and relative densitometric bar graphs of phosphorylated-AMPK/AMPK (f) and phosphorylated-PKC/PKC (g) in H9c2 cells exposed to hypoxia for 1 h and reoxygenation for 4 h (H1R4) were shown. The data were presented as the mean ± SD of three biological replicates at three separate times. Representative Western blot image (h) and protein expression levels of phosphorylated-AMPK, AMPK, phosphorylated-PKC, and PKC in ventricular tissue from sham control, ischemia/reperfusion (I/R), and I/R plus DAPA treatment animals, eight animals in each group, were shown (i, j). (* indicating p < 0.05 compared with the control group; # indicating p < 0.05 compared to H1R4 condition or I/R without DAPA treatment)
Fig. 2
Fig. 2
Dapagliflozin (DAPA) prevents the upregulation of ROS via AMPK/ PKC/ NADPH oxidase signaling. Representative Western blot images (a) and relative densitometric bar graphs of Nox-2/Na/K ATPase (b) and Rac-1/Na/K ATPase (c) in H9c2 cells exposed to hypoxia for 1 h and reoxygenation for 4 h (H1R4) were shown. Protein expression levels of Nox-2 and β-actin in ventricular tissue from sham control, ischemia/reperfusion (I/R), and I/R plus DAPA treatment animals (d, e), eight animals in each group, were examined. Activity of NADPH oxidase in H9c2 cells (f) and primary cardiomyocytes (g) was measured. ROS generation was measured using a flow cytometry to examine the fluorescent intensity of H9c2 cells (h) and primary cardiomyocytes (i). For in vitro experiments, the data were presented as the mean ± SD of three biological replicates at three separate times. (* indicating p < 0.05 compared with the control group; # indicating p < 0.05 compared to H1R4 condition or I/R without DAPA treatment; & indicating p < 0.05 compared with the DAPA-treated cells in H1R4 condition)
Fig. 3
Fig. 3
Dapagliflozin (DAPA) prevents hypoxia/reoxygenation-induced PGC-1α downregulation and dysfunction of mitochondrial biogenesis. Representative Western blot images (a) and relative densitometric bar graphs (b) of PGC-1α/β-actin in H9c2 cells exposed to hypoxia for 1 h and reoxygenation for 4 hr (H1R4) were shown. In some cases, cells were transfected with AMPK siRNA 48 hr or pretreated with DPI before exposure to hypoxia/reoxygenation. Representative Western blot images (c) and protein expression levels of PGC-1α and β-actin in ventricular tissue from sham control, ischemia/reperfusion (I/R), and I/R plus DAPA treatment animals (d), eight animals in each group, were shown. Percentage of cells expressing JC-1 monomers (green fluorescence; FL1) (e) and JC-1 aggregates (red fluorescence; FL2) (f) were assessed using flow cytometry. Mitochondrial DNA copy numbers were examined after stimulation of hypoxia/reoxygenation in H9c2 cells (g) and primary cardiomyocytes (h). Mitochondrial DNA copy numbers in ventricular tissue from sham control, ischemia/reperfusion (I/R), and I/R plus DAPA treatment animals, eight animals in each gruop, were measured (i). For in vitro experiments, the data were presented as the mean ± SD of three biological replicates at three separate times. (* indicating p < 0.05 compared with the control group; # indicating p < 0.05 compared to H1R4 condition or I/R without DAPA treatment; & indicating p < 0.05 compared with the DAPA-treated cells in H1R4 condition)
Fig. 4
Fig. 4
Dapagliflozin (DAPA) prevents hypoxia/reoxygenation-induced apoptosis through AMPK-modulated mitochondrial-dependent pathway. Representative Western blot images (a) and relative densitometric bar graphs (b–e) of mitochondrial-dependent apoptotic markers in H9c2 cells exposed to hypoxia for 1 h and reoxygenation for 4 h (H1R4) were shown. Antiapoptotic effect of DAPA was further confirmed by caspase 3 activity (f). TUNEL assay was used for investigating apoptotic cells in H9c2 cells (g) and primary cardiomyocytes (h). The data were presented as the mean ± SD of three biological replicates at three separate times (* indicating p < 0.05 compared with the control group; # indicting p < 0.05 compared to H1R4 condition without DAPA treatment; & indicating p < 0.05 compared the DAPA-treated cells in H1R4 condition)
Fig. 5
Fig. 5
Dapagliflozin (DAPA) protects heart function on ischemia/reperfusion injury. Measurement in echocardiography, including (a) ejection fraction (EF), (b) fractional shortening (FS), (c) left ventricular end-diastolic volume (LV Vol d), (d) left ventricular end-systolic volume (LV Vol s), (e) left ventricular internal dimension at end-diastole (LVIDd), and (f) left ventricular internal dimension at end-systole (LVIDs), were shown. (g) Representative echocardiographic M-mode images from animals in different conditions revealed reduced motion of anterior wall of left ventricle caused by ischemia/reperfusion (I/R) (middle panel), which was attenuated by DAPA treatment (lower panel). Red arrows indicate the motion of anterior wall of left ventricle. The data were presented as the mean ± SD in sham control, ischemia/reperfusion (I/R), and I/R plus DAPA treatment animals, eight animals in each group (* indicating p < 0.05 compared with the control group; # indicating p < 0.05 compared to I/R without DAPA treatment)
Fig. 6
Fig. 6
Anti-apoptotic effect of dapagliflozin (DAPA) on ischemia/reperfusion injured animals. Representative images of TUNEL staining of the cardiac tissues (a) and quantification of the apoptotic areas (b) were shown. The plasma concentration of myocardial damage markers, lactate dehydrogenase (LDH) (c) and creatine kinase-MB (CK-MB) (d), in control animals and ischemia/reperfusion (I/R) animals with or without DAPA treatment were checked. The data were presented as the mean ± SD of eight animals in each group. (* indicating p < 0.05 compared with the control group; # indicating p < 0.05 compared to I/R without DAPA treatment)
Fig. 7
Fig. 7
Schematic diagram showing protective signaling of dapagliflozin (DAPA) on ischemia/reperfusion-caused cardiac dysfunction

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