Empagliflozin inhibits excessive autophagy through the AMPK/GSK3β signaling pathway in diabetic cardiomyopathy

Background and aims: Sodium-glucose cotransporter 2 (SGLT2) inhibitors have beneficial effects on heart failure and cardiovascular mortality in diabetic and nondiabetic patients, with unclear mechanisms. Autophagy is a cardioprotective mechanism under acute stress conditions, but excessive autophagy accelerates myocardial cell death leading to autosis. We evaluated the protective role of empagliflozin (EMPA) against cardiac injury in murine diabetic cardiomyopathy. Methods and results: Male mice, rendered diabetics by one single intraperitoneal injection of streptozotocin and treated with EMPA (30 mg/kg/day) had fewer apoptotic cells (4.9 ± 2.1 vs 1 ± 0.5 TUNEL-positive cells %, p < 0.05), less senescence (10.1 ± 2 vs 7.9 ± 1.2 β-gal positivity/tissue area, p < 0.05), fibrosis (0.2 ± 0.05 vs 0.15 ± 0.06, p < 0.05 fibrotic area/tissue area), autophagy (7.9 ± 0.05 vs 2.3 ± 0.6 fluorescence intensity/total area, p < 0.01), and connexin (Cx)-43 lateralization compared with diabetic mice. Proteomic analysis showed a downregulation of the 5' adenosine monophosphate-activated protein kinase (AMPK) pathway and upstream activation of sirtuins in the heart of diabetic mice treated with EMPA compared with diabetic mice. Because sirtuin activation leads to modulation of cardiomyogenic transcription factors, we analyzed the DNA binding activity to serum response elements (SRE) of serum response factor (SRF) by electromobility shift assay. Compared with diabetic mice (0.5 ± 0.01 densitometric units, DU), nondiabetic mice treated with EMPA (2.2 ± 0.01 DU, p < 0.01) and diabetic mice treated with EMPA (2.0 ± 0.1 DU, p < 0.01) significantly increased SRF binding activity to SRE, paralleled by increased cardiac actin expression (4.1 ± 0.1 vs 2.2 ± 0.01 target protein/β-actin ratio, p < 0.01). EMPA significantly reversed cardiac dysfunction on echocardiography in diabetic mice and inhibited excessive autophagy in high-glucose-treated cardiomyocytes by inhibiting the autophagy inducer GSK3β, leading to reactivation of cardiomyogenic transcription factors. Conclusions: Taken together, our results describe a novel paradigm in which EMPA inhibits hyperactivation of autophagy through the AMPK/GSK3β signaling pathway in the context of diabetes.

Aims Sodium-glucose cotransporter 2 inhibitors have beneficial effects on heart failure and cardiovascular mortality in diabetic and non-diabetic patients, with unclear mechanisms. Autophagy is a cardioprotective mechanism under acute stress conditions, but excessive autophagy accelerates myocardial cell death leading to autosis. We evaluated the protective role of empagliflozin (EMPA) against cardiac injury in murine diabetic cardiomyopathy. Methods and results Male mice, rendered diabetics by one single intraperitoneal injection of streptozotocin and treated with EMPA (30 mg/kg/day), had fewer apoptotic cells (4.9 +/- 2.1 vs. 1 +/- 0.5 TUNEL-positive cells %, P < 0.05), less senescence (10.1 +/- 2 vs. 7.9 +/- 1.2 beta-gal positivity/tissue area, P < 0.05), fibrosis (0.2 +/- 0.05 vs. 0.15 +/- 0.06, P < 0.05 fibrotic area/tissue area), autophagy (7.9 +/- 0.05 vs. 2.3 +/- 0.6 fluorescence intensity/total area, P < 0.01), and connexin (Cx)-43 lateralization compared with diabetic mice. Proteomic analysis showed a down-regulation of the 5 ' adenosine monophosphate-activated protein kinase (AMPK) pathway and upstream activation of sirtuins in the heart of diabetic mice treated with EMPA compared with diabetic mice. Because sirtuin activation leads to the modulation of cardiomyogenic transcription factors, we analysed the DNA binding activity to serum response elements (SRE) of serum response factor (SRF) by electromobility shift assay. Compared with diabetic mice [0.5 +/- 0.01 densitometric units (DU)], non-diabetic mice treated with EMPA (2.2 +/- 0.01 DU, P < 0.01) and diabetic mice treated with EMPA (2.0 +/- 0.1 DU, P < 0.01) significantly increased SRF binding activity to SRE, paralleled by increased cardiac actin expression (4.1 +/- 0.1 vs. 2.2 +/- 0.01 target protein/beta-actin ratio, P < 0.01). EMPA significantly reversed cardiac dysfunction on echocardiography in diabetic mice and inhibited excessive autophagy in high-glucose-treated cardiomyocytes by inhibiting the autophagy inducer glycogen synthase kinase 3 beta (GSK3 beta), leading to reactivation of cardiomyogenic transcription factors. Conclusion Taken together, our results describe a novel paradigm in which EMPA inhibits hyperactivation of autophagy through the AMPK/GSK3 beta signalling pathway in the context of diabetes.