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

BACKGROUNDDiabetic cardiomyopathy (DCM) increases the risk of hospitalization for heart failure (HF) and mortality in patients with diabetes mellitus. However, no specific therapy to delay the progression of DCM has been identified. Mitochondrial dysfunction, oxidative stress, inflammation, and calcium handling imbalance play a crucial role in the pathological processes of DCM, ultimately leading to cardiomyocyte apoptosis and cardiac dysfunctions. Empagliflozin, a novel glucose-lowering agent, has been confirmed to reduce the risk of hospitalization for HF in diabetic patients. Nevertheless, the molecular mechanisms by which this agent provides cardioprotection remain unclear.AIMTo investigate the effects of empagliflozin on high glucose (HG)-induced oxidative stress and cardiomyocyte apoptosis and the underlying molecular mechanism.METHODSTwelve-week-old db/db mice and primary cardiomyocytes from neonatal rats stimulated with HG (30 mmol/L) were separately employed as in vivo and in vitro models. Echocardiography was used to evaluate cardiac function. Flow cytometry and TdT-mediated dUTP-biotin nick end labeling staining were used to assess apoptosis in myocardial cells. Mitochondrial function was assessed by cellular ATP levels and changes in mitochondrial membrane potential. Furthermore, intracellular reactive oxygen species production and superoxide dismutase activity were analyzed. Real-time quantitative PCR was used to analyze Bax and Bcl-2 mRNA expression. Western blot analysis was used to measure the phosphorylation of AMP-activated protein kinase (AMPK) and myosin phosphatase target subunit 1 (MYPT1), as well as the peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1 alpha) and active caspase-3 protein levels.RESULTSIn the in vivo experiment, db/db mice developed DCM. However, the treatment of db/db mice with empagliflozin (10 mg/kg/d) for 8 wk substantially enhanced cardiac function and significantly reduced myocardial apoptosis, accompanied by an increase in the phosphorylation of AMPK and PGC-1 alpha protein levels, as well as a decrease in the phosphorylation of MYPT1 in the heart. In the in vitro experiment, the findings indicate that treatment of cardiomyocytes with empagliflozin (10 mu M) or fasudil (FA) (a ROCK inhibitor, 100 mu M) or overexpression of PGC-1 alpha significantly attenuated HG-induced mitochondrial injury, oxidative stress, and cardiomyocyte apoptosis. However, the above effects were partly reversed by the addition of compound C (CC). In cells exposed to HG, empagliflozin treatment increased the protein levels of p-AMPK and PGC-1 alpha protein while decreasing phosphorylated MYPT1 levels, and these changes were mitigated by the addition of CC. Adding FA and overexpressing PGC-1 alpha in cells exposed to HG substantially increased PGC-1 alpha protein levels. In addition, no sodium-glucose cotransporter (SGLT)2 protein expression was detected in cardiomyocytes.CONCLUSIONEmpagliflozin partially achieves anti-oxidative stress and anti-apoptotic effects on cardiomyocytes under HG conditions by activating AMPK/PGC-1 alpha and suppressing of the RhoA/ROCK pathway independent of SGLT2.