Empagliflozin Mitigates High Glucose-Disrupted Mitochondrial Respiratory Function in H9c2 Cardiomyoblasts: A Comparative Study with NHE-1 and ROCK Inhibition.

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Hyperglycemia in patients with Diabetes Mellitus (DM) increases the risk of developing cardiomyopathy and heart failure. Elevation of sodium/proton exchanger-1 (NHE-1) expression and activity in cardiomyocytes leads to greater sensitivity to neurohormonal stimulation and cardiomyopathy, whereas inhibition of Sodium-Glucose Cotransporter 2 (SGLT2) clinically benefits DM population in reducing heart failure risk. This study characterized the expression profiles of NHE-1 and SGLT2 in H9c2 cardiomyoblasts under High Glucose (HG) exposure and examined the effects of Empagliflozin (EMPA), an SGLT2 inhibitor, on the HG-induced cardiomyoblasts deterioration, in comparison with NHE-1 specific inhibitor cariporide and Rho/ROCK inhibitor hydroxy fasudil. Western blotting and immunofluorescent staining were used to monitor protein expression and subcellular location, respectively. Reactive Oxygen Species (ROS) production and mitochondrial membrane potential were measured by flow cytometry. Kinetic mitochondrial oxygen consumption rate and respiratory function were monitored by a real-time cell metabolic analyzer. HG treatment upregulated SGLT2 and NHE-1 expression and RhoA/ROCK activity in H9c2 cardiomyoblasts. The HG-upregulated NHE-1 is localized in actin-rich cortical cytoplasm, implicating its involvement in cell shape and adhesion alterations. Treatment with NHE-1 and ROCK inhibitors, but not EMPA, significantly attenuated the HG-induced ROS overproduction and mitochondrial membrane potential elevation. However, EMPA treatment restored the HG-suppressed mitochondrial maximal respiration, spare respiratory capacity, and non-mitochondrial oxygen consumption rate. In comparison, Rho/ROCK and NHE-1 inhibitions effectively prevent ROS overproduction, while SGLT2 inhibition rescues the deteriorated mitochondrial respiratory function under diabetogenic conditions. Blockade of SGLT2, NHE-1, or Rho/ROCK activity is useful for the prevention of diabetic cardiomyopathy.