Empagliflozin prevents oxidative stress induced by acute hyperglycemia in cardiac myocytes.

Abstract

Empagliflozin, a sodium-glucose cotransporter-2 (SGLT-2) inhibitor, has shown to improve cardiovascular outcomes in patients with diabetes and heart failure. Empagliflozin has also been shown to provide advantageous effects in excised hearts and isolated cardiomyocytes despite SGLT-2 expression lacking in cardiac myocytes. This suggests empagliflozin may have important off-target effects. Previous studies suggested that empagliflozin effects may include attenuation of both reactive oxygen species (ROS) and Ca/calmodulin-dependent kinase II activation, but exact signaling details remained unclear. Using transgenic mice expressing glutaredoxin 1-coupled redox-sensitive green fluorescent protein 2 (Grx1-roGFP2) sensor localized to either the cytosol or mitochondria, we tested for empagliflozin effect on oxidative stress in myocytes bathed in high-glucose (30 mmol/L) containing Tyrode's solution. To assess the changes in redox potential, we exposed each cell to H2O2 and dithiothreitol to calibrate the sensor. We found acute hyperglycemia induced significant increases in ROS over a 3-5min of perfusion, specifically in the cytosolic compartment. This is in line with previous report showing that hyperglycemia activates CaMKII by intracellular O-GlcNAcylation, then the active CaMKII promotes cytosolic ROS production via NAPDH oxidase 2 (NOX2). Importantly, cells preincubated with empagliflozin were fully protected against the hyperglycemia-induced increase in ROS. We also extended our studies on angiotensin II (AngII)-dependent ROS production because AngII activates NOX2, and the excess ROS promotes oxidation- dependent CaMKII activation. Myocytes pretreated with the selective CaMKII inhibitor autocamtide-2-relative inhibitory peptide (cell-permeable myristoylated form) were used to test the role of CaMKII in ROS production by either hyperglycemia or Ang-II. These studies could reveal whether empagliflozin acts on glucose uptake, CaMKII activation, or via direct antioxidant mechanisms. Our findings may provide a mechanistic basis for the positive effects of empagliflozin in myocytes under conditions relevant to heart disease.