UC Davis

In this dissertation I focus on a sodium glucose cotransporter-2 (SGLT2) inhibitor, empagliflozin (EMPA), which is used to treat type 2 diabetes mellitus (T2DM) and more recently heart failure (HF). In clinical trials SGLT2 inhibitors improved cardiovascular outcomes in HF patients regardless of their diabetes status. Preclinical studies demonstrated EMPA’s effects also in ex vivo perfused hearts and isolated cardiomyocytes. However, cardiomyocytes do not express SGLT2, thus the mechanism of EMPA’s direct effects in cardiac myocytes is unknown. EMPA has been shown to inhibit the late sodium current (INaL) in a transverse aortic constriction (TAC) model of HF with reduced ejection fraction (HFrEF). Cardiomyocytes from a murine model of HF with preserved ejection fraction (HFpEF) also exhibited enhancement of the INaL, and importantly, was reversed with EMPA pre-treatment (~4hr), but interestingly this was not observed with acute (~3min) treatment. The same effects were also seen when a calcium/calmodulin dependent protein kinase II (CaMKII) inhibitor and reactive oxygen species (ROS) inhibitor was used. These findings point towards EMPA affecting upstream regulators of the INaL. CaMKII is an important upstream regulator of the INaL and it has increased activity in disease states such as HF. CaMKII can enhance ROS production via NADPH oxidase 2 (NOX2) and increased levels of ROS can oxidize an already active CaMKII to further enhance its activity. Moreover, a shift in cardiac fuel utilization in HF can increase glucose uptake into cardiomyocytes and can lead to an increase in ROS production. An additional modification of CaMKII that enhances its activity upon elevated glucose levels is O-linked-N-acetylglucosamine, or O-GlcNAcylation, where its attachment and detachment are controlled by two enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. Thus, we hypothesized that EMPA is modulating one or more of these processes (CaMKII, ROS, NOX2, OGA, OGT), which have downstream effects on INaL. Therefore, the work in this dissertation addressed this hypothesis by testing how EMPA affects hyperglycemia-induced myocyte ROS production in comparison to CaMKII inhibition in control and HFpEF myocytes, as well whether EMPA directly affects the activities of NOX2, OGA, and OGT. To test whether EMPA has effects on ROS we utilized isolated cardiomyocytes from transgenic mouse lines expressing the fluorescent ROS sensor Grx1-roGFP2 sensor, localized either to the cytosol or the mitochondria, allowing us to measure compartment-specific ROS. Acute hyperglycemia increases cytosolic ROS production and EMPA pretreatment (1 hr) was able to reduce the rate of production, however this was not seen in the mitochondria where hyperglycemia did not increase mitochondrial ROS. This was seen also in HFpEF mice and their aged-matched controls. We also tested whether CaMKII inhibition had similar effects to EMPA treatment and saw that CaMKII inhibition was able to attenuate the hyperglycemia-induced ROS production in the cytosol. Additional tests were done with heart tissue lysates to compare EMPA’s effect on NOX2, O-GlcNAcase, and O-GlcNAc transferase with known inhibitors of these enzymes. No direct effect was seen on the activity of NOX2, O-GlcNAcase, and O-GlcNAc transferase by EMPA. This data suggests EMPA target works within this connected pathway, but not directly with NOX2, O-GlcNAcase, and O-GlcNAc inhibition, as seen by its ability to reduce the rate of hyperglycemia-induced ROS production, but not to the same degree of reduction as CaMKII inhibition. Since ROS and CaMKII are altered in disease, this may provide insight into EMPA’s SGLT2-independent and cardiomyocyte-specific targets.EMPA has received considerable attention over recent years for its unexpected and direct cardiovascular effects. Hopefully, continued research discoveries contribute to better understanding of HF disease progression and elucidate EMPA’s cardiac-specific target that ultimately translated into new therapies for HF patients.