Purpose: Empagliflozin (EMP) is a member of the gliflozin class of drugs used to treat patients with type 2 diabetes mellitus (T2DM). The presumed mechanism of action is the inhibition of the sodium glucose co-transporter 2 (SGLT-2) and glucose disposal via the kidneys. There is considerable interest in the growing evidence that select anti-diabetic drugs such as empagliflozin may have a cardioprotective effect independent of glycemic control manifested as a reduction in the risk of developing a major adverse cardiovascular event (MACE). The mechanism(s) underlying this effect are unknown. The purpose of this work is to test the hypothesis that the beneficial cardiac effects of EMP may be mediated by enhancing mitochondrial turnover, specifically enhanced mitophagy and mitochondrial biogenesis.
Methods: We treated 5 day differentiated H9C2 rat ventricular cardiomyocytes with 100nM EMP for 24h. Cells were then subjected to subcellular fractionation to obtain post nuclear supernatant and nuclear fractions. Western blot analysis was performed to examine several markers of mitochondria and mitochondrial turnover. Seahorse respirometry was also performed on these cells wherein they were subject to a mitochondrial stress test using 1uM oligomycin, 1uM FCCP and a combination of 0.5uM antimycin and 0.5uM rotenone. To determine if the stimulation of mitochondrial biogenesis may be related to the SGLT-2 receptor we interrogated its protein expression in several tissues of the mouse and in our differentiated H9C2 cardiomyocytes.
Results: As shown in the figures, we found that EMP increases several markers of mitochondria mass (Tom40, Tom70, Cox4, and OXPHOS components) and mitochondrial turnover (Mfn2 and Opa1) assessed by western blot analysis (Fig. 1A). These findings were associated with increased translocation of PGC-1α to the nucleus suggesting that EMP-mediated mitochondrial biogenesis is PGC-1α-dependent (Fig. 1B). Seahorse respirometry revealed that this rise in mitochondria translated to an increase in basal and maximal respiratory capacity of the cardiomyocytes which were treated by EMP (Fig. 2). We detected protein expression of the SGLT-2 receptor in mouse heart, liver, adipose, kidney, lung, spleen, and in our differentiated H9C2 cardiomyocytes (Fig. 3).
Conclusion: Whether EMP-mediated stimulation of mitochondrial biogenesis is related to SGLT-2 receptor inhibition, or if this process is linked to its ability to improve cardiac function remains to be determined. Our findings suggest however that a possible mechanism underlying the reduction in MACE and improvements in cardiac function associated with EMP treatment may be due in part to its effect on mitochondrial turnover. Understanding the mechanism and utility of EMP may help lead to future studies that can be implemented in the clinical setting to treat heart failure.