Empagliflozin and sacubitril/valsartan reverse methotrexate cardiotoxicity by repressing oxidative stress and hypoxia in heart embryonic H9c2 cardiomyocytes-the role of morphology of mitochondria observed on electron microscopy

OBJECTIVE: Oxidative stress and hypoxia play an important role in the pathogenesis of various cardiovascular dis-eases. We aimed to evaluate the effectiveness of sacubitril/valsartan (S/V) and Empaglifloz-in (EMPA) on hypoxia-inducible factor-1a (HIF-1a) and oxidative stress in H9c2 rat embryonic cardiomyocyte cells.MATERIALS AND METHODS: BH9c2 car-diomyocyte cells were treated with methotrex-ate (MTX) (10-0.156 pM), empagliflozin (EMPA; 10-0.153 pM) and sacubitril/valsartan (S/V; 100-1.062 pM) for 24, 48 and 72 h. The half maximum inhib-itory concentration (IC50) and half maximum exci-tation concentration (EC50) values of MTX, EMPA and S/V were determined. The cells under investi-gation were exposed to 2.2 pM MTX before treat-ment with 2 pM EMPA and 25 pM S/V. The cell vi-ability, lipid peroxidation, oxidation of proteins and antioxidant parameters were measured while morphological changes were also observed by transmission electron microscopy (TEM).RESULTS: The results showed that treatment with 2 pM EMPA, 25 pM S/V or their combina-tion produced a protective effect against the re-duction in cell viability caused by 2.2 pM MTX. While HIF-1a levels plunged to their lowest with S/V treatment, oxidant parameters dipped, and antioxidant parameters soared to their high -est level with S/V and EMPA combination treat-ment. A negative correlation was found be-tween HIF-1a and total antioxidant capacity in the S/V treatment group.CONCLUSIONS: A significant decrease in HIF-1a and oxidant molecules together with an enhancement in antioxidant molecules and nor-malization of the mitochondria morphology as observed on electron microscopy in S/V and EMPA-treated cells were detected. Although S/V and EMPA have both protective effects against cardiac ischemia and oxidative damage, this ef-fect may be increased more with S/V treatment alone compared to combined treatment.