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 diseases. We aimed to evaluate the effectiveness of sacubitril/valsartan (S/V) and Empagliflozin (EMPA) on hypoxia-inducible factor-1α (HIF-1α) and oxidative stress in H9c2 rat embryonic cardiomyocyte cells.

Materials And Methods: BH9c2 cardiomyocyte cells were treated with methotrexate (MTX) (10-0.156 μM), empagliflozin (EMPA; 10-0.153 µM) and sacubitril/valsartan (S/V; 100-1.062 µM) for 24, 48 and 72 h. The half maximum inhibitory concentration (IC50) and half maximum excitation concentration (EC50) values of MTX, EMPA and S/V were determined. The cells under investigation were exposed to 2.2 μM MTX before treatment with 2 μM EMPA and 25 μM S/V. The cell viability, 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 µM EMPA, 25 µM S/V or their combination produced a protective effect against the reduction in cell viability caused by 2.2 µM MTX.  While HIF-1α levels plunged to their lowest with S/V treatment, oxidant parameters dipped, and antioxidant parameters soared to their highest level with S/V and EMPA combination treatment. A negative correlation was found between HIF-1α and total antioxidant capacity in the S/V treatment group.

Conclusions: A significant decrease in HIF-1α and oxidant molecules together with an enhancement in antioxidant molecules and normalization 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 effect may be increased more with S/V treatment alone compared to combined treatment.