Assessment of the Effects of Methylene Blue on Cellular Bioenergetics in H9c2 Cells

Methylene blue (MB), a tricyclic phenothiazine compound, has been reported to exert beneficial effects in experimental models of cerebral, hepatic, and intestinal ischemia/reperfusion injury via the modulation of mitochondrial function and the promotion of cell survival. The present study was purported to assess the acute effects of MB on the bioenergetic profile of the H9c2 rat cardiomyoblast cells. To this aim, H9c2 cells were incubated for 60 minutes with progressive concentrations of MB (0.05, 0.1, 1, 5, and 10 mu M respectively). Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using the Seahorse Bioscience XF-24e extracellular flux analyzer. Analysis of mitochondrial function was performed in the presence of the classic modulators of the electron transport chain: oligomycin, FCCF: and antimycin. All OCR linked parameters (i.e., basal respiration, proton leak, ATP turnover, maximal respiration, spare reserve capacity), as well as ECAR were increased by 0.1 mu M MB and decreased by the higher tested concentrations; the lowest concentration tested (0.05 mu M) had no effect on respiratory and metabolic parameters. In conclusion, methylene blue in submicromolar concentrations elicited an overall improvement of cellular bioenergetics in the H9c2 cell line.