Use of the MTT assay in adult ventricular cardiomyocytes to assess viability: Effects of adenosine and potassium on cellular survival

This study used the colorimetric MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide)] assay to assess cell viability in isolated quiescent adult guinea-pig ventricular myocytes exposed to different insults or cardioprotective conditions, including adenosine and hyperkalemic-cardioplegia. Optical density (OD), reflecting intracellular reduction of MTT into formazan pigment formation, was a function of the number of viable cells (coefficient of linear correlation ~0.99), with MTT reduction preferentially carried out by rod-shaped cardiomyocytes (absorbance at 1.009 ± 0.013 and 0.006 ± 0.001 OD units for populations containing 50 and 0% of rod-shaped cells). Following prolonged mechanical (pressure of 1 lb/min for 40 min), chemical (10% DMSO or ethanol) or hypoxic injury (N₂-saturated solution), the MTT reductase activity reflected reduction in the number of viable cells by 87%, > 50%, and 77%, respectively. In cardiomyocytes exposed to a 40 min hypoxia (with CO₂), the MTT reductase activity was 0.056 ± 0.009 in the absence, and 0.074 ± 0.008 OD units in the presence of adenosine (1 mM), i.e. adenosine reduced the number of non-viable cells. Also, the MTT assay revealed that the effect of potassium-containing solutions (16 and 32 mMK⁺) on cellular viability may depend on the extent of insult imposed on cardiomyocytes; i.e. a ~24% and 49% increase under mild hypoxia (0.03% CO₂), or an 18% decrease in cell viability under severe hypoxia (N₂) in pre-injured cells. Thus, the MTT assay used to assess viability of isolated adult cardiomyocytes revealed a direct cytoprotective effect of adenosine and hyperkalemic-cardioplegia by promoting cell survival under certain conditions in vitro.