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 approximately 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 mM K+) on cellular viability may depend on the extent of insult imposed on cardiomyocytes; i.e. a approximately 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.
Gomez
L A
LA
Department of Medicine and Pharmacology, Mayo Clinic, Mayo Foundation, Rochester, MN 55905, USA.
Alekseev
A E
AE
Aleksandrova
L A
LA
Brady
P A
PA
Terzic
A
A
D000241:Adenosine / Q000494:pharmacology*; D000367:Age Factors; D000818:Animals; D002314:Cardioplegic Solutions / Q000494:pharmacology; D002448:Cell Adhesion; D016923:Cell Death / Q000187:drug effects; D004121:Dimethyl Sulfoxide / Q000633:toxicity; D006168:Guinea Pigs; D006352:Heart Ventricles / Q000166:cytology* / Q000187:drug effects / Q000293:injuries; D000860:Hypoxia / Q000139:chemically induced / Q000378:metabolism; D009584:Nitrogen / Q000633:toxicity; D010084:Oxidation-Reduction; D010641:Phenotype; D011188:Potassium / Q000494:pharmacology*; D013053:Spectrophotometry / Q000379:methods*; D013314:Stress, Mechanical; D013778:Tetrazolium Salts / Q000378:metabolism*
D002314:Cardioplegic Solutions; D013778:Tetrazolium Salts; D000241:Adenosine; D009584:Nitrogen; D011188:Potassium; D004121:Dimethyl Sulfoxide