Metallothionein-overexpressing neonatal mouse cardiomyocytes are resistantto H2O2 toxicity

To study cellular and molecular events of cardiac protection by metallothio nein (MT) from oxidative injury, a primary neonatal cardiomyocyte culture w as established from a specific cardiac MT-overexpressing transgenic mouse m odel. Ventricular cardiomyocytes were isolated from 1- to 3-day-old neonata l mice and cultured in an Eagle's minimum essential medium supplemented wit h 20% fetal bovine serum under an atmosphere of 5% CO2-95% air at 37 degree s C. Forty-eight hours after plating was completed, the purity of such cult ures was 95% myocytes, assessed by an immunocytochemical assay. Over 80% of the cardiomyocytes beat spontaneously on the first day of culture and sync hronously in a confluent monolayer after the sixth day of culture. Cellular MT concentrations in the transgenic cardiomyocytes before culturing and on the sixth day postculturing were about seven- and twofold higher than nont ransgenic controls, respectively. However, there were no significant differ ences in cell morphology, glutathione content, and antioxidant enzymatic ac tivities between these two types of cardiomyocytes. When these cells were c hallenged by H2O2, the transgenic cardiomyocytes displayed a significant re sistance to the toxic effect of this oxidant, as measured by cell viability , lactate dehydrogenase leakage, and morphological alterations. In addition , the transgenic cells were highly protected from H2O2-induced lipid peroxi dation. These observations demonstrate that MT protects the cultured cardio myocytes from H2O2 toxicity by preventing its interaction with macromolecul es such as lipids, and this cultured primary neonatal mouse cardiomyocyte s ystem provides a valuable tool to directly study cellular and molecular eve nts of MT in cardiac protection against oxidative injury.