Metallothionein-overexpressing neonatal mouse cardiomyocytes are resistant to H2O2 toxicity

To study cellular and molecular events of cardiac protection by metallothionein (MT) from oxidative injury, a primary neonatal cardiomyocyte culture was established from a specific cardiac MT-overexpressing transgenic mouse model. Ventricular cardiomyocytes were isolated from 1- to 3-day-old neonatal mice and cultured in an Eagle's minimum essential medium supplemented with 20% fetal bovine serum under an atmosphere of 5% CO2-95% air at 37 degreesC. Forty-eight hours after plating was completed, the purity of such cultures was 95% myocytes, assessed by an immunocytochemical assay. Over 80% of the cardiomyocytes beat spontaneously on the first day of culture and synchronously 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 nontransgenic controls, respectively. However, there were no significant differences in cell morphology, glutathione content, and antioxidant enzymatic activities between these two types of cardiomyocytes. When these cells were challenged by H2O2, the transgenic cardiomyocytes displayed a significant resistance 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 peroxidation. These observations demonstrate that MT protects the cultured cardiomyocytes from H2O2 toxicity by preventing its interaction with macromolecules such as lipids, and this cultured primary neonatal mouse cardiomyocyte system provides a valuable tool to directly study cellular and molecular events of MT in cardiac protection against oxidative injury.