Seenarain V., Viola H.M., Ravenscroft G., Casey T.M., Lipscombe R.J., Ingley E., Laing N.G., Bringans S.D. and Hool L.C. (2010) Evidence for alterations in protein expression and function after 5 minutes exposure of ventricular myocytes to H2O2. Journal of Proteome Research, 9(4), 1985-94.
Oxidative stress and alterations in cellular calcium homeostasis are associated with the development of cardiac hypertrophy. However, the early cellular mechanisms for the development of hypertrophy are not well understood. Guinea pig ventricular myocytes were exposed to 30 µM H2O2 for 5 min followed by 10 units/mL catalase to degrade the H2O2, and effects on protein expression were examined 48 h later. Transient exposure to H2O2 increased the level of protein synthesis more than 2-fold, assessed as incorporation of [3H]leucine (n ) 12; p < 0.05). Cell size was increased slightly, but there was no evidence of major cytoskeletal disorganization assessed using fluorescence microscopy. Changes in the expression of individual proteins were assessed using iTRAQ protein labeling followed by mass spectrometry analysis (LC-MALDI-MSMS); 669 proteins were identified, and transient exposure of myocytes to H2O2 altered expression of 35 proteins that were predominantly mitochondrial in origin, including TCA cycle enzymes and oxidative phosphorylation proteins. Consistent with changes in the expression of mitochondrial proteins, transient exposure of myocytes to H2O2 increased the magnitude of the mitochondrial NADH signal 10.5 ( 2.3% compared to cells exposed to 0 µM H2O2 for 5 min followed by 10 units/mL catalase (n ) 8; p < 0.05). In addition, metabolic activity was significantly increased in the myocytes 48 h after transient exposure to H2O2, assessed as formation of formazan from tetrazolium salt. We conclude that a 5 min exposure of ventricular myocytes to 30 µM H2O2 is sufficient to significantly alter protein expression, consistent with the development of hypertrophy in the myocytes. Changes in mitochondrial protein expression and function appear to be early sequelae in the development of hypertrophy.