Characterisation of changes in the cardiac proteome after transient exposure of myocytes to hydrogen peroxide

We have shown previously that exposure of guinea pig ventricular myocytes to 30 μM hydrogen peroxide (H2O2) for 5 min followed by 10 U/ml catalase to degrade the H2O2 results in a persistent increase in superoxide production by the mitochondria. This leads to a 2-fold increase in protein synthesis measured as [3H] leucine incorporation. We characterised the effect of a transient exposure of cardiac myocytes to H2O2 on the proteome. Myocytes were exposed to 0 μM or 30 μM H2O2 for 5 min followed by 10U/ml catalase. Protein was extracted, labelled with iTRAQ reagents and the peptides were analysed by LC–MALDI-TOF/TOF mass spectrometry and identified against SwissProt and Ludwig NR databases. More than 900 proteins were identified. Transient exposure of the myocytes to H2O2 resulted in altered expression of 62 proteins. 40% of proteins identified with altered expression were mitochondrial. Consistent with our in vitro data, mitochondria complex I and complex III subunit expression was upregulated while downregulation of complex IV and ATP synthase subunits suggested compromised ATP production. We also detected an increase in sarcomere associated proteins including Troponin C, Tropomyosin I alpha isoform 6 and Myosin light chain 2 and 3. Prohibitin was downregulated and calcireticulin expression was upregulated, consistent with promotion of cellular growth. We are currently validating the findings with in vitro functional studies in the myocytes. These data suggest that transient exposure to cardiac myocytes is sufficient to significantly alter expression of proteins and provides insight into early mechanisms of development of cardiac hypertrophy