Redox proteomics identification of oxidatively modified myocardial proteins in human heart failure: implications for protein function.
Bottom Line: The levels of carbonylated proteins were significantly higher in the HF patients than in the controls (p<0.01).Exposure of cardiomyocytes to angiotensin II and norepinephrine led to ROS generation and M-CK carbonylation with loss of its enzymatic activity.Our findings indicate that protein carbonylation is increased in the myocardium during HF and that these oxidative changes may help to explain the decreased CK activity and consequent defects in energy metabolism observed in HF.
Affiliation: Centro Cardiologico Monzino IRCCS, Milan, Italy.
Increased oxidative stress in a failing heart may contribute to the pathogenesis of heart failure (HF). The aim of this study was to identify the oxidised proteins in the myocardium of HF patients and analyse the consequences of oxidation on protein function. The carbonylated proteins in left ventricular tissue from failing (n = 14) and non-failing human hearts (n = 13) were measured by immunoassay and identified by proteomics. HL-1 cardiomyocytes were incubated in the presence of stimuli relevant for HF in order to assess the generation of reactive oxygen species (ROS), the induction of protein carbonylation, and its consequences on protein function. The levels of carbonylated proteins were significantly higher in the HF patients than in the controls (p<0.01). We identified two proteins that mainly underwent carbonylation: M-type creatine kinase (M-CK), whose activity is impaired, and, to a lesser extent, α-cardiac actin. Exposure of cardiomyocytes to angiotensin II and norepinephrine led to ROS generation and M-CK carbonylation with loss of its enzymatic activity. Our findings indicate that protein carbonylation is increased in the myocardium during HF and that these oxidative changes may help to explain the decreased CK activity and consequent defects in energy metabolism observed in HF.
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Mentions: As ROS can oxidatively modify proteins altering their biological activity, ,  we investigated the relationship between the oxidative modification and CK catalytic activity, which was assessed in HL-1 cells treated with angiotensin II or phenylephrine in the absence or presence of antioxidants. The two agents inhibited enzymatic activity, whereas pre-treatment with N-acetyl-l-cysteine (NAC) blocked the decrease induced by both compounds (Fig. 6A). We next confirmed that the oxidative modification directly altered the activity of purified CK in in vitro conditions: H2O2 reduced the activity of purified human M-CK, in the absence but not in the presence of catalase (Fig. 6B). Taken together, these data suggest that the oxidative modification may be responsible for the loss of M-CK activity in cardiomyocytes exposed to stimuli relevant to HF. The xanthine oxidase inhibitor allopurinol, the NADPH-oxidase inhibitor diphenyliodonium (DPI), and the mitochondrial complex I inhibitor rotenone completely prevented the inhibition of CK activity induced by phenylephrine (Fig. 6C). By contrast the complex III inhibitor antimycin and the complex II inhibitor 4,4,4,-trifluoro-1[2-thienyl]-1,3-butanediol did not recover the loss of CK activity (Fig. 6C).