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Redox proteomics identification of oxidatively modified myocardial proteins in human heart failure: implications for protein function.

Brioschi M, Polvani G, Fratto P, Parolari A, Agostoni P, Tremoli E, Banfi C - PLoS ONE (2012)

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.

View Article: PubMed Central - PubMed

Affiliation: Centro Cardiologico Monzino IRCCS, Milan, Italy.

ABSTRACT
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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ELISA-measured protein carbonyl levels in the myocardium of 14 HF patients and 13 controls.*p<0.01 vs controls.
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pone-0035841-g001: ELISA-measured protein carbonyl levels in the myocardium of 14 HF patients and 13 controls.*p<0.01 vs controls.

Mentions: Carbonylated protein levels measured by ELISA were significantly higher in the hearts of the HF patients than in the control hearts (p<0.01, Wilcoxon Mann-Whitney analysis) (Fig. 1), but the difference between the ischemic and idiopathic patients was not significant (0.60±0.32 nmoles/mg protein vs 0.87±0.24 nmoles/mg protein; p = 0.34). The carbonylated proteins were then identified by means of 2-DE combined with the high specificity of immunoblotting after the DNPH derivatisation of the carbonyl groups. Immunoblotting with anti-DNP antibody, performed on 14 left ventricular tissue samples from HF patients and 13 from healthy controls, showed that different isoforms of two proteins were carbonylated to a greater extent in the myocardium of the patients (Fig. 2 and Table S1). The carbonylated protein spots were excised from the 2-DE gel, digested with trypsin, and analysed by means of LC-MS/MS, which identified them as α-cardiac actin (ACTC) and creatine kinase M-type (M-CK), which is only detectable in its oxidised form in failing hearts (Table S1). The analysis of CK activity revealed that it was significantly reduced in the myocardium of HF patients than in controls (88.9±10.4 U/mg protein vs 175±9.6 U/mg protein, p = 0.0001, respectively).


Redox proteomics identification of oxidatively modified myocardial proteins in human heart failure: implications for protein function.

Brioschi M, Polvani G, Fratto P, Parolari A, Agostoni P, Tremoli E, Banfi C - PLoS ONE (2012)

ELISA-measured protein carbonyl levels in the myocardium of 14 HF patients and 13 controls.*p<0.01 vs controls.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3351458&req=5

pone-0035841-g001: ELISA-measured protein carbonyl levels in the myocardium of 14 HF patients and 13 controls.*p<0.01 vs controls.
Mentions: Carbonylated protein levels measured by ELISA were significantly higher in the hearts of the HF patients than in the control hearts (p<0.01, Wilcoxon Mann-Whitney analysis) (Fig. 1), but the difference between the ischemic and idiopathic patients was not significant (0.60±0.32 nmoles/mg protein vs 0.87±0.24 nmoles/mg protein; p = 0.34). The carbonylated proteins were then identified by means of 2-DE combined with the high specificity of immunoblotting after the DNPH derivatisation of the carbonyl groups. Immunoblotting with anti-DNP antibody, performed on 14 left ventricular tissue samples from HF patients and 13 from healthy controls, showed that different isoforms of two proteins were carbonylated to a greater extent in the myocardium of the patients (Fig. 2 and Table S1). The carbonylated protein spots were excised from the 2-DE gel, digested with trypsin, and analysed by means of LC-MS/MS, which identified them as α-cardiac actin (ACTC) and creatine kinase M-type (M-CK), which is only detectable in its oxidised form in failing hearts (Table S1). The analysis of CK activity revealed that it was significantly reduced in the myocardium of HF patients than in controls (88.9±10.4 U/mg protein vs 175±9.6 U/mg protein, p = 0.0001, respectively).

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.

View Article: PubMed Central - PubMed

Affiliation: Centro Cardiologico Monzino IRCCS, Milan, Italy.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus