Oxidative changes in the cardiac proteome during ischemia/reperfusion
Myocardial ischemia is associated with coronary artery disease. Prolonged ischemia may lead to loss of viability of myocardial cells and reperfusion is imperative for the reversal of changes. However, reperfusion is associated with tissue injury. There is strong evidence for reactive oxygen species being involved in ischemia/reperfusion injury. Major targets of these reactive oxygen species are redox sensitive thiols. They have important regulatory, catalytic and structural significance in proteins involved in vital functions of heart. This study is aimed at developing and applying redox proteomic approaches to identify oxidative changes in protein thiols, and establishing how thiol protein changes relate to general oxidative stress during ischemia/reperfusion in isolated mouse heart using a Langendorff perfusion system. Mouse hearts were subjected to 20 min ischemia with or without aerobic reperfusion for 5 or 30 min, or 30 min perfusion for controls. Two stages blocking with N-ethylmaleimide (NEM) just after excision and during protein extraction was performed. To study global changes two approaches were adopted: 1) oxidized thiols were labelled with thiol specific fluorescent tag and changes were measured as difference in fluorescence intensity between control and ischemia/reperfusion samples separated on 2 dimensional gels and 2) a global quantitative proteomics approach called Isotope Coded Affinity Tags (ICAT) modified to look at redox changes used by exploiting thiol specific nature of iodoacetamide (IAM) based isotopic tag i.e. a tag with same physical properties except mass. Oxidized thiols in control and treated groups were labelled with the light and heavy tags respectively and the relative proportions of heavy and light isotope labelled peaks for specific peptides used to quantify oxidative changes between the control and treated groups. Oxidative changes in specific proteins were monitored by labelling oxidized thiols with deuterium based NEM and free thiols with unlabelled NEM, then quantifying the ratio of reduced to oxidized peptide by mass spectrometry. To establish how thiol protein changes relate to general oxidative stress, oxidation of redox sensor peroxiredoxins, protein kinase G and changes in the oxidative biomarkers glutathione and protein carbonyls in the heart during ischemia/reperfusion were measured. Oxidative stress was evident from the protein carbonyl accumulation during ischemia/reperfusion. Global analysis using ICAT strategy picked up 18 changes out of more than 100 peptides identified, mostly at redox sensitive sites (identified as undergoing oxidative change in other situations). Thiol oxidation occurred mainly during ischemia or early reperfusion followed by reversal with longer reperfusion. The study documented thiol oxidation in some of the proteins involved in vital heart functions. These included mitochondrial electron transport complexes namely complex I, II, III and ATP synthase crucial for energy generation, carnitine palmitoyltransferase II, long-chain specific acyl-CoA dehydrogenase crucial for fatty acid metabolism and sarcoplasmic/endoplasmic reticulum calcium ATPase crucial for cardiac contraction. In addition, its degradation was also noticed and was probably due to activation of protease during ischemia/reperfusion. Mitochondria appeared to be a main target of oxidative stress. Further evidence for this was the detection of reversible oxidation of mitochondrial peroxiredoxin 3 during ischemia with no changes in cytosolic peroxiredoxin 1 and 2. Oxidation activation of protein kinase G, a cardioprotective protein, during reperfusion was also noticed. Changes were modest in comparison to literature studies where external oxidants were used. In conclusion most of the oxidative changes during ischemia/reperfusion remain reversible up to certain duration; activation of cardioprotective system might be reason for that. However changes like protein carbonyl formation and, SERCA degradation could be deleterious for the ischemic heart subjected to reperfusion.
Advisor: Winterbourn , Christine; Hampton, Mark
Degree Name: Doctor of Philosophy
Degree Discipline: Pathology UOC
Publisher: University of Otago
Keywords: cardiac ischemia reperfusion; oxidative stress; peroxiredoxins; redox proteomics; ICAT
Research Type: Thesis