Proteomics study of oxidative stress and Src kinase inhibition in H9C2 cardiomyocytes: a cell model of heart ischemia-reperfusion injury and treatment.
Protein phosphorylation plays a crucial role in the signal transduction pathways that regulate gene expression, metabolism, cell adhesion, and cell survival in response to oxidative stress. In this study, we have used hydrogen peroxide treatment of H9C2 rat cardiomyocytes as a model of oxidative stress in heart ischemia-reperfusion injury. We show that oxidative stress induces a robust tyrosine phosphorylation of multiple proteins in this cell type. A phosphoproteomics approach using anti-phosphotyrosine affinity purification and LC-MS/MS was then used to identify the protein targets of this stress-induced phosphorylation. Twenty-three tyrosine-phosphorylated proteins were identified, with the majority known to be associated with cell-cell junctions, the actin cytoskeleton, and cell adhesion. This suggested that oxidative stress may have a profound effect on intercellular connections and the cytoskeleton to affect cell adhesion, morphology, and survival. Importantly, Src kinase was shown to be a major upstream regulator of these events. Immunofluorescence studies, fluorescence-activated cell sorting, and cell-based assays were used to demonstrate oxidative stress-induced modification of cell adhesion structures and the cytoskeleton, induced de-adhesion, and increased apoptosis, which were reversed by treatment with the Src kinase inhibitor PP1. These data demonstrate the critical role of Src kinase in oxidative stress-induced phosphorylation and cell damage in cardiomyocytes and suggest that targeting this kinase may be an effective strategy for preventing ischemia-reperfusion injury in the heart.
Chou
Hsiu-Chuan
HC
Tissue Regeneration Bio-Device Tech Lab, Medical Electronics and Device Technology Center, Industrial Technology Research Institute, Hsinchu 31040, Taiwan.
Chen
Yi-Wen
YW
Lee
Tian-Ren
TR
Wu
Fen-Shiun
FS
Chan
Hsin-Tsu
HT
Lyu
Ping-Chiang
PC
Timms
John F
JF
Chan
Hong-Lin
HL
D000818:Animals; D017209:Apoptosis; D002460:Cell Line; D002846:Chromatography, Affinity; D003599:Cytoskeleton; D006801:Humans; D007365:Intercellular Junctions; D015428:Myocardial Reperfusion Injury / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology / Q000503:physiopathology; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D018384:Oxidative Stress; D010766:Phosphorylation; D040901:Proteomics; D011720:Pyrazoles / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D051381:Rats; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics
C108123:4-amino-5-(4-methylphenyl)-7-(tert-butyl)pyrazolo(3,4-d)pyrimidine; D011720:Pyrazoles; D011743:Pyrimidines; D019061:src-Family Kinases