Focal complex formation in adult cardiomyocytes is accompanied by the activation of beta3 integrin and c-Src.
In pressure-overloaded myocardium, our recent study demonstrated cytoskeletal assembly of c-Src and other signaling proteins which was partially mimicked in vitro using adult feline cardiomyocytes embedded in three-dimensional (3D) collagen matrix and stimulated with an integrin-binding Arg-Gly-Asp (RGD) peptide. In the present study, we improved this model further to activate c-Src and obtain a full assembly of the focal adhesion complex (FAC), and characterized c-Src localization and integrin subtype(s) involved. RGD dose response experiments revealed that c-Src activation occurs subsequent to its cytoskeletal recruitment and is accompanied by p130Cas cytoskeletal binding and focal adhesion kinase (FAK) Tyr925 phosphorylation. When cardiomyocytes expressing hexahistidine-tagged c-Src via adenoviral gene delivery were used for RGD stimulation, the expressed c-Src exhibited relocation: (i) biochemical analysis revealed c-Src movement from the detergent-soluble to the -insoluble cytoskeletal fraction and (ii) confocal microscopic analysis showed c-Src movement from a nuclear/perinuclear to a sarcolemmal region. RGD treatment also caused sarcolemmal co-localization of FAK and vinculin. Characterization of integrin subtypes revealed that beta3, but not beta1, integrin plays a predominant role: (i) expression of cytoplasmic domain of beta1A integrin did not affect the RGD-stimulated FAC formation and (ii) both pressure-overloaded myocardium and RGD-stimulated cardiomyocytes exhibited phosphorylation of beta3 integrin at Tyr773/785 sites but not beta1 integrin at Thr788/789 sites. Together these data indicate that RGD treatment in cardiomyocytes causes beta3 integrin activation and c-Src sarcolemmal localization, that subsequent c-Src activation is accompanied by p130Cas binding and FAK Tyr925 phosphorylation, and that these events might be crucial for growth and remodeling of hypertrophying adult cardiomyocytes.
Willey
Christopher D
CD
Cardiology Division, Department of Medicine, Gazes Cardiac Research Institute, Medical University of South Carolina, 114 Doughty Street, SC 29425 2221, Charleston, USA.
Balasubramanian
Sundaravadivel
S
Rodríguez Rosas
María C
MC
Ross
Robert S
RS
Kuppuswamy
Dhandapani
D
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D015153:Blotting, Western; D002415:Cats; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006639:Histidine / Q000737:chemistry; D006984:Hypertrophy; D019012:Integrin beta1 / Q000378:metabolism; D039661:Integrin beta3 / Q000378:metabolism*; D016023:Integrins / Q000378:metabolism; D018613:Microscopy, Confocal; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D009842:Oligopeptides / Q000494:pharmacology; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011506:Proteins; D050717:Retinoblastoma-Like Protein p130; D015398:Signal Transduction; D013997:Time Factors; D014443:Tyrosine / Q000737:chemistry; D016596:Vinculin / Q000378:metabolism; D019061:src-Family Kinases / Q000378:metabolism*
D019012:Integrin beta1; D039661:Integrin beta3; D016023:Integrins; D009842:Oligopeptides; D010750:Phosphoproteins; D011506:Proteins; D050717:Retinoblastoma-Like Protein p130; D016596:Vinculin; D014443:Tyrosine; D006639:Histidine; C047981:arginyl-glycyl-aspartic acid; D003094:Collagen; D011505:Protein-Tyrosine Kinases; D051416:Focal Adhesion Protein-Tyrosine Kinases; D019061:src-Family Kinases