ABSTRACT
AIMS: The highly expressed cell adhesion receptor CD29 (ß(1)-integrin) is essential for cardiomyocyte growth and survival, and its loss of function causes severe heart disease. However, CD29-induced signalling in cardiomyocytes is ill defined and may involve reactive oxygen species (ROS). A decisive source of cardiac ROS is the abundant NADPH oxidase (NOX) isoform NOX2. Because understanding of NOX-derived ROS in the heart is still poor, we sought to test the role of ROS and NOX in CD29-induced survival signalling in cardiomyocytes. METHODS AND RESULTS: In neonatal rat ventricular myocytes, CD29 activation induced intracellular ROS formation (oxidative burst) as assessed by flow cytometry using the redox-sensitive fluorescent dye dichlorodihydrofluorescein diacetate. This burst was inhibited by apocynin and diphenylene iodonium. Further, activation of CD29 enhanced NOX activity (lucigenin-enhanced chemiluminescence) and activated the MEK/ERK and PI3K/Akt survival pathways. CD29 also induced phosphorylation of the inhibitory Ser9 on the pro-apoptotic kinase glycogen synthase kinase-3ß in a PI3K/Akt- and MEK-dependent manner, and improved cardiomyocyte viability under conditions of oxidative stress. The ROS scavenger MnTMPyP or adenoviral co-overexpression of the antioxidant enzymes superoxide dismutase and catalase inhibited CD29-induced pro-survival signalling. Further, CD29-induced protective pathways were lost in mouse cardiomyocytes deficient for NOX2 or functional p47(phox), a regulatory subunit of NOX. CONCLUSION: p47(phox)-dependent, NOX2-derived ROS are mandatory for CD29-induced pro-survival signalling in cardiomyocytes. These findings go in line with a growing body of evidence suggesting that ROS can be beneficial to the cell and support a crucial role for NOX2-derived ROS in cell survival in the heart.
Subject(s)
Integrin beta1/metabolism , Membrane Glycoproteins/metabolism , Myocytes, Cardiac/cytology , Myocytes, Cardiac/enzymology , NADPH Oxidases/metabolism , Signal Transduction/physiology , Acetophenones/pharmacology , Animals , Antioxidants/pharmacology , Cell Survival/drug effects , Cell Survival/physiology , Glycogen Synthase Kinase 3/metabolism , Glycogen Synthase Kinase 3 beta , Integrin beta1/genetics , Membrane Glycoproteins/genetics , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Myocytes, Cardiac/drug effects , NADPH Oxidase 2 , NADPH Oxidases/genetics , Onium Compounds/pharmacology , Rats , Rats, Sprague-Dawley , Rats, Wistar , Reactive Oxygen Species/metabolism , Signal Transduction/drug effectsABSTRACT
ß(1)-Integrin mediates cardiomyocyte growth and survival and its proper regulation is essential for the structural and functional integrity of the heart. ß(1)-Integrin expression is enhanced in hypertrophy, but the mechanism and significance of its up-regulation are unknown. Because reactive oxygen species (ROS) are important mediators of myocardial remodeling we examined their role in regulated ß(1)-integrin expression. Hypertrophy was induced in neonatal cardiomyocytes by endothelin-1 (ET-1), which activated the regulatory NADPH oxidase subunit Rac1, evoked ROS, and enhanced fetal gene expression and cardiomyocyte size. ET-1 also enhanced cell adhesion and FAK phosphorylation and inhibited oxidative stress-induced cardiomyocyte apoptosis. Further, ET-1 increased ß(1)-integrin mRNA and protein expression via Rac1-ROS-dependent MEK/ERK and EGF receptor-PI3K/Akt activation as shown by adenoviral dominant-negative Rac1 or overexpression of copper/zinc-superoxide dismutase. The relevance of regulated ß(1)-integrin expression was examined in cardiomyocytes, in which targeting siRNA impeded the ET-1-induced ß(1)-integrin up-regulation. In these cells, ET-1-induced cell adhesion, FAK phosphorylation, and hypertrophic response were significantly blunted, whereas its antiapoptotic effect was predominantly unchanged, suggesting at least partial dissociation of prohypertrophic and prosurvival signaling elicited by ET-1. In conclusion, ß(1)-integrin up-regulation in response to ET-1 is mediated via Rac1-ROS-dependent activation of prohypertrophic pathways and is mandatory for ET-1-induced FAK activation, cell adhesion, and hypertrophic response.
Subject(s)
Hyperplasia/metabolism , Integrin beta1/metabolism , Myocardium/pathology , Myocytes, Cardiac/metabolism , Reactive Oxygen Species/metabolism , rac1 GTP-Binding Protein/metabolism , Animals , Apoptosis/drug effects , Apoptosis/genetics , Cell Adhesion/drug effects , Cell Adhesion/genetics , Cells, Cultured , Endothelin-1/pharmacology , Gene Expression Regulation, Developmental/drug effects , Hyperplasia/genetics , Integrin beta1/genetics , Mutation/genetics , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/pathology , RNA, Small Interfering/genetics , Rats , Rats, Wistar , Signal Transduction/drug effects , Transgenes/genetics , Up-Regulation/drug effects , Ventricular Remodeling/genetics , rac1 GTP-Binding Protein/geneticsABSTRACT
The totality of functional cardiomyocytes and an intact cardiac progenitor cell pool are key players in the myocardial cell homeostasis. Perturbation of either one may compromise the structural and functional integrity of the heart and lead to heart failure. Reactive oxygen/nitrogen species (ROS/RNS) are important regulators of cardiomyocyte viability; more recently, the interrelation between ROS and progenitor cell behavior and fate has moved into the spotlight. Increasing evidence suggests not only that ROS participate in the regulation of cardiac progenitor cell survival but also that they likewise affect their functional properties in terms of self-proliferation and differentiation. The apparent dichotomy of ROS/RNS effects with their adaptive and regulatory character on the one hand and their maladaptive and damaging features on the other pose a great challenge in view of the therapeutic exploitation of their role in the regulation of the myocardial cell homeostasis. In this article, mechanisms and potential significance of ROS/RNS action in the regulation of the myocardial cell homeostasis, in particular with respect to the preservation of viable cardiomyocytes and the maintenance of a functional cardiac progenitor cell pool, will be discussed.
