Matrix metalloproteinases mediate beta-adrenergic receptor-stimulated apoptosis in adult rat ventricular myocytes.
Changes in the synthesis and activity of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) are associated with myocardial remodeling. Here we measured the expression and activity of MMPs and TIMPs, and tested the hypothesis that increased MMP activity plays a proapoptotic role in beta-adrenergic receptor (beta-AR)-stimulated apoptosis of adult rat ventricular myocytes (ARVMs). beta-AR stimulation (isoproterenol, 24 h) increased mRNA levels of MMP-2 and TIMP-1 while it decreased TIMP-2 mRNA levels as analyzed by real-time PCR. Western blot analysis, immunocytochemical analysis, in-gel zymography, and MMP-2 activity assay confirmed beta-AR-stimulated increases in MMP-2 protein levels and activity. Inhibition of MMPs using GM-6001 (a broad-spectrum inhibitor of MMPs), SB3CT (inhibitor of MMP-2), and purified TIMP-2 inhibited beta-AR-stimulated apoptosis as determined by TdT-mediated dUTP nick end labeling staining. Treatment with active MMP-2 alone increased the number of apoptotic cells. This increase in MMP-2-mediated apoptosis was inhibited by GM-6001 and SB3CT pretreatment. Coimmunoprecipitation studies indicated increased physical association of MMP-2 with beta1-integrins after beta-AR stimulation. Inhibition of MMP-2 using SB3CT or stimulation of beta1-integrin signaling using laminin inhibited the increased association of MMP-2 with beta1-integrins. beta-AR stimulation increased poly-ADP-ribose-polymerase cleavage, which was inhibited by inhibition of MMP-2. These data suggest the following: 1) beta-AR stimulation increases MMP-2 expression and activity and inhibits TIMP-2 expression; 2) inhibition of MMPs, most likely MMP-2, inhibits beta-AR-stimulated apoptosis; and 3) the apoptotic effects of MMP-2 may be mediated, at least in part, via its interaction with beta1 integrins and poly-ADP-ribose-polymerase cleavage.
Menon
Bindu
B
Department of Physiology, James H. Quillen College of Medicine, East Tennessee State University, PO Box 70576, Johnson City, Tennessee 37614, USA.
Singh
Mahipal
M
Singh
Krishna
K
D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D004151:Dipeptides / Q000494:pharmacology; D019012:Integrin beta1 / Q000378:metabolism; D007545:Isoproterenol / Q000494:pharmacology; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism / Q000502:physiology*; D061965:Matrix Metalloproteinase Inhibitors; D032383:Myocytes, Cardiac / Q000502:physiology*; D011480:Protease Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000037:antagonists & inhibitors; D051381:Rats; D011943:Receptors, Adrenergic, beta / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000037:antagonists & inhibitors / Q000235:genetics; D016276:Ventricular Function
D000318:Adrenergic beta-Agonists; D004151:Dipeptides; D019012:Integrin beta1; D061965:Matrix Metalloproteinase Inhibitors; C078131:N-(2(R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl)-L-tryptophan methylamide; D011480:Protease Inhibitors; D012333:RNA, Messenger; D011943:Receptors, Adrenergic, beta; D019716:Tissue Inhibitor of Metalloproteinase-2; D020778:Matrix Metalloproteinase 2; D007545:Isoproterenol