Matrix metalloproteinases mediate β-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 beta(1)-integrins after beta-AR stimulation. Inhibition of MMP-2 using SB3CT or stimulation of beta(1)-integrin signaling using laminin inhibited the increased association of MMP-2 with beta(1)-integrins. beta-AR stimulation increased poly-DP- 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-timulated apoptosis; and 3) the apoptotic effects of MMP-2 may be mediated, at least in part, via its interaction with beta(1) integrins and poly-ADP-ribose-polymerase cleavage.