Tumor necrosis factor alpha (TNF alpha) is associated with a higher risk of cardiovascular disease. Matrix metalloproteinase-2 (MMP-2) has been implicated in the pathophysiology of ischemic heart disease. However, the role of interactions between MMP-2 and TNF alpha, associated with cardiac apoptosis, is unknown. We hypothesized that MMP-2 will contribute to TNF alpha-induced myocardial apoptosis. After treatment with TNF alpha (1-20 ng/ml) for 24 h, or with TNF alpha (10 ng/ml) for 0, 6, 12, 24, or 48 h, MMP-2 activity, percent of TUNEL-positive myocytes, and DNA fragmentation dose, and time-dependently increased compared to control. However, TNF alpha blockade (neutralizing antibodies against human TNF alpha, 25 mu g/ml) significantly reduced the activity of MMP-2 and markers of apoptosis induced by TNF alpha. Interestingly, MMP-2 antibody (30 mu g/ml), or the MMP-2 inhibitors Doxycycline (Dox, 1-50 mu mol/l) or GM6001 (GM, 10 mu mol/l), prior to TNFa insult, decreased myocardial MMP-2 activity and reduced the percent of TUNEL-positive myocytes and DNA fragmentation. Moreover, MMP-2 inhibition reduced Bax expression and caspase3 activity, as well as increasing BcI2 expression. MMP-2 inhibition was associated with decreased cardiac MMP-2 activity and decreased myocardial apoptosis induced by TNF alpha. These results suggest that MMP-2 contributes to TNF alpha-induced apoptosis in cultured rat cardiac myocytes. (c) 2006 Elsevier Inc. All rights reserved.