Human heart matrix metalloproteinases (MMP) are present in the latent form and activated in the failing heart. To examine whether the MMP activation was due to gene and/or post-translational modification, we analysed tissue from 10 explanted hearts due to coronary heart disease (CHD) and five normal left atrial tissue from donor hearts. Based on in situ immunolabeling MMP-1, tissue inhibitor of metalloproteinase (TIMP-1) and collagen were co-localized in the interstitial tissue. Based on sandwich ELISA, TIMP-1 and MMP-1 levels were 37 +/- 8 ng/mg and 9 +/- 2 ng/mg in normal tissue (P < 0.01) and 12 +/- 5 ng/mg and 75 +/- 11 ng/mg in the infarcted tissue (P < 0.01), respectively. These levels suggest repression of TIMP-1 during myocardial infarction. Northern blot analysis indicated that the mRNAs for both MMP-1 and TIMP-1 were increased three-to four-fold in the infarcted tissue as compared to the normal tissue, suggesting upregulation of MMP and TIMP gene transcription following infarction. Based on in situ tissue overlay zymography, the generalized activation of MMP was observed in the interstitium of the infarcted heart. Zymographic and immunoblot analysis demonstrated the presence of one band at 66 kDa (MMP-2) in the normal tissue and several bands at 92 (MMP-9), 66 (MMP-2) and 54 kDa (MMP-1) in the infarcted heart. Incubation of the zymographic gel with metal chelator (phenanthroline) abolished bands at 92 kDa and 54 kDa but phenanthroline did not abolish the lytic band at 66 kDa. The 66 kDa band was completely abolished in the presence of phenanthroline and phenyl methyl sulfonyl fluoride (PMSF). 2D-zymographic analysis suggested that the lytic band at 66 kDa was a mixture of two neutral proteinases with different isoelectric point. Plasminogen/gelatin zymographic analysis of infarcted tissue extract indicated that the band at 66 kDa was plasmin generated due to increased expression of tissue plasminogen activator (tPA) activity. In relation to increased expression of gelatinase in the infarcted tissue, our data suggest that gelatinase B (92 kDa) is induced in diseased heart. The results suggest that tPA converts plasminogen to plasmin which, in turn, activates MMPs and inactivates TIMP-1 post-translationally following ischemic cardiomyopathy.