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Gelatin zymography, first described by Heussen and Dowdle in the 1980s, is a widely used valuable tool in research and diagnostics. The technique identifies gelatinases by the degradation of their preferential substrate as well as by their molecular weight (kDa). We here describe detailed methodology for the detection of pro- and active- forms of both MMP-2 (gelatinase A) and MMP-9 (gelatinase B) in cells using norepinephrine-stimulated H9c2 cardiomyoblasts as model. An easy to follow step-by-step protocol has been carefully written for reliable results. We also suggest an acceptable method for quantification of gelatin zymograms.

Zymography has long been used for the detection of substrate-specific enzyme activity. In situ zymography (ISZ), an adaptation from the conventional substrate zymography, is a widely employed technique useful for the detection, localization, and estimation of enzyme-substrate interactions in tissues. Here, we describe a protocol to detect 'in position' matrix metalloproteinase (MMP) activity in cells utilizing H9c2 cardiomyoblasts as a model. This technique is primarily adopted from the method used for histological sections and is termed as 'Cell in situ Zymography'. It is a simple, sensitive, and quantifiable methodology to assess the functional activity of an enzyme 'on site/in position' in cell culture.

Matrix metalloproteinases (MMPs), a family of proteolytic enzymes important in the degradation and turnover of extracellular matrix (ECM) components, result in geometric and structural changes of the myocardium which has become a major mechanism for the progression of various cardiovascular pathologies including atherosclerosis, hypertension and end-organ damage, myocardial infarction, ischemia-reperfusion injury, and hypertrophic cardiomyopathies which ultimately result in heart failure. The natural MMP inhibitors known as Tissue Inhibitors of Matrix Metalloproteinases (TIMPs) also play a major role in regulating the myocardial remodeling by working in a balance with MMPs. An imbalance between MMPs and TIMPs results in disease condition. Restoration of this balance is therefore a promising therapeutic strategy for the treatment of cardiac diseases. Use of synthetic inhibitors to regulate the MMP activity serves as emerging candidates of clinical utility in order to design effective therapeutic strategies. In this chapter we summarize about the MMP family, their involvement in various cardiovascular disease conditions along with the role of MMP inhibitors. We have also mentioned different techniques which can be useful in identification of MMPs and TIMPs and help unveil the mechanisms related to them in disease conditions.