| Regulation of cardiac responses to increased load: Role of endothelin-1, angiotensin II and collagen XV | ||
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After the exercise protocol the left and right ventricles were separated and the left ventricles cut into three pieces for histological, mRNA and biochemical analysis. The possibility of cardiac injury was studied by analyzing the extent of apoptosis, the activities of matrix metalloproteinase 2 (proMMP-2) and β -glucuronidase and the ANP mRNA levels.
DNA fragmentation (terminal deoxyribonucleotidyl transferase -mediated dUTP nick end labeling, TUNEL assay) was detected from cryostat sections stained with the In Situ Cell Death Detection Kit (Boehringer Mannheim) according to the manufacturer’s protocol. For quantitative analysis, the mean number of TUNEL-positive nuclei was counted in four sections on different depths in each sample.
A frozen sample was placed in buffer (1:10 w/v in homogenization buffer: 0.2 M NaCl, 0.1% TritonX-100, 0.02 M Tris) and homogenized by hand using a glass probe. 50 µl of homogenate was taken for the β -glucuronidase activity assay and the rest centrifuged for 20 minutes at 13000 rpm for zymography.
ProMMP-2 activity was measured by zymography. 7.5% running gels containing 1 mg/ml gelatin were overlaid with 4% stacking gels. The samples (cardiac homogenates mixed with a 1/1 volume of sample buffer: 0.4 M Tris, pH 6.8, 2% SDS, 20% glycerol and 0.03% bromphenol blue) were loaded into the gel and electrophoresis was carried out first at 16 mA for 1 hour and then at 24 mA until the dye front ran off the gels. The gels were incubated for 30 min in a solution containing 2.5% Tween 80 and 50 nM Tris, pH 7.5, and then at 37°C for 18 h in a solution containing 50 mM Tris, pH 7.5, 5 mM CaCl2 and 10 mM ZnCl2. Gelatinase activity was revealed by negative staining with Coomassie Brilliant. Purified proMMP-2 was used for identifying the enzyme activity. The degree of digestion was quantified by densitometry and area analysis.
β -glucuronidase activity was measured in a muscle homogenate. Briefly, 450 µl of 0.1M acetate buffer (pH 4.2) was added to 50 µl cardiac homogenate. After five minutes of preincubation at 37°C, 250 µl of substrate (5 mM p-nitrophenyl-β D-glucuronidase, Sigma) was added, and incubated overnight at 37°C. The reaction was stopped by adding 1.5 ml of cold glycine buffer (0.1 M pH 10.8), followed by centrifugation at 3000 rpm for 10 minutes, after which β -glucuronidase activity was calculated based on absorbance at 420 nm.