Generation of nitrotyrosine precedes activation of metalloproteinase in myocardium of hyperhomocysteinemic rats

The hypothesis is that homocysteine decreases endothelial nitric oxide (NO) availability by generating nitrotyrosine. In the absence of NO, and in an attempt to reduce endocardial load by dilatation, the matrix metalloproteinase (MMP) is activated. To address this hypothesis, homocysteine (0.67 mg/ml) was administered in drinking water of Sprague-Dawley rats for 8 weeks. To elicit the reversible effects of homocysteine, homocysteine was removed from the water after 8 weeks. The plasma levels of homocysteine were 2.79 +/- 0.5 muM in control (n = 6), measured by spectrofluorometry. The levels of homocysteine increased to 22 +/- 1.3 and 17 +/- 2.8 muM following 4 (n = 6) and 8 (n = 6) weeks of homocysteine treatment, respectively. The level of homocysteine decreased to 5.8 +/- 1.0 muM (n = 6) when homocysteine was removed from the drinking water. The mean arterial pressure (MAP) of control rats was 108 +/- 10 mm Hg and increased to 128 +/- 2 and 130 +/- 3 mm Hg following 4 and 8 weeks of homocysteine treatment, respectively. When homocysteine was removed from the drinking water, the MAP was decreased to 118 +/- 3 mm Hg. Left ventricle (LV) parameters were measured by a catheter in the LV through right common carotid artery in anesthetized rats. The LV tissue was analyzed for MMP activity by zymography. Levels of nitrotyrosine and cardiospecific tissue inhibitor of metalloproteinase-4 (TIMP-4/CIMP) were measured by western blot analysis using the respective antibodies. The specific bands in zymographic gel and western blot were scanned and normalized with beta-actin. The results suggest a continuous increase in nitrotyrosine levels at 4 and 8 weeks after homocysteine administration. The removal of homocysteine did not decrease the levels of nitrotyrosine. The zymographic analysis revealed a temporal increase in MMP-2 activity from 4 to 8 weeks post bornocysteine administration. However, removal of homocysteine did not decrease the MMP-2 activity. The cardiac active diastolic function, -dP/dt, was decreased at 4 weeks and stayed depressed up to 12 weeks. The end-diastolic pressure started increasing at 8 weeks; at this point the MMP-2 activity was also increased. The results suggest that in the absence of endothelial NO, and in an attempt to reduce LV load, MMP-2 is activated and CIMP is inactivated, by increasing nitrotyrosine.