Increased activity of inducible nitric oxide synthase (iNOS) is an important factor contributing to the occurrence of apoptosis in the heart in various disorders. The present study was designed to evaluate the correlation of these changes in acute Chagasic myocarditis. Dogs (n=6) were sacrificed 33 days after inoculation with the 21-SF strain of Trypanosoma cruzi. Sections of formalin-fixed, paraffin-embedded myocardium were used for the detection of apoptosis (nick end labeling with a rhodamine conjugate) and then immunostained with polyclonal antibodies against iNOS and nitrotyrosine (a product of the reaction between NO and tissue proteins), followed by secondary antibodies conjugated with fluorescein isothiocyanate. Sections were then counterstained with DAPI to demonstrate the DNA in tissue cells and T. cruzi and examined by confocal microscopy. Apoptosis was most common in macrophages and lymphocytes, but was also observed in endothelial cells, as well as in infected and uninfected myocytes and dendritic cells in areas of inflammation. The parasites within some of the infected myocytes gave a positive reaction for apoptosis. Reactivity for iNOS and nitrotyrosine was strong in myocytes and macrophages. In myocytes, the extent of apoptosis in intracellular T. cruzi correlated with the intensity of staining for iNOS. iNOS is greatly increased in areas of inflammation in acute Chagasic myocarditis, suggesting that it plays a role (by generating NO) in inducing apoptosis in T. cruzi and host cells.
