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Differential regulation of nitric oxide synthase isoforms in experimental acute chagasic cardiomyopathy
B Chandrasekar1, P C Melby, D A Troyer
1Medical Service, Department of Veterans Affairs Medical Center, and Departments of Medicine and Pathology, The University of Texas Health Science Center, San Antonio, TX 78229-3900, USA.
Insights
Early in Chagas
Area of Science:
- Cardiovascular Research
- Immunology
- Parasitology
Background:
- Previous studies demonstrated induction of IL-1beta, IL-6, and TNF-alpha in the myocardium during acute experimental Trypanosoma cruzi infection (Chagas' disease).
- These cytokines are known to mediate myocardial depressive effects partly through nitric oxide synthase (NOS) induction, nitric oxide (NO) production, and peroxynitrite formation.
Purpose of the Study:
- To investigate the expression, activity, and localization of NOS isoforms in the myocardium during experimental T. cruzi infection.
- To quantify levels of NO, malondialdehyde (oxidative stress marker), and peroxynitrite in the myocardium at various time points post-infection.
Main Methods:
- Experimental infection of rats with T. cruzi trypomastigotes.
- Assessment of myocardial inflammatory infiltrate and amastigote nests.
- Measurement of nitrate + nitrite levels, NOS2 and NOS3 mRNA expression, NOS isoform protein levels, NOS enzyme activity, malondialdehyde, and nitrotyrosine immunoreactivity.
Main Results:
- Myocardial inflammatory infiltrate and amastigote nests increased over the infection course.
- Significant increases in nitrate + nitrite, NOS2 mRNA, and NOS2 activity were observed at all time points in infected rats.
- Constitutive NOS activity, malondialdehyde, and NOS3 mRNA levels showed transient increases; protein levels paralleled mRNA expression. Nitrotyrosine levels increased over time, indicating peroxynitrite formation.
Conclusions:
- The myocardium expresses high levels of NOS and NO metabolites as early as 1.5 days post-infection, even before detectable parasite or immune cell infiltration.
- Early NO production in the myocardium is insufficient to clear T. cruzi parasites.
- These findings highlight the complex interplay between inflammation, oxidative stress, and NO metabolism in the early stages of Chagas' disease.
Abstract:
We have previously demonstrated induction and high level expression of IL-1beta, IL-6 and tumour necrosis factor-alpha in the myocardium during the acute stage of experimental Trypanosoma cruzi infection (Chagas' disease). The myocardial depressive effects of these cytokines are mediated in part by the induction of nitric oxide synthase (NOS), production of nitric oxide (NO) and formation of peroxynitrite. In this study we investigated the expression, activity and localization of NOS isoforms, and the levels of NO, malondialdehyde (a measure of oxidative stress), and peroxynitrite in rats at 1.5, 5, 10 and 15 days after infection with T. cruzi trypomastigotes. The myocardial inflammatory infiltrate and number of amastigote nests increased over the course of infection. A significant increase in tissue nitrate + nitrite levels, NOS2 mRNA, and NOS2 enzyme activity was observed at all time points in the infected compared with uninfected animals. The enzyme activity of constitutive NOS, tissue malondialdehyde levels, and NOS3 mRNA levels was only transiently increased after infection. The protein levels of the NOS isoforms paralleled their mRNA expression. While no positive nitrotyrosine immunoreactivity was detected in control myocardium, its levels increased in infected animals over time. Thus, by 1.5 days post-infection, when no parasite or immune cell infiltration could be detected, the myocardium expressed high levels of NOS and NO metabolites. Nevertheless, the early production of NO in the myocardium was not sufficient to clear the parasites.