Differential regulation of nitric oxide synthase isoforms in experimental acute chagasic cardiomyopathy

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.

Fig. 1

Nitric oxide synthase isoform mRNA expression in myocardium from Trypanosoma cruzi-infected rats. Each lane represents RNA from an individual animal. Thirty micrograms of total RNA per lane were electrophoresed, electroblotted onto nitrocellulose, and fixed by UV irradiation. The blot was reprobed after stripping off previous probe. mRNA size was determined in comparison with the relative mobility of 28S and 18S rRNA and with that of the mRNA ladder (0·2–9·5 kb). 28S rRNA probe was used as an internal control and indicates equal levels of RNA loading in all lanes of the gel. The autoradiographic time was 4 days for NOS2 and 3, and 12 h for 28S. Even after 8 days of exposure, no signal was detected in lanes that did not have detectable levels of mRNA.

Fig. 2

Densitometric analysis. The autoradiographic bands shown in Figs 1 and 3 are semiquantified by videoimage analysis. The values are mean ± s.e.m. of arbitrary numbers obtained. For mRNA expression, the numbers are represented as a ratio of specific gene to that of 28S rRNA to compensate for any loading differences. NOS2, *P < 0·01; **P < 0·0001; NOS3, †P < 0·05; ††P < 0·0001 (versus respective control).

Fig. 3

NOS protein levels (Western blot analysis) in myocardium from Trypanosoma cruzi-infected rats. Each lane represents protein homogenate from an individual animal. Sixty micrograms of protein per lane were electrophoresed using glycine−16·5% SDS–PAGE. The separated proteins were electroblotted onto nitrocellulose. After blocking with 10% normal goat serum, membranes were sequentially incubated with the primary, secondary and ¹²⁵I-Protein A. Autoradiographic exposure time was 4 days. Protein molecular weights were determined in comparison with respective recombinant cytokines, and prestained low molecular weight standards (shown on the left). Arrow indicates the relative position of protein detected by the respective antibody.

Fig. 4

Enzyme activity. Enzyme activity was determined by conversion of ³H-arginine to ³H-citrulline in the absence (constitutive; NOS1 and 3) or presence (inducible; NOS2) of calcium chelators EGTA + EDTA. Values are mean ± s.e.m. of six animals at each time period. *P < 0·025; **P < 0·005; and †P < 0·0001 (versus control).

Fig. 5

Malondialdehyde (MDA) levels. As a measure of lipid peroxidation, MDA levels were determined in control and infected animals. Values are mean ± s.e.m. of six animals at each time period. *P < 0·01; **P < 0·005; ***P < 0·0001 (versus control).

Fig. 6

(See previous page.) Localization of peroxynitrite and NOS isoforms. H–E staining of control (A) and Trypanosoma cruzi-infected (B) myocardium at 15 days post-infection. (C) No specific immunoreactivity in an infected animal when primary antibody is omitted. While no nitrotyrosine immunoreactivity was detected in a control animal (data not shown), positive immunoreactivity was readily detected in infected animal at 1·5 days post-infection localized to the edges of myocardial islands (D); its levels increased with majority of cardiomyocytes showing positive staining by 15 days p.i. (E). While weak NOS2 immunoreactivity was detected in blood vessels in control animals (F), intense and diffuse NOS2 staining was detected in both blood vessels and cardiomyocytes in an infected animal at 15 days p.i. (G). NOS1 was not detected in either control (H) or in infected animals at 1·5 days p.i. (data not shown), but a weak staining was observed at 15 days p.i. (I). Weak NOS3 immunoreactivity in controls (J), and intense staining in an infected animal at 1·5 days p.i (K). Presence of amastigotes in the cardiomyocytes is indicated by arrows. (Mag. × 250.)