Role of NO synthase in the development of Trypanosoma cruzi-induced cardiomyopathy in mice

Abstract

Trypanosoma cruzi infection results in an increase in myocardial NO and intense inflammation. NO modulates the T. cruzi-induced myocardial inflammatory reaction. NO synthase (NOS)1-, NOS2-, and NOS3-null mice were infected with T. cruzi (Brazil strain). Infected NOS1-null mice had increased parasitemia, mortality, and left ventricular inner diameter (LVID). Chronically infected NOS1- and NOS2-null and wild-type mice (WT) exhibited increased right ventricular internal diameter (RVID), although the fold increase in the NOS2-null mice was smaller. Infected NOS3-null mice exhibited a significant reduction both in LVID and RVID. Reverse transcriptase-polymerase chain reaction showed expression of NOS2 and NOS3 in hearts of infected NOS1-null and WT mice, whereas infected NOS2-null hearts showed little change in expression of other NOS isoforms. Infected NOS3-null hearts showed an increase only in NOS1 expression. These results may indicate different roles for NOS isoforms in T. cruzi-induced cardiomyopathy.

FIGURE 1

Representative myocardial sections of uninfected and T. cruzi–infected NOS-null (KO) mice 21 days post-infection stained with H&E (original magnification, ×20). Uninfected NOS1-null mice showed fibrosis, whereas infected NOS1-null mice showed marked inflammation and numerous pseudocysts (arrow). Infected NOS2-null mice had minimal inflammation, whereas in infected NOS3-null mice, there was inflammation and scattered areas of myonecrosis in the myocardium. Uninfected NOS2- and NOS3-null mice had no significant pathology. This figure appears in color at www.ajtmh.org.

FIGURE 2

Semi-quantitative RT-PCR for NOS isoforms in murine heart of control and T. cruzi–infected mice from wild-type and various NOS-null (KO) backgrounds at day 30 after infection. Representative gels showing NOS isoform expression in wild-type control (Con) and infected (Inf) mice (A). Representative RT-PCR gels showing NOS2 and NOS3 in NOS1-null mice (B), NOS1 and NOS3 in NOS2-null mice (C), and NOS1 and NOS2 in NOS3-null (D) mice. Data from two independent control (Con1 and Con2) and infected (Inf1 and Inf2) mice from wild-type and each null background are shown. In B–D, the respective NOS-null isoform was also amplified as a negative control. E, GAPDH RT-PCR as an internal control (N = 4 for each group).

FIGURE 3

Densitometry of the semi-quantitative RT-PCR data for NOS isoforms in T. cruzi–infected (Inf) murine hearts. In infected WT mice, there was an increase in the expression of all NOS isoforms compared with control (Con). In infected NOS1-null (KO) mice, expression of both NOS2 and NOS3 was significantly increased. In infected NOS2 KO mice, there was no significant increase in NOS1 or NOS3 isoforms, whereas infected NOS3 KO mice showed increased expression of NOS1 isoform. N = 3 for each group and column values represents the average, error bars, and the maximum and minimum values.

FIGURE 4

MRI measurements of end-diastolic right ventricle inner diameter (RVID) of chronically (6 months) infected and uninfected WT (C57BL/6J), NOS1-null (KO), NOS2-null and NOS3-null mice. N values are stated in Table 1.

FIGURE 5

Transverse cardiac gated end-diastolic MR images of hearts of infected and uninfected WT and NOS1- and NOS3-null (KO) mice. Images of the NOS2-null mice are not shown, because they were similar to WT mice, although with a less robust increase in RVID. Left (LV) and right ventricle (RV) are indicated by arrows. Images were acquired with a spin-echo sequence, FOV of 40 mm, and with TE = 18 ms, TR = 100–200 ms, number of transients = 4, and 128 × 256 matrix size interpolated to 256 × 256.