Gene expression analysis in mitochondria from chagasic mice: alterations in specific metabolic pathways

Abstract

Cardiac hypertrophy and remodelling in chagasic disease might be associated with mitochondrial dysfunction. In the present study, we characterized the cardiac metabolic responses to Trypanosoma cruzi infection and progressive disease severity using a custom-designed mitoarray (mitochondrial function-related gene array). Mitoarrays consisting of known, well-characterized mitochondrial function-related cDNAs were hybridized with 32P-labelled cDNA probes generated from the myocardium of mice during immediate early, acute and chronic phases of infection and disease development. The mitoarray successfully identified novel aspects of the T. cruzi-induced alterations in the expression of the genes related to mitochondrial function and biogenesis that were further confirmed by real-time reverse transcriptase-PCRs. Of note is the up-regulation of transcripts essential for fatty acid metabolism associated with repression of the mRNAs for pyruvate dehydrogenase complex in infected hearts. We observed no statistically significant changes in mRNAs for the enzymes of tricarboxylic acid cycle. These results suggest that fatty acid metabolism compensates the pyruvate dehydrogenase complex deficiencies for the supply of acetyl-CoA for a tricarboxylic acid cycle, and chagasic hearts may not be limited in reduced energy (NADH and FADH2). The observation of a decrease in mRNA level for several subunits of the respiratory chain complexes by mitoarray as well as global genome analysis suggests a limitation in mitochondrial oxidative phosphorylation-mediated ATP-generation capacity as the probable basis for cardiac homoeostasis in chagasic disease.

Figure 1. Mitoarray image of the gene expression profile in T. cruzi-infected murine hearts

C3H/HeN mice were infected by intraperitoneal injection with culture-derived trypomastigotes of T. cruzi. Heart ventricle sections were obtained at various time intervals post-infection, and total RNA was isolated and then used for prototype customized mitoarray analysis as described in the Materials and methods section. Replicate phosphorimages of the mitoarrays hybridized with P³²-labelled cDNA probes generated from total RNA of the murine hearts killed at 0, 5, 25, 40, 130 and 180 dpi are shown. Selected genes whose expression was increased or decreased in infected murine hearts relative to normal controls are enclosed by a rectangle or oval structure respectively.

Figure 2. Traditional RT–PCR analysis of the selected genes in heart tissue samples from T. cruzi-infected mice

Total RNA was isolated from the heart tissue of normal (N) mice and mice killed during the immediate early (IE, 3–5 dpi), acute (A, 25–40 dpi) and chronic (C, 130–180 dpi) phases of disease progression. First-strand cDNA was synthesized from 2 μg of the total RNA samples in a 20 μl reaction mixture. Subsequently, PCR was performed for 28–32 cycles using 2 μl of the 10-fold diluted cDNA as template and gene-specific primers. The ethidium bromide-stained gel images were quantified on a FluorChem 8800 Image Analyzing System. The quantitative results represent the mean values obtained from three independent experiments. The S.D. for all the data points was < 12%. All gene names are defined in Table 1. Results from traditional RT–PCR are shown in the left panels. The densitometric quantification of the mitoarray results is shown in the right panels.

Figure 3. Confirmation of mitoarray expression results by real-time PCR analysis

Total RNA was isolated from the heart tissue of normal and infected mice and first-strand cDNA synthesized (as in Figure 2). Subsequently, real-time PCR was performed using 2 μl of the 10-fold diluted cDNA as template and gene-specific primers. The results were normalized to GAPDH and represent mean values obtained from two independent experiments using heart tissue of at least three mice/experiment. The S.D. for all the data points was < 12%. All gene names are defined in Table 1. The bar graphs on the left show the relative mRNA level for target genes determined by real-time RT–PCR. 1/2^Ct values were plotted on the y-axis. Densitometric quantifications of the mitoarray results are shown in the right panels.

Figure 4. Enzymic activity of PDC is reduced in the myocardium of T. cruzi-infected mice

C3H/HeN mice were infected by intraperitoneal injection with T. cruzi. Enzymic activities of PDC, SDH and CS were measured in cardiac mitochondria isolated from normal mice and T. cruzi-infected mice killed at various time points post-infection. Specific activity [mmol·min⁻¹·(mg of protein)⁻¹] was calculated as means±S.D. obtained from duplicates estimated in three independent experiments.