In this study, we evaluated the activities of respiratory chain complexes and oxidative phosphorylation (OXPHOS) capacity of the heart to gain insights into the pathological significance of mitochondrial dysfunction in chagasic cardiomyopathy (CCM). In a murine model of Trypanosoma cruzi infection, biochemical and histochemical analysis of the cardiac mitochondria revealed deficiency of the respiratory chain complexes (CI-CV) in infected mice; the inhibition of CI activity was more pronounced in the acute infection phase, CIII was constitutively repressed throughout the infection and disease phase, and the CV defects appeared in chronic phase only. A substantial decline in cardiac mtDNA content (54-60%) and mitochondria-encoded transcripts (50-65%) with disease development indicated that the alterations in mtDNA contribute to the quantitative deficiencies in respiratory chain activity in chagasic hearts. The observations of a selective inhibition of redox-sensitive CI and CIII complexes that are also the site of free radical generation in mitochondria, and the decline in cardiac mtDNA content in infected mice, all support the free radical hypothesis of mitochondria dysfunction in CCM. Consequently, OXPHOS-mediated ATP synthesis capacity of the cardiac mitochondria in infected mice was substantially reduced (37-50%), suggesting an energy homeostasis in the affected tissue.

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.

SummarySeveral murine models demonstrate that mammalian longevity can be increased by single gene mutations affecting endocrine signalling, particularly via the GH/IGF-1 axis. In this study, we identify age-independent patterns of hepatic gene expression characteristic of long-lived Snell ( Pit1

Ultra-thin-layer SDS gel electrophoresis in conjunction with automated laser-induced fluorescence detection is a novel and powerful method for the analysis of fluorophore-labeled proteins. The technique described in this paper employs instant, noncovalent fluorophore labeling by the addition of a fluorescent staining dye to the sample proteins either during or immediately prior to the sample loading process. Thus, the method does not require time-consuming post- or preseparation staining/labeling. By combining the multilane format of SDS polyacrylamide slab gel electrophoresis and the high separation efficiency of capillary SDS gel electrophoresis, ultra-thin-layer SDS gel electrophoresis features rapid, high-throughput, and high-resolution analysis of proteins in the molecular mass range of 14-116 kDa. The good heat dissipation inherent to the ultrathin format enables the use of agarose and agarose-based composite separation matrixes, which can be easily replaced within the separation platform. Labeling efficiency as a function of the concentration of the staining dye, SDS, and proteins is thoroughly discussed. Detection sensitivity of the method was found to be at the low-femtomole level (1.25 ng/band), determined by analyzing a set of serial dilutions of standard proteins. Practical example of molecular mass determination and characterization of a complex protein mixture are also shown.

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