Chagasic cardiomyopathy emerges in 30-40% of the patients infected by Trypanosoma cruzi, and accounts for >50,000 deaths and the loss of 2.74 million disability-adjusted life years per year. By the time most patients are diagnosed with Chagas disease, heart dysfunction has been progressing for many years. Available anti-parasite drug therapies are toxic and largely ineffective during the clinical disease phase. We do not yet know the molecular mechanisms that are disturbed in susceptible patients who will proceed to develop clinical cardiac disease. In recent preclinical and clinical studies, we have shown that T. cruzi-infected hosts sustain an oxidative stress of an inflammatory and mitochondrial origin, and oxidative pathology of the heart plays an important role in eliciting cardiac dysfunction during progressive Chagas disease. Further, we have found that distinct plasma proteins are oxidized/nitrated in acute and chronic chagasic animals, and these proteins are different from those modified in the plasma of animals with cardiomyopathy of other etiologies. These observations support our hypothesis that pathological processes leading to the development of chagasic cardiomyopathy in patients would cause characteristic changes in the concentration/oxidation of proteins in the blood and generate a detectable disease-specific molecular phenotype. In this study, we propose to develop a plasma oxidative proteome in chagasic patients.
In aim 1, we would utilize a ProteomeLab PF2D liquid chromatography system to identify the changes in the plasma proteome of chagasic subjects and compare them to findings in normal/healthy and other cardiomyopathy subjects.
In aim 2, we would identify the oxidative modifications in the plasma proteome of Chagasic patients. For this purpose, we have enhanced the PF2D system by adding an in-line photodiode array spectrophotometer that allows us to visualize the post-translational modifications at their signature wavelength. Proteins that are differentially expressed/oxidized in a disease-specific manner will be identified by LC-MS/MS and validated by western blotting. Upon completion of these studies, we anticipate identifying molecular markers that will be useful in classification of the Chagas disease state and diagnosis of those asymptomatic individuals who are at risk of developing chronic, dilated cardiomyopathy. By biological analysis of the differentially expressed/modified proteome data, we will identify the interlocking pathways and co-regulated sub-networks that are dysregulated during progression from asymptomatic to symptomatic clinical disease. These disturbed networks will suggest why and when infected individuals become susceptible to Chagas disease and yield novel targets for engineering the combination therapies for the prevention and treatment of chagasic, and possibly other cardiomyopathies.
The studies in this project are aimed at developing the Chagas disease-associated plasma oxidative proteome. Upon completion of these studies, we will identify molecular markers of patient's disease state and patient's susceptibility to develop Chagas disease and the molecular disturbances that enhance the risk of clinical disease development in asymptomatic individuals. These studies will yield potential targets for the development of new drug therapies. ? ? ?
