Pathway analysis of dilated cardiomyopathy using global proteomic profiling and enrichment maps

Global protein expression profiling can potentially uncover perturbations associated with common forms of heart disease. We have used shotgun MS/MS to monitor the state of biological systems in cardiac tissue correlating with disease onset, cardiac insufficiency and progression to heart failure in a time-course mouse model of dilated cardiomyopathy. However, interpreting the functional significance of the hundreds of differentially expressed proteins has been challenging. Here, we utilize improved enrichment statistical methods and an extensive collection of functionally related gene sets, gaining a more comprehensive understanding of the progressive alterations associated with functional decline in dilated cardiomyopathy. We visualize the enrichment results as an Enrichment Map, where significant gene sets are grouped based on annotation similarity. This approach vastly simplifies the interpretation of the large number of enriched gene sets found. For pathways of specific interest, such as Apoptosis and the MAPK (mitogen-activated protein kinase) cascade, we performed a more detailed analysis of the underlying signaling network, including experimental validation of expression patterns

In utero exposure to butyl benzyl phthalate induces modifications in the morphology and the gene expression profile of the mammary gland: an experimental study in rats

<p>Abstract</p> <p>Background</p> <p>Environmental estrogens are exogenous estrogen-mimicking compounds that can interfere with endogenous endocrine systems. Several of these endocrine disruptors have been shown to alter normal development and influence tumorigenesis in experimental models. N-butyl benzyl phthalate (BBP), a widely used plasticizer, is a well-known endocrine disruptor. The aim of this study was to elucidate the effect of prenatal exposure to BBP on the morphology, proliferative index, and genomic signature of the rat mammary gland at different ages.</p> <p>Methods</p> <p><it>In utero </it>exposure was performed by gavage of pregnant Sprague Dawley CD rats with 120mg or 500mg BBP/kg/day from day 10 post-conception to delivery. Female litters were euthanized at 21, 35, 50 and 100 days. The morphology and proliferative index of the mammary gland were studied from whole mount preparations and BrdU incorporation, respectively. Gene expression profile was assessed by microarrays. Several genes found differentially expressed and related to different functional categories were further validated by real time RT-PCR.</p> <p>Results</p> <p>Prenatal exposure of BBP induced delayed vaginal opening and changes in the post-natal mammary gland long after the end of the treatment, mainly by 35 days of age. Exposure to the high dose resulted in modifications in architecture and proliferative index of the mammary gland, mostly affecting the undifferentiated terminal end buds. Moreover, the expression profiles of this gland in the exposed rats were modified in a dose-dependent fashion. Analysis of functional categories showed that modified genes were related to immune function, cell signaling, proliferation and differentiation, or metabolism.</p> <p>Conclusions</p> <p>Our data suggest that <it>in utero </it>exposure to BBP induced a delayed pubertal onset and modified morphology of the mammary gland. These alterations were accompanied by modifications in gene expression previously associated with an increased susceptibility to carcinogenesis.</p

A Mighty Small Heart: The Cardiac Proteome of Adult Drosophila melanogaster

Drosophila melanogaster is emerging as a powerful model system for the study of cardiac disease. Establishing peptide and protein maps of the Drosophila heart is central to implementation of protein network studies that will allow us to assess the hallmarks of Drosophila heart pathogenesis and gauge the degree of conservation with human disease mechanisms on a systems level. Using a gel-LC-MS/MS approach, we identified 1228 protein clusters from 145 dissected adult fly hearts. Contractile, cytostructural and mitochondrial proteins were most abundant consistent with electron micrographs of the Drosophila cardiac tube. Functional/Ontological enrichment analysis further showed that proteins involved in glycolysis, Ca2+-binding, redox, and G-protein signaling, among other processes, are also over-represented. Comparison with a mouse heart proteome revealed conservation at the level of molecular function, biological processes and cellular components. The subsisting peptidome encompassed 5169 distinct heart-associated peptides, of which 1293 (25%) had not been identified in a recent Drosophila peptide compendium. PeptideClassifier analysis was further used to map peptides to specific gene-models. 1872 peptides provide valuable information about protein isoform groups whereas a further 3112 uniquely identify specific protein isoforms and may be used as a heart-associated peptide resource for quantitative proteomic approaches based on multiple-reaction monitoring. In summary, identification of excitation-contraction protein landmarks, orthologues of proteins associated with cardiovascular defects, and conservation of protein ontologies, provides testimony to the heart-like character of the Drosophila cardiac tube and to the utility of proteomics as a complement to the power of genetics in this growing model of human heart disease

Thermal injury increases TMR induced angiogenesis in the ischemic myocardium

Background. A growing number of patients suffering from ischemic cardiomyopathy are not eligible for conventional revascularization. This has prompted new research in the field of angiogenesis. This study hypothesized that since inflammation is probably the mechanism behind TMR induced angiogenesis; a larger inflammatory response induced by thermal injury may lead to increased angiogenesis.Methods. The model used for this study was coronary artery ligation in the Rat. Four groups of animals were used to compare the novel experimental approach with conventional TMR and with ischemia alone. Neovascularization was determined by immunohistochemical techniques using anti-Factor VIII antibody. Evaluation of VEGF, Ang-1 and Ang-2 expression was also carried out using immunohistochemistry.Results. The experimental "HOT" TMR technique resulted in significantly increased angiogenesis presumably due to the thermal injury induced by the novel technique. Also a significant increase in VEGF expression was observed in all ischemic groups. Ang-1 expression was decreased in the experimental group while it was similar in the other groups. Finally Ang-2 was induced by ischemia as evidenced by increased expression among all ischemic groups. However Ang-2 expression did not significantly vary among ischemic groups.Conclusions. The addition of thermal injury by heating of the needle led to an increased angiogenic response compared to ischemia alone and compared to conventional TMR. This increased angiogenesis was associated with increased VEGF expression at one week, however there was a significant inverse correlation between VEGF expression and angiogenesis among the ischemic groups. Also angiopoietin expression was in agreement with expression characteristics described in the literature

The role of urotensin-II in atherosclerosis and ischemic cardiomyopathy /

Atherosclerosis, a vascular disease which may lead to coronary artery occlusion and consequent myocardial infarction is primarily caused by dyslipidemia. Ischemic cardiomyopathy due to atherosclerosis is the leading cause of morbidity and mortality in the western world today. Vasoactive factors are increasingly being recognized not only as contributors to atherosclerotic plaque formation, but also to cardiac function and remodeling following ischemic cardiac injury. Urotensin II (UII) is one such vasoactive factor. UII possesses a wide range of cardiovascular effects, from contraction of the rat aorta to complete cardiovascular collapse in cynomolgus monkeys. UII binds a seven transmembrane spanning G-protein coupled receptor termed UT. Expression of UII is significantly elevated in the hearts of patients with congestive heart failure (CHF). Recent reports have also shown increased plasma levels of the peptide in patients with CHF, and these levels correlated with the severity of the disease. This project was designed to investigate the role of UII and UT in both atherosclerosis and CHF. To this end, UII expression was evaluated both in a model of CHF in the rat, and in human atherosclerosis of the carotid arteries and aortae. Furthermore, the pathophysiological role of urotensin-I1 in CHF was investigated, with the use of a selective UII antagonist, SB-611812. Finally, genetically modified mice deficient in either ApoE, UT, or both genes, were evaluated to study the role of UII/UT signaling in a model of atherosclerosis. We found that UII and its receptor, UT, were both significantly elevated in a model of CHF induced by coronary artery ligation. UII antagonism significantly attenuated mortality, cardiac dysfunction, and hypertrophy. This was associated with a significant decrease in cardiac fibrosis. We next went on to demonstrate that UII and UT were significantly elevated in human atherosclerotic carotid arteries and aortae. Finally, we demonstrated that deletion of the UT gene in mice deficient for ApoE exacerbates atherosclerosis of the aorta. Furthermore, this was associated with significantly increased hyperlipidemia and organ hypertrophy as well as significantly reduced body mass, liver mass, and hepatic steatosis.In conclusion we were the first to demonstrate a pathophysiological role for UII in cardiovascular diseases which may lead to a breakthrough in the management of CHF and may also give more insight into the pathogenesis of atherosclerosis

Lipotoxic Palmitate Impairs the Rate of β-Oxidation and Citric Acid Cycle Flux in Rat Neonatal Cardiomyocytes

Background/Aims: Diabetic hearts exhibit intracellular lipid accumulation. This suggests that the degree of fatty acid oxidation (FAO) in these hearts is insufficient to handle the elevated lipid uptake. We previously showed that palmitate impaired the rate of FAO in primary rat neonatal cardiomyocytes. Here we were interested in characterizing the site of FAO impairment induced by palmitate since it may shed light on the metabolic dysfunction that leads to lipid accumulation in diabetic hearts. Methods: We measured fatty acid oxidation, acetyl-CoA oxidation, and carnitine palmitoyl transferase (Cpt1b) activity. We measured both forward and reverse aconitase activity, as well as NAD+ dependent isocitrate dehydrogenase activity. We also measured reactive oxygen species using the 2', 7'-Dichlorofluorescin Diacetate (DCFDA) assay. Finally we used thin layer chromatography to assess diacylglycerol (DAG) levels. Results: We found that palmitate significantly impaired mitochondrial β-oxidation as well as citric acid cycle flux, but not Cpt1b activity. Palmitate negatively affected net aconitase activity and isocitrate dehydrogenase activity. The impaired enzyme activities were not due to oxidative stress but may be due to DAG mediated PKC activation. Conclusion: This work demonstrates that palmitate, a highly abundant fatty acid in human diets, causes impaired β-oxidation and citric acid cycle flux in primary neonatal cardiomyocytes. This metabolic defect occurs prior to cell death suggesting that it is a cause, rather than a consequence of palmitate mediated lipotoxicity. This impaired mitochondrial metabolism can have important implications for metabolic diseases such as diabetes and obesity