Dataset Information

Reproducible ion-current-based approach for 24-plex comparison of the tissue proteomes of hibernating versus normal myocardium in swine models.

ABSTRACT: Hibernating myocardium is an adaptive response to repetitive myocardial ischemia that is clinically common, but the mechanism of adaptation is poorly understood. Here we compared the proteomes of hibernating versus normal myocardium in a porcine model with 24 biological replicates. Using the ion-current-based proteomic strategy optimized in this study to expand upon previous proteomic work, we identified differentially expressed proteins in new molecular pathways of cardiovascular interest. The methodological strategy includes efficient extraction with detergent cocktail; precipitation/digestion procedure with high, quantitative peptide recovery; reproducible nano-LC/MS analysis on a long, heated column packed with small particles; and quantification based on ion-current peak areas. Under the optimized conditions, high efficiency and reproducibility were achieved for each step, which enabled a reliable comparison of 24 the myocardial samples. To achieve confident discovery of differentially regulated proteins in hibernating myocardium, we used highly stringent criteria to define "quantifiable proteins". These included the filtering criteria of low peptide FDR and S/N > 10 for peptide ion currents, and each protein was quantified independently from ?2 distinct peptides. For a broad methodological validation, the quantitative results were compared with a parallel, well-validated 2D-DIGE analysis of the same model. Excellent agreement between the two orthogonal methods was observed (R = 0.74), and the ion-current-based method quantified almost one order of magnitude more proteins. In hibernating myocardium, 225 significantly altered proteins were discovered with a low false-discovery rate (?3%). These proteins are involved in biological processes including metabolism, apoptosis, stress response, contraction, cytoskeleton, transcription, and translation. This provides compelling evidence that hibernating myocardium adapts to chronic ischemia. The major metabolic mechanisms include a down-regulation of mitochondrial respiration and an increase in glycolysis. Meanwhile, cardioprotective and cytoskeletal proteins are increased, while cardiomyocyte contractile proteins are reduced. These intrinsic adaptations to regional ischemia maintain long-term cardiomyocyte viability at the expense of contractile function.

SUBMITTER: Qu J

PROVIDER: S-EPMC4015685 | biostudies-literature | 2014 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Reproducible ion-current-based approach for 24-plex comparison of the tissue proteomes of hibernating versus normal myocardium in swine models.

Qu Jun J Young Rebeccah R Page Brian J BJ Shen Xiaomeng X Tata Nazneen N Li Jun J Duan Xiaotao X Fallavollita James A JA Canty John M JM

Journal of proteome research 20140404 5

Hibernating myocardium is an adaptive response to repetitive myocardial ischemia that is clinically common, but the mechanism of adaptation is poorly understood. Here we compared the proteomes of hibernating versus normal myocardium in a porcine model with 24 biological replicates. Using the ion-current-based proteomic strategy optimized in this study to expand upon previous proteomic work, we identified differentially expressed proteins in new molecular pathways of cardiovascular interest. The ...[more]

PMID: 24697261

Similar Datasets

Project description:RationaleAllogeneic bone marrow-derived mesenchymal stem cells (MSCs) and cardiosphere-derived cells (CDCs) have each entered clinical trials, but a direct comparison of these cell types has not been performed in a large animal model of hibernating myocardium.ObjectiveUsing completely blinded methodology, we compared the efficacy of global intracoronary allogeneic MSCs (icMSCs, ≈35×10(6)) and CDCs (icCDCs, ≈35×10(6)) versus vehicle in cyclosporine-immunosuppressed swine with a chronic left anterior descending coronary artery stenosis (n=26).Methods and resultsStudies began 3 months after instrumentation when wall thickening was reduced (left anterior descending coronary artery % wall thickening [mean±SD], 38±11% versus 83±26% in remote; P<0.01) and similar among groups. Four weeks after treatment, left anterior descending coronary artery % wall thickening increased similarly after icCDCs and icMSCs, whereas it remained depressed in vehicle-treated controls (icMSCs, 51±13%; icCDCs, 51±17%; vehicle, 34±3%, treatments P<0.05 versus vehicle). There was no change in myocardial perfusion. Both icMSCs and icCDCs increased left anterior descending coronary artery myocyte nuclear density (icMSCs, 1601±279 nuclei/mm(2); icCDCs, 1569±294 nuclei/mm(2); vehicle, 973±181 nuclei/mm(2); treatments P<0.05 versus vehicle) and reduced myocyte diameter (icMSCs, 16.4±1.5 μm; icCDCs, 16.8±1.2 μm; vehicle, 20.2±3.7 μm; treatments P<0.05 versus vehicle) to the same extent. Similar changes in myocyte nuclear density and diameter were observed in the remote region of cell-treated animals. Cell fate analysis using Y-chromosome fluorescent in situ hybridization demonstrated rare cells from sex-mismatched donors.ConclusionsAllogeneic icMSCs and icCDCs exhibit comparable therapeutic efficacy in a large animal model of hibernating myocardium. Both cell types produced equivalent increases in regional function and stimulated myocyte regeneration in ischemic and remote myocardium. The activation of endogenous myocyte proliferation and regression of myocyte cellular hypertrophy support a common mechanism of cardiac repair.

Project description:The time course and extent of recovery after revascularization of viable dysfunctional myocardium are variable. Although fibrosis is a major determinant, myocyte structural and molecular remodeling may also play important roles.This study sought to determine whether persistent myocyte loss and/or irreversibility of protein changes that develop in hibernating myocardium have an impact on functional recovery in the absence of infarction.Swine implanted with a chronic left anterior descending artery (LAD) stenosis to produce hibernating myocardium underwent percutaneous revascularization, with serial functional recovery evaluated for 1 month (n = 12). Myocardial tissue was evaluated to assess myocyte size, nuclear density, and proliferation indexes in comparison with those of normal animals and nonrevascularized controls. Proteomic analysis by 2-dimensional differential in-gel electrophoresis was used to determine the reversibility of molecular adaptations of hibernating myocytes.At 3 months, physiological features of hibernating myocardium were confirmed, with depressed LAD wall thickening and no significant infarction. Revascularization normalized LAD flow reserve, with no immediate change in LAD wall thickening. Regional LAD wall thickening slowly improved but remained depressed 1 month post-percutaneous coronary intervention. Surprisingly, revascularization was associated with histological evidence of myocytes re-entering the growth phase of the cell cycle and increases in the number of c-Kit(+) cells. Myocyte nuclear density returned to normal, whereas regional myocyte hypertrophy regressed. Proteomic analysis demonstrated heterogeneous effects of revascularization. Up-regulated stress and cytoskeletal proteins normalized, whereas reduced contractile and metabolic proteins persisted.Delayed recovery of hibernating myocardium in the absence of scar may reflect persistent reductions in the amounts of contractile and metabolic proteins. Although revascularization appeared to stimulate myocyte proliferation, the persistence of small immature myocytes may have contributed to delayed functional recovery.

Project description:BACKGROUND:Protein post-translational modifications (PTMs) are an important aspect of protein regulation. The number of PTMs discovered within the human proteome, and other proteomes, has been rapidly expanding in recent years. As a consequence of the rate in which new PTMs are identified, analysis done in one year may result in different conclusions when repeated in subsequent years. Among the various functional questions pertaining to PTMs, one important relationship to address is the interplay between modifications and mutations. Specifically, because the linear sequence surrounding a modification site often determines molecular recognition, it is hypothesized that mutations near sites of PTMs may be more likely to result in a detrimental effect on protein function, resulting in the development of disease. METHODS AND RESULTS:We wrote an application programming interface (API) to make analysis of ProteomeScout, a comprehensive database of PTMs and protein information, easy and reproducible. We used this API to analyze the relationship between PTMs and human mutations associated with disease (based on the 'Clinical Significance' annotation from dbSNP). Proteins containing pathogenic mutations demonstrated a significant study bias which was controlled for by analyzing only well-studied proteins, based on their having at least one pathogenic mutation. We found that pathogenic mutations are significantly more likely to lie within eight amino acids of a phosphoserine, phosphotyrosine or ubiquitination site when compared to mutations in general, based on a Fisher's Exact test. Despite the skew of pathogenic mutations occurring on positively charged arginines, we could not account for this relationship based only on residue type. Finally, we hypothesize a potential mechanism for a pathogenic mutation on RAF1, based on its proximity to a phosphorylation site, which represents a subtle regulation difference that may explain why its biochemical effect has failed to be uncovered previously. The combination of the API and a dynamically expanding PTM database will make the reanalysis of this question and other systems-level questions easier in the future.

Dataset Information

Reproducible ion-current-based approach for 24-plex comparison of the tissue proteomes of hibernating versus normal myocardium in swine models.

Publications

Reproducible ion-current-based approach for 24-plex comparison of the tissue proteomes of hibernating versus normal myocardium in swine models.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets