Unknown,Transcriptomics,Genomics,Proteomics

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Expression data from cultured rat right ventricular papillary muscles

ABSTRACT: Several different mechanical signals have been proposed to control the extent and pattern of myocardial growth and remodeling, though this has largely been studied using in vitro model systems that are not representative of intact myocardium or in vivo models in which isolating the effects of individual candidate stimuli is exceedigly difficult. We used a unique tissue culture system that allows the simultaneous control of multiple mechanical inputs and other potentially confounding stimuli (e.g., hormonal). Following a 12 hour culture period under prescribed mechanics, we used microarrays to identify genes that are up- or down-regulated in response to different amounts of mean stretch and cyclic shortening. Muscles were dissected (one from each of 12 different male LBN-F1 rats) and cultured for 12 hours in a pseudo-sterile muscle culture system under one of four mechanical input schemes (i.e., three biological replicates per mechanical input group). We prescribed low or high values of both time averaged stretch and cyclic shortening. Specifically, we targeted 4% or 16% mean stretch (from slack length) and 4% of 16% cylic shortening (% of slack length) over the 12 hour culture period to give the following four groups: high mean stretch x high shortening (group A); high mean stretch x low shortening (group B); low mean stretch x low shortening (group C); low mean stretch x high shortening (group D). This 2 x 2 factorial design allowed us to identify individual genes and/or molecular pathways that might be regulated by one or both of these mechanical inputs indpendent from other candidate mechanical or hormonal stimuli.

ORGANISM(S): Rattus norvegicus

SUBMITTER: Charles Haggart

PROVIDER: E-GEOD-45250 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Acute respiratory distress syndrome (ARDS) is a catastrophic form of acute lung injury (ALI). The necessity for mechanical ventilation (MV) renders patients at risk for ventilator induced lung injury (VILI). Exposure to repetitive cyclic stretch (CS) and/or over-inflation exacerbates injury. Reducing tidal volume (VT) is the only therapeutic strategy shown to mitigate morbidity and mortality. Cyclic stretch has been shown to differentially regulate gene expression in part through the activation of mammalian mitogen-activated protein kinase (MAPK). Although these studies have shown both molecular and cellular alterations, no unifying hypothesis to explain MV-induced lung injury has emerged. In the current study, we hypothesized that coordinated expression of cyclic stretch (CS)-responsive genes relies on the presence of common CS-sensitive regulatory elements. To identify CS-responsive genes, we undertook a comparative examination of the gene expression profile of human bronchial epithelial airway (Beas-2B) cells in response to various injurious stimuli involved in the pathogenesis of acute lung injury (ALI)/Ventilator induced lung injury (VILI): cyclic stretch, tumor necrosis factor alpha (TNF-a), and lipopolysaccharide (LPS). Experiment Overall Design: Human Bronchial Epithelial Cells (Beas-B2) cells grown on silicon elastic plates coated with Type I collagen (Flexercell International, McKeesport, PA) were exposed to six regiments for 4 h: 1) control (static, [control]); 2) mechanical stretch (25 PKa, 30 cycles per min, [stretch]); 3) LPS (1 mcg/ml [LPS]); 4) TNF-Î± (20 ng/ml; [TNF]); 5) mechanical stretch plus LPS [LPS+S], and 6) mechanical stretch plus TNF-Î± [TNF+S]. Total RNA (duplicate experiments) was extracted using TRIZOL reagent (as per manufactures specifications) and purified using Qiagen mRNA purification Kit (as per manufacturers specifications). mRNA was hybridized to Affymetrix Human U133plus2.0 chips. Probe based analysis, background reduction, and quantile data normalization was performed in MeV 4.0 of TM4 using Robust Multi-array Average (RMA).

Project description:Engineered cardiac tissues (ECTs) are platforms to investigate cardiomyocyte maturation and functional integration, to evaluate the feasibility of generating implantable tissues for cardiac repair and regeneration, and may be useful models for pharmacology and toxicology bioassays. These ECTs rapidly mature in vitro to acquire the features of functional cardiac muscle and respond to mechanical load with increased proliferation and maturation. ECTs can be generated from various immature cardiac cell sources and little is known regarding the broad changes in regulatory transcript expression that occur in these in vitro tissues during normal maturation and in response to mechanical or pharmacologic interventions. We tested the hypothesis that global ECT gene expression patterns are sensitive to mechanical loading conditions and tyrosine kinase inhibitors, similar to the maturing myocardium. We generated 3D ECTs from day 14.5 rat embryo ventricular cells, as previously published, and then treated constructs after 5 days in culture for 48 hours with mechanical stretch (5%, 0.5 Hz) and/or the p38MAPK (p38 mitogen-activated protein kinase) selective inhibitor BIRB796. RNA was isolated from 3 sets of experiments and assayed using a standard Agilent rat 4x44k V3 microarray and Pathway Analysis software for transcript expression fold changes and changes in regulatory molecules and networks. At the threshold of a 1.5 fold change in expression, mechanical stretch altered 1,559 transcripts, versus 1,411 for BIRB796, and 1,846 for stretch plus BIRB796. As anticipated, top pathways altered in response to these stimuli include Cellular Development, Cellular Growth and Proliferation; Tissue Development; Cell Death, Cell Signaling, and Small Molecule Biochemistry as well as numerous other pathways. Changes in transcript expression were confirmed by quantitative-PCR for selected regulatory molecules. Thus, ECTs display a broad spectrum of altered gene expression in response to mechanical load and/or tyrosine kinase inhibition, reflecting the complex regulation of proliferation, differentiation, and architectural alignment that occurs during ECT maturation and adaptation. This approach can now be used to test the role of individual molecules and pathways on the regulation of ECT maturation and remodeling. 7 and 4 biological replicates with four groups (control, mechanical stretch, BIRB and mechanical stretch with BIRB)

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Expression data from cultured rat right ventricular papillary muscles

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