Project description:Here we examine key regulatory pathways underlying the transition from compensated hypertrophy (HYP) to decompensated heart failure (HF) and sudden cardiac death (SCD) in a guinea pig model by integrated multi-ome analysis. Relative protein abundances from sham-operated, HYP and HF hearts were assessed by iTRAQ shotgun LC-MS/MS. Metabolites were quantified by LC-MS/MS or GC-MS. Transcriptome profiles were obtained using DNA microarrays. The guinea pig HF proteome exhibited classic biosignatures of cardiac HYP, left ventricular dysfunction, fibrosis, inflammation and extravasation. Fatty acid metabolism, mitochondrial transcription/translation factors, antioxidant enzymes, and other mitochondrial processes, were downregulated in HF, but not HYP. Proteins upregulated in HF implicate extracellular matrix remodeling, cytoskeletal remodeling, and acute phase inflammation markers. Among metabolites, downregulation of acyl-carnitines was observed in HYP, while fatty acids accumulated in HF. Correlation of transcript and protein changes in HF is weak (R2=0.23), suggesting transcript/proteome divergence may reveal post-transcriptional gene regulation in HF. Proteome/Metabolome integration suggests metabolic bottlenecks in fatty acyl-CoA processing by carnitine palmitoyl transferase (CPT1B) as well as TCA cycle inhibition. We present a model by which acute signaling in HF, including Ca2+ dysregulation and low cAMP levels, is coupled to mitochondrial metabolic and antioxidant defects, through a CREB/PGC1 transcriptional axis. Animal Model The guinea pig model of heart failure and sudden cardiac death has been described previously. Briefly, the HF and SCD guinea pig model was produced by combining ascending aortic constriction (AC) and daily isoproterenol challenge (ACi model). Specifically, Hartley guinea pigs (~250 g; Hilltop Lab Animals) were anesthetized with 4% isoflurane in a closed box for 4min, and then intubated and ventilated with oxygen and 2% isoflurane. Ascending aortic constriction (AC) was produced by tying a suture around the ascending aorta using an 18‐gauge needle as a spacer, which was then removed. For sham operations the procedure was identical though the suture was not tied. After the procedure, bupronex (0.05 mg/kg) was administered via intramuscular injection for analgesia and animals were observed until full recovery. Isoproterenol was administered daily by intra peritoneal injection at 1 mg/kg for the first week after surgery and at 2 mg/kg for a subsequent 3 weeks. As characterized previously (1), cardiac function of ACi animal is well compensated in the first 2 weeks (HYP) but declined rapidly thereafter (HF). Hypertrophic heart was collected between 1-2 weeks post-surgery (ACi-2w), whereas failing heart was collected at 4 weeks after surgery (ACi-4w). Following retrograde perfusion with 20ml Tyrode’s solution, excised hearts were Snap-frozen in liquid N2 and stored at -80°C Experimental Design The experiment consisted of 3 treatment groups: 1) HYP (ACi-2wk), 2) HF (ACi-4wk) 3) sham-operated animals with daily administration for 4 weeks (Shami-4w). 1 heart from each group was included in an iTRAQ 4-plex experiment wherein peptides from each heart are subjected to reaction with an isobaric label. The experiment was repeated twice, yielding a total of 3 independent experiments quantifying the peptides from 9 hearts. ITRAQ reagents were shuffled among treatment groups for each experiment to minimize labeling bias.

Project description:Background: Heart failure with preserved ejection fraction (HFpEF) constitutes more than half of all heart failure but has few effective therapies. Recent human myocardial transcriptomics and metabolomics have revealed major differences between HFpEF, HF with reduced EF (HFrEF), and controls. How this translates at the protein level is currently unknown. Methods: Myocardial tissue from patients with HFpEF and non-failing donor controls was analyzed by data-dependent (DDA, n=10 HFpEF, n=9 controls) and data-independent (DIA, n=44 HFpEF, n=5 controls) mass spectrometry-based proteomics. Previously reported myocardial proteomic data from end-stage HFrEF and controls were also used. Differential protein expression analysis, machine learning and pathway enrichment were integrated with clinical characteristics and myocardial transcriptomics. Results: DDA-MS proteomics identified 88 significantly upregulated and 248 down-regulated proteins in HFpEF vs controls, out of 1996 identified proteins. Principal component analysis of DDA-MS proteomics found HFpEF was separated into 2 sub-groups: one being similar to controls the other quite disparate. Top proteins contributing to the separation of HFpEF subgroups were enriched in actin/myosin binding, regulation of DNA replication/repair, transcription, and translation. Downregulated proteins in HFpEF vs controls were enriched in pathways related to ribosome structure, transmembrane transporters, metabolic enzymes, and oxidative phosphorylation (OxPhos) proteins. Enriched pathways for proteins upregulated in HFpEF related to actin and phospholipid binding, growth factor signaling, kinase regulation, and glycolysis. Ingenuity pathway analysis predicted downregulation of protein translation, mitochondrial function, and glucose and fat metabolism in HFpEF. OxPhos gene (increased) versus protein (decreased) expression was discordant in HFpEF. The second DIA proteomic analysis also yielded two HFpEF sub-groups; the one most different from controls also having reduced OxPhos and protein translation pathways. A higher proportion of these patients also had severe obesity. Conclusions: Integrative proteomics, transcriptomics, and pathway analysis supports a translational defect particularly involving mitochondrial, ribosomal and protein translation proteins in HFpEF. Patients with more distinct proteomic signatures from control were more often very obese. The results support therapeutic efforts targeting metabolism, mitochondrial function, and protein translation in this subgroup.

Project description:This series is a supplementary data set for the manuscript titled "Cohesin relocation from sites of chromosomal loading to places of convergent transcription" All data shown in the paper were registered (30 ChIP-chip and one transcription data). Experimental design: 1. Type of experiment: a. ChIP (Chromatin immunoprecipitation) analysis hybridised to a high density oligonucleotide array. The S. cerevisiae chromosome VI array described by Katou et al. (2003) Nature 424, 1078-83 has been used. Newly developed S. cerevisiae chromosomes III-V and S. pombe chromosomes 2-3 oligonucleotide arrays produced by Affymetrix have also been used. b. Gene expression profile analysis of S. cerevisiae. 2. Experimental factors: a. Distribution of different proteins related to sister chromatid cohesion or condensation during the cell cycle, mainly in G1 (alpha-factor arrested cells), S-phase (HU arrested cells) and metaphase (Nocodazole arrested cells). b. Gene expression profile analysis of logarithmically growing cells. 3. Number of hybridizations performed: a. ChIP analysis: 32 b. Gene expression: 1 4. Hybridisation design: ChIP analysis: comparison of ChIP fraction with SUP (supernatant) fraction 5. Quality control: Duplication, confirmation using different tags, different subunits of the same complex, and different yeast strain backgrounds (W303, BY4741, and SK1). Checking of the ChIP fraction by Western blotting. Checking amount of mRNA by RT-PCR. 6. URL of supplemental website: GEO Accession number: GSE1461 Web site: http://chromosomedynamics.bio.titech.ac.jp Samples used, extract preparation and labeling: 1. Origin of the biological sample Budding yeast (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe) 2. Manipulation of the samples and the protocols used: a. Mitotic budding yeast cells were synchronised in G1 with alpha-factor (2uM) and then released at 23ÂºC into medium containing 200 mM HU or 7.5 ug/ml Nocodazole for 60 min and 150 min, respectively. scc2-4 cells were shifted to 37ÂºC 60 min before alpha-factor release. Heat-shock treatment has been done at 37ÂºC for 15 minutes. b. Meiotic budding yeast cells were collected 4 hours after meiosis induction at 23ÂºC. c. Mitotic fission yeast cells were grown logarithmically in minimum medium (MM). Cells were fixed over night in 1% formaldehyde at 4ÂºC. 3. Protocols for preparing extracts: a. ChIP analysis: Extract preparation was processed following the Shirahige lab protocol (http://chromosomedynamics.bio.titech.ac.jp ). 5x10^8 cells were disrupted using Multi-Beads Shocker (MB400U, YASUI KIKAI, Osaka). Whole cell extract was sonicated to obtain 400-600 bp genomic DNA fragments. Anti-HA monoclonal antibody (clone 16B12, CRP Inc., Denver, PA) and anti-Flag monoclonal antibody M2 (Sigma-Aldrich Co., St Louis, MO) coupled to Dynabeads (Dynal, protein A Dynabeads) were used for chromatin immunoprecipitation. The immunprecipates were eluted and incubated over night at 65ÂºC to reverse the cross-link. Immunoprecipitated genomic DNA was incubated with proteinase K, extracted 2 times with phenol/chloroform/isoamylalcohol, precipitated, resuspended in TE and incubated with RnaseA. The DNA was then purified using the Qiagen PCR purification kit, and concentrated by ethanol precipitation. The DNA was amplified by PCR after random priming. 10 ug of amplified DNA was digested with Dnase I to a mean size of 100 bp. After Dnase I inactivation at 95ÂºC, fragments were labelled as detailled below. b. Gene expression analysis: Total RNA was prepared using FastRNA Pro Red Kit (BIO 101 Systems) according to the manufacturerâs instructions. 10 ug of total RNA was reverse transcribed according to the protocol recommended by Affymetrix. In vitro transcription was performed on 1 ug of cDNA. cRNA was cleaned and fragmented by heating. 20 ug of fragmented cRNA were hybridised. 4. Labelling protocol DNA fragments were end-labelled by addition of 25 U of Terminal Transferase and 1 nmol Biotin-N6ddATP (NEN) for 1 hour at 37ÂºC as previously described by Winzeler et al. (Science. 281, 1194-1197, 1998). The entire sample was used for hybridization. Hybridization procedures and parameters: Hybridization, blocking and washing were carried out as previously described (http://chromosomedynamics.bio.titech.ac.jp). Each sample was hybridized to the array in 150 ul containing 6xSSPE; 0.005% TritonX-100; 15 ug fragmented denatured salmon sperm DNA (Gibco-BRL); 1 nmole 3âbiotin labelled control oligonucleotide (oligo B2, Affymetrix). Samples were denatured at 100ÂºC for 10 minutes, and then put on ice before being hybridized for 16 hours at 42ÂºC in an hybridization oven (GeneChip Hybri. Oven 320, Affymetrix). Washing and scanning protocol (FlexMidi-euk2v3-450) provided by Affymetrix was performed automatically on a fluidics station (GeneChip fluidics station 400, Affymetrix). Measurement data and specifications: S. cerevisiae arrays were scanned using the HP GeneArray Scanner (Affymetrix) at an emission wavelength of 560 nm, resolution 7.5 uM. S. pombe arrays were scanned by GeneChip Scanner 3000 (Affymetrix) at an emission wavelength of 570 nm, resolution 2.5 uM. Grids were aligned to scans following the library array description. Primary data analyses were carried out using Affymetrix Microarray Suite Ver.5.0 software to obtain signal intensity, fold change value, change p-value and detection p-value. The detailed information of this analysis is available at http://www.affymetrix.com/support/technical/whitepapers/sadd_whitepaper.pdf and http://www.affymetrix.com/support/technical/technotes/statistical_reference_guide.pdf To discriminate significant signals for binding to DNA (dark grey signals), signals from the ChIP fraction scan were compared to the SUP (supernatant) fraction scan using the 3 following criteria: first, the signal reliability was judged by the detection p-value of each locus (p-value â¤0.025); secondly, reliability of binding ratio was judged by change p-value (p-value â¤0.025); thirdly, only clusters of at least 3 contiguous loci responding to the 2 previous criteria were selected as significantly enriched locus. The light grey signals represent the statistically not significantly enriched signals. The software to present the result (chr6viewer) is available at http://everythingchromovi.gsc.riken.go.jp. The raw and transformed data files are available at GEO database website and at our web site http://chromosomedynamics.bio.titech.ac.jp. Array Design 1. General array design: in situ synthesized arrays by Affymetrix 2. Availability of arrays: commercially available from Affymetrix 3. Location and ID of each spot on arrays: available at our web site (http://chromosomedynamics.bio.titech.ac.jp) and from Affymetrix on request 4. Probe type: oligonucleotide 5. The arrays used in this study can be purchased from Affymetrix: Chromosome VI S.cerevisiae: rikDACF, P/N# 510636 Chromosome III,IV,V S.cerevisiae: SC3456a520015F, P/N# 520015 Chromosome II,III S.pombe: S_pombea520106F, P/N# 520106 YGS98 GeneChipsT oligonucleotide arrays: P/N# 900256 Keywords = Yeast Cohesin Condensin Loading Distribution Keywords: other

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M.musculus - Heart, SC (Kruger,cell,2008)

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