Summary of study ST000925

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR000641. The data can be accessed directly via it's Project DOI: 10.21228/M8T973 This work is supported by NIH grant, U2C- DK119886.

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Study IDST000925
Study TitleMuRF1-Related Metabolomic Changes in Stretched and Unloaded HL-1 Cells
Study SummaryIntroduction: Left ventricular assist devices (LVADs) provide significant pressure and volume unloading, which reverse key structural features of heart failure, including hypertrophy, fibrosis, and altered sympathetic innervation. This has led to LVADs increasing utilization as both a bridge or destination therapy for heart failure. While distinct metabolic changes occur in the human myocardium with LVAD placement, the specific molecular mechanisms underlying these changes have not been identified. Objectives: To identify the role of MuRF1 in the metabolic changes in cardiomyocytes unloading in vitro. Methods: HL-1 atrial cardiomyocyte cells were plated on silastic membranes coated with gelatin/fibronectin and transduced with AdshRNA MuRF1 (or AdshRNA Scramble control) to knock-down MuRF1 protein to <25% of controls and subjected to 15% biaxial stretch at 1 Hz using the Flexcell FX-5000™ Compression System in serum free DMEM (or no-stretch). After 6 hours stretch, media was collected in parallel with time-matched no-stretch controls, followed by serial collections at 1, 3, 6, and 12 hours unloading (i.e. after termination of stretch). Media was analyzed by untargeted metabolomics using GC-MS. Results: After 6 hours stretch, control HL-1 cell media (AdshRNA Scramble) had 19 significantly altered metabolites compared to non-stretched control cell media (AdshRNA Scramble) by t-test, involved in: 1) glyoxylate and dicarboxylate metabolism (p=0.00087043, FDR=0.071375), 2) methane metabolism (p=0.0041113, FDR=0.089824), 3) glycine, serine and threonine metabolism (p=0.0043816, FDR=0.089824), and 4) aminoacyl-tRNA biosynthesis(p=0.0060141, FDR=0.09863). To determine the role of MuRF1 in stretch, we additionally compared the AdshRNA Scramble groups to AdshRNA MuRF1 +/- stretch at 6 hours and identified 41 significant metabolites (of 79 identified) by ANOVA, involved in: 1) phenylalanine, tyrosine, and tryptophan biosynthesis (p=0.0036482, FDR=0.05983), 2) aminoacyl-tRNA biosynthesis (p=3.74E-07, FDR=3.06E-05), and 3) valine, leucine, and isoleucine biosynthesis (p=0.0021624, FDR=0.053255). We next compared the 6 hour stretch time point of the four groups to the 1, 3, 6, and 12 hours unloading conditions to identify MuRF1-associated metabolites during the unloading period. At 12 hours of unloading (representative 1, 3, and 6 hours unloading), MuRF1 knock-down cell media had 35 (of 82 named metabolites) significantly different metabolites by ANOVA involved in 1) phenylalanine, tyrosine and tryptophan biosynthesis (p=0.0023668, FDR=0.048519), 2) aminoacyl-tRNA biosynthesis (p=0.0011403, FDR=0.0032631), 3) phenylalanine metabolism (P= 0.0011403, FDR 0.042673),and arginine and proline metabolism (p=0.0015612, FDR 0.042673). A final analysis comparing all four groups across all five time points identified 21 significant metabolites. When MuRF1 was knocked down (no stretch), these 21 metabolites were significantly increased without stretch. In response to stretch and unloading, these metabolites were further decreased in AdshRNA Scramble (control) cell media decreased. In contrast, these stretch and unloading induced decreases were attenuated in AdshRNA MuRF1 cell media. These metabolites included nine (9) amino acids (citrulline, phenylalanine, tyrosine, asparagine, 2-ketovaline, and -ketoleucine/ketoisoleucine, threonine, isoleucine, leucine), three (3) metabolic co-factors (Pantothenic acid, Myoinositol, 5,6-Dihydrouracil), and one fatty acid (stearic acid). Conclusion: These studies identify for the first time a role for MuRF1 in generating key amino acids recently reported in LVAD unloading in human myocardium using an in vitro model of atrial cardiomyocyte unloading.
Institute
University of North Carolina at Chapel Hill
DepartmentMcAllister heart Institute, Department of Internal medicine
LaboratoryMultiple Centers
Last NameWillis
First NameMonte
Address111 Mason Farm road, Chapel Hill, North Carolina, 27599-7126, USA
Emailmonte_willis@med.unc.edu
Phone(984) 999-5431
Submit Date2017-12-25
Study CommentsCell culture media
Raw Data AvailableYes
Raw Data File Type(s) .M, .D, .ms,.val,.mac, .MS, .inf, .txt, .p,etc
Analysis Type DetailGC-MS
Release Date2019-01-22
Release Version1
Monte Willis Monte Willis
https://dx.doi.org/10.21228/M8T973
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:


Project:

Project ID:PR000641
Project DOI:doi: 10.21228/M8T973
Project Title:The Multi-Faceted Roles of MuRF1 in Unloaded HL-1 Cardiomyocyte-Derived Cell Metabolism Identified by Untargeted Metabolomics
Project Type:GC-MS Non-targeted analysis
Project Summary:Stretch-induced alterations in metabolites (+/- MuRF1 knock-down) in HL-1 cardiomyocyte-derived cell line
Institute:University of North Carolina at Chapel Hill
Department:McAllister heart Institute, Dept. Pathology & Lab Medicine, Dept. Pharmacology
Laboratory:Multiple Centers
Last Name:Willis
First Name:Monte
Address:111 Mason Farm road, Chapel Hill, North Carolina, 27599-7126, USA
Email:monte_willis@med.unc.edu
Phone:(984) 999-5431
Funding Source:NIH, Leducq Foundation

Subject:

Subject ID:SU000963
Subject Type:Animal
Subject Species:Mus musculus
Taxonomy ID:10090

Factors:

Subject type: Animal; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id local_sample_id Sample type Stretch Cycle
SA05527520Ad.shRNA MuRF1 6 hours stretch
SA05527621Ad.shRNA MuRF1 6 hours stretch
SA05527724Ad.shRNA MuRF1 6 hours stretch
SA05527819Ad.shRNA MuRF1 6 hours stretch
SA05527923Ad.shRNA MuRF1 6 hours stretch
SA05528022Ad.shRNA MuRF1 6 hours stretch
SA055287118Ad.shRNA MuRF1 6 hours stretch, 12 hours unloading
SA055288119Ad.shRNA MuRF1 6 hours stretch, 12 hours unloading
SA055289120Ad.shRNA MuRF1 6 hours stretch, 12 hours unloading
SA055290115Ad.shRNA MuRF1 6 hours stretch, 12 hours unloading
SA055291117Ad.shRNA MuRF1 6 hours stretch, 12 hours unloading
SA055292116Ad.shRNA MuRF1 6 hours stretch, 12 hours unloading
SA05528146Ad.shRNA MuRF1 6 hours stretch, 1 hour unloading
SA05528245Ad.shRNA MuRF1 6 hours stretch, 1 hour unloading
SA05528344Ad.shRNA MuRF1 6 hours stretch, 1 hour unloading
SA05528443Ad.shRNA MuRF1 6 hours stretch, 1 hour unloading
SA05528548Ad.shRNA MuRF1 6 hours stretch, 1 hour unloading
SA05528647Ad.shRNA MuRF1 6 hours stretch, 1 hour unloading
SA05529369Ad.shRNA MuRF1 6 hours stretch, 3 hours unloading
SA05529467Ad.shRNA MuRF1 6 hours stretch, 3 hours unloading
SA05529570Ad.shRNA MuRF1 6 hours stretch, 3 hours unloading
SA05529672Ad.shRNA MuRF1 6 hours stretch, 3 hours unloading
SA05529771Ad.shRNA MuRF1 6 hours stretch, 3 hours unloading
SA05529868Ad.shRNA MuRF1 6 hours stretch, 3 hours unloading
SA05529996Ad.shRNA MuRF1 6 hours stretch, 6 hours unloading
SA05530095Ad.shRNA MuRF1 6 hours stretch, 6 hours unloading
SA05530191Ad.shRNA MuRF1 6 hours stretch, 6 hours unloading
SA05530292Ad.shRNA MuRF1 6 hours stretch, 6 hours unloading
SA05530393Ad.shRNA MuRF1 6 hours stretch, 6 hours unloading
SA05530494Ad.shRNA MuRF1 6 hours stretch, 6 hours unloading
SA05530515Ad.shRNA MuRF1 Non-stretched
SA05530613Ad.shRNA MuRF1 Non-stretched
SA05530714Ad.shRNA MuRF1 Non-stretched
SA05530816Ad.shRNA MuRF1 Non-stretched
SA05530917Ad.shRNA MuRF1 Non-stretched
SA05531018Ad.shRNA MuRF1 Non-stretched
SA05531110Ad.shRNA Scramble 6 hours stretch
SA0553129Ad.shRNA Scramble 6 hours stretch
SA05531311Ad.shRNA Scramble 6 hours stretch
SA0553148Ad.shRNA Scramble 6 hours stretch
SA0553157Ad.shRNA Scramble 6 hours stretch
SA05531612Ad.shRNA Scramble 6 hours stretch
SA055323103Ad.shRNA Scramble 6 hours stretch, 12 hours unloading
SA055324106Ad.shRNA Scramble 6 hours stretch, 12 hours unloading
SA055325107Ad.shRNA Scramble 6 hours stretch, 12 hours unloading
SA055326108Ad.shRNA Scramble 6 hours stretch, 12 hours unloading
SA055327104Ad.shRNA Scramble 6 hours stretch, 12 hours unloading
SA055328105Ad.shRNA Scramble 6 hours stretch, 12 hours unloading
SA05531734Ad.shRNA Scramble 6 hours stretch, 1 hour unloading
SA05531835Ad.shRNA Scramble 6 hours stretch, 1 hour unloading
SA05531933Ad.shRNA Scramble 6 hours stretch, 1 hour unloading
SA05532032Ad.shRNA Scramble 6 hours stretch, 1 hour unloading
SA05532131Ad.shRNA Scramble 6 hours stretch, 1 hour unloading
SA05532236Ad.shRNA Scramble 6 hours stretch, 1 hour unloading
SA05532955Ad.shRNA Scramble 6 hours stretch, 3 hours unloading
SA05533059Ad.shRNA Scramble 6 hours stretch, 3 hours unloading
SA05533157Ad.shRNA Scramble 6 hours stretch, 3 hours unloading
SA05533256Ad.shRNA Scramble 6 hours stretch, 3 hours unloading
SA05533360Ad.shRNA Scramble 6 hours stretch, 3 hours unloading
SA05533458Ad.shRNA Scramble 6 hours stretch, 3 hours unloading
SA05533582Ad.shRNA Scramble 6 hours stretch, 6 hours unloading
SA05533679Ad.shRNA Scramble 6 hours stretch, 6 hours unloading
SA05533784Ad.shRNA Scramble 6 hours stretch, 6 hours unloading
SA05533883Ad.shRNA Scramble 6 hours stretch, 6 hours unloading
SA05533981Ad.shRNA Scramble 6 hours stretch, 6 hours unloading
SA05534080Ad.shRNA Scramble 6 hours stretch, 6 hours unloading
SA0553413Ad.shRNA Scramble Non-stretched
SA0553422Ad.shRNA Scramble Non-stretched
SA0553434Ad.shRNA Scramble Non-stretched
SA0553446Ad.shRNA Scramble Non-stretched
SA0553451Ad.shRNA Scramble Non-stretched
SA0553465Ad.shRNA Scramble Non-stretched
Showing results 1 to 72 of 72

Collection:

Collection ID:CO000957
Collection Summary:Cell culture media was collected and immediately stored at -80C.
Sample Type:cardiomyocyte cells

Treatment:

Treatment ID:TR000977
Treatment Summary:No further treatment prior to extraction was performed.

Sample Preparation:

Sampleprep ID:SP000970
Sampleprep Summary:The samples were crash deprotonized by methanol precipitation and spiked with D27-deuterated myristic acid (D27-C14:0) as an internal standard for retention-time locking and dried. The trimethylsilyl-D27-C14:0 standard retention time (RT) was set at 16.727 min. Reactive carbonyls were stabilized at 50C with methoxyamine hydrochloride in dry pyridine. Metabolites were made volatile with TMS groups using N-methyl N-(trimethylsilyl) trifluoroacetamide or MSTFA with catalytic trimethylchlorosilane at 50C.

Combined analysis:

Analysis ID AN001518
Analysis type MS
Chromatography type GC
Chromatography system Agilent 6890N
Column Agilent DB5-MS (15m × 0.25mm, 0.25um)
MS Type EI
MS instrument type Single quadrupole
MS instrument name Agilent 5975
Ion Mode POSITIVE
Units Peak values (Log transformed)

Chromatography:

Chromatography ID:CH001071
Chromatography Summary:GC/MS methods follow previous studies using a 6890 N GC connected to a 5975 Inert single quadrupole MS (Agilent Technologies, Santa Clara, CA) (Bonikos et al. 1975; Fiehn 2008; Kind et al. 2009). The two wall-coated, open-tubular GC columns connected in series are both from J&W/Agilent (part 122-5512), DB5-MS, 15 meters in length, 0.25 mm in diameter, with an 0.25-l m luminal film. Positive ions generated with conventional electron ionization at 70 eV are scanned broadly from 600 to 50 m/z in the detector throughout the 45 min cycle time.
Instrument Name:Agilent 6890N
Column Name:Agilent DB5-MS (15m × 0.25mm, 0.25um)
Chromatography Type:GC

MS:

MS ID:MS001401
Analysis ID:AN001518
Instrument Name:Agilent 5975
Instrument Type:Single quadrupole
MS Type:EI
Ion Mode:POSITIVE
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