Summary of study ST000924

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 PR000640. The data can be accessed directly via it's Project DOI: 10.21228/M8Z40Z This work is supported by NIH grant, U2C- DK119886.

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Study IDST000924
Study TitleMuRF1-Related Metabolic Alterations in HL-1 Cardiomyocyte Induced by Cyclic Stretch
Study TypeCardiomyocyte cell culture
Study SummaryWe collected cell media and performed GC-MS non-targeted metabolomics to identify the role of MuRF1 in the dynamic metabolic changes in cardiomyocytes.
Institute
University of North Carolina at Chapel Hill
DepartmentPathology & Laboratory Medicine
LaboratoryWillis
Last NameWillis
First NameMonte
Address111 Mason Farm Road
Emailmonte_willis@med.unc.edu
Phone9849995431
Submit Date2017-10-30
Num Groups6
Total Subjects32
Raw Data AvailableYes
Raw Data File Type(s) .M, .D, .ms,.val,.mac, .MS, .inf, .txt, .p,etc
Analysis Type DetailLC-MS
Release Date2018-12-11
Release Version1
Monte Willis Monte Willis
https://dx.doi.org/10.21228/M8Z40Z
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR000640
Project DOI:doi: 10.21228/M8Z40Z
Project Title:MuRF1-Related Metabolic Alterations in HL-1 Cardiomyocyte Induced by Cyclic Stretch
Project Type:Cardiomyocyte cell culture
Project Summary:We collected cell media and performed GC-MS non-targeted metabolomics to identify the role of MuRF1 in the dynamic metabolic changes in cardiomyocytes.
Institute:University of North Carolina at Chapel Hill
Department:Pathology & Laboratory Medicine
Laboratory:Willis
Last Name:Willis
First Name:Monte
Address:111 Mason Farm Road
Email:monte_willis@med.unc.edu
Phone:9849995431
Funding Source:National Institutes of Health R01HL104129
Contributors:Wei Tang, PhD1,*, Jessica E. Rodríguez, PhD2,*, Amro Ilaiwy, MD3,4, James R. Bain, PhD3,4, Michael J. Muehlbauer, PhD3, Megan T. Quintana, MD5, Alexa Hartman, BS6, Cam Patterson, MD1,7, Janet Rubin, MD8, Monte S. Willis, MD, PhD1,2,9

Subject:

Subject ID:SU000962
Subject Type:Cell culture
Subject Species:Mus musculus
Taxonomy ID:10090
Cell Strain Details:C57BL/6
Cell Primary Immortalized:AT1
Cell Counts:~60% confluency 6 well plate

Factors:

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

mb_sample_id local_sample_id type Stretch Cycle
SA05525118Ad.shMuRF1 15 min cyclic stretch
SA05525230Ad.shMuRF1 15 min cyclic stretch
SA0552536Ad.shMuRF1 15 min cyclic stretch
SA05525431Ad.shMuRF1 30 min cyclic stretch
SA0552557Ad.shMuRF1 30 min cyclic stretch
SA05525619Ad.shMuRF1 30 min cyclic stretch
SA05525720Ad.shMuRF1 60 min cyclic stretch
SA0552588Ad.shMuRF1 60 min cyclic stretch
SA05525932Ad.shMuRF1 60 min cyclic stretch
SA05526029Ad.shMuRF1 No stretch
SA05526117Ad.shMuRF1 No stretch
SA0552625Ad.shMuRF1 No stretch
SA05526314Ad.shRNA Scramble 15 min cyclic stretch
SA0552642Ad.shRNA Scramble 15 min cyclic stretch
SA05526526Ad.shRNA Scramble 15 min cyclic stretch
SA0552663Ad.shRNA Scramble 30 min cyclic stretch
SA05526727Ad.shRNA Scramble 30 min cyclic stretch
SA05526815Ad.shRNA Scramble 30 min cyclic stretch
SA05526916Ad.shRNA Scramble 60 min cyclic stretch
SA05527028Ad.shRNA Scramble 60 min cyclic stretch
SA0552714Ad.shRNA Scramble 60 min cyclic stretch
SA05527225Ad.shRNA Scramble No stretch
SA05527313Ad.shRNA Scramble No stretch
SA0552741Ad.shRNA Scramble No stretch
Showing results 1 to 24 of 24

Collection:

Collection ID:CO000956
Collection Summary:Media harvested at 14, 30, and 60 min post stretch (1Hz, 15%)
Collection Protocol ID:None
Collection Protocol Comments:Non-targeted metabolomics analysis. After HL-1 cells were cultured for 16 h in specialized 6well BioFlex culture plates in Claycomb medium, medium was changed to DMEM supplemented 1% penicillin-streptomycin and 10% serum. Transient knockdown of MuRF1 was carried out using recombinant Ad.shRNA MuRF1 at MOI 30. After transduction for 48 h, HL-1 cells were used for mechanical stretch at 15% strain and for different times (15, 30, and 60 min) in a computer-regulated Flexcell FX-5000 Compression System (Flexcell International Corporation, Hillsborough, NC). Non-stretch cells (0 min) were used as the control. After HL-1 cells were stretched, HL-1 cells (at 0 and 60 min) and DMEM medium (at 0, 15, 30, and 60 min) were collected for GC/MS measurement of metabolites, and samples were placed on dry ice/stored at -80C. Samples were then analyzed by GC/MS, as previously described [24]. The raw, transformed, and sorted data used for each of the three comparisons in the metabolomics analyses can be found in Supplemental Table 1. Up to 1 missing value per group was imputed using lowest value in the same group, with groups missing 2 or more excluded from the analysis. The data obtained in this study will be accessible at the NIH Common Fund’s Data Repository and Coordinating Center (supported by NIH grant, U01-DK097430) website, http://www.metabolomicsworkbench.org.
Sample Type:Media
Collection Method:Pipette
Collection Location:Tissue culture.
Collection Frequency:q 15-30 minutes
Collection Duration:1 hour
Volumeoramount Collected:10 ul
Storage Conditions:-80C
Collection Vials:Cryovials
Storage Vials:Cryovials
Collection Tube Temp:Ice

Treatment:

Treatment ID:TR000976
Treatment Summary:Cell culture, adenovirus transduction, and MuRF1 knock-down. The cardiomyocyte-derived HL-1 cell line was maintained as previously described [11-13]. Briefly, cells were split and cultured to ~90% confluency in 6-well BioFlex culture plates (Flexcell International Corporation, Hillsborough, NC) coated with 0.02% gelatin (wt/vol) and 0.5% fibronectin (vol/vol) for ∼30 min and grown in complete Claycomb media containing 10% serum (Cat. #51800C, Sigma-Aldrich; St. Louis, MO), as previously described [13]. Media was changed to serum-free Dulbecco’s modified Eagle’s medium (DMEM) supplemented 1% penicillin-streptomycin. HL-1 cells were transduced with 30 MOI adenovirus expressing shRNA MuRF1 (Ad.shRNA MuRF1) or shRNA scramble (Ad.shRNA Scr) control (custom made by Vector Biolabs, Philadelphia, PA) to transiently knock down MuRF1, as previously described [13].
Treatment:Ad.shRNA MuRF1 (or Ad.shRNA Scramble) treatment
Treatment Compound:Adenovirus
Treatment Route:Media
Treatment Dose:30 MOI
Treatment Dosevolume:<10 ul
Treatment Doseduration:48 hours
Treatment Vehicle:DMEM
Cell Media:DMEM / No serum
Cell Envir Cond:Stretch
Cell Pct Confluence:~60%
Cell Media Lastchanged:At start of stretch period.

Sample Preparation:

Sampleprep ID:SP000969
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 (TMS)-D27-C14:0 standard retention time (RT) was set at ~16.727 min. Reactive carbonyls were stabilized at 50 °C 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 50 °C. GC/MS methods generally follow those of Roessner et al. (2000), Fiehn et al. (2008), and Kind et al. (2009), and used a 6890 N GC connected to a 5975 Inert single-quadrupole MS (Agilent Technologies, Santa Clara, CA). The two wall-coated, open-tubular (WCOT) 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-μm luminal film. Positive ions generated with conventional electron-ionization (EI) at 70 eV are scanned broadly from 600 to 50 m/z in the detector throughout the 45 min cycle time.

Combined analysis:

Analysis ID AN001517
Analysis type MS
Chromatography type GC
Chromatography system Agilent 6890N
Column GC columns connected in series are both from J&W/Agilent (part 122-5512)
MS Type ESI
MS instrument type Single quadrupole
MS instrument name Agilent 5975
Ion Mode POSITIVE
Units Peak values (Log transformed)

Chromatography:

Chromatography ID:CH001070
Chromatography Summary:Metabolites were made volatile with trimethylsilyl (TMS) groups using N-methyl- N-(trimethylsilyl) trifluoroacetamide or MSTFA with catalytic trimethylchlorosilane at 50 C. GC/MS methods generally follow those of Roessner et al. (Roessner et al. 2000), Fiehn et al. (2008), and Kind et al. (2009), and used a 6890N GC connected to a 5,975 inert single-quadrupole MS (Agilent Technologies, Santa Clara, CA). The two wall-coated, open-tubular (WCOT) GC columns connected in series are both from J&W/Agilent (part 122-5512), DB5- MS, 15 m in length, 0.25 mm in diameter, with an 0.25-lm luminal film. Positive ions generated with conventional electron-ionization (EI) 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:GC columns connected in series are both from J&W/Agilent (part 122-5512)
Chromatography Type:GC

MS:

MS ID:MS001400
Analysis ID:AN001517
Instrument Name:Agilent 5975
Instrument Type:Single quadrupole
MS Type:ESI
Ion Mode:POSITIVE
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