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MB Sample ID: SA055260
| Local Sample ID: | 29 |
| 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 |
Select appropriate tab below to view additional metadata details:
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:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| 29 | SA055260 | FL010116 | Ad.shMuRF1 | type |
| 29 | SA055260 | FL010116 | No stretch | Stretch Cycle |
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 | GCs 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: | GCs 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 |
