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.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
| Study ID | ST000925 |
| Study Title | MuRF1-Related Metabolomic Changes in Stretched and Unloaded HL-1 Cells |
| Study Summary | Introduction: 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 |
| Department | McAllister heart Institute, Department of Internal medicine |
| Laboratory | Multiple Centers |
| Last Name | Willis |
| First Name | Monte |
| Address | 111 Mason Farm road, Chapel Hill, North Carolina, 27599-7126, USA |
| monte_willis@med.unc.edu | |
| Phone | (984) 999-5431 |
| Submit Date | 2017-12-25 |
| Study Comments | Cell culture media |
| Raw Data Available | Yes |
| Raw Data File Type(s) | d |
| Analysis Type Detail | GC-MS |
| Release Date | 2019-01-22 |
| Release Version | 1 |
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 |
|---|---|---|---|
| SA055275 | 20 | Ad.shRNA MuRF1 | 6 hours stretch |
| SA055276 | 21 | Ad.shRNA MuRF1 | 6 hours stretch |
| SA055277 | 24 | Ad.shRNA MuRF1 | 6 hours stretch |
| SA055278 | 19 | Ad.shRNA MuRF1 | 6 hours stretch |
| SA055279 | 23 | Ad.shRNA MuRF1 | 6 hours stretch |
| SA055280 | 22 | Ad.shRNA MuRF1 | 6 hours stretch |
| SA055287 | 118 | Ad.shRNA MuRF1 | 6 hours stretch, 12 hours unloading |
| SA055288 | 119 | Ad.shRNA MuRF1 | 6 hours stretch, 12 hours unloading |
| SA055289 | 120 | Ad.shRNA MuRF1 | 6 hours stretch, 12 hours unloading |
| SA055290 | 115 | Ad.shRNA MuRF1 | 6 hours stretch, 12 hours unloading |
| SA055291 | 117 | Ad.shRNA MuRF1 | 6 hours stretch, 12 hours unloading |
| SA055292 | 116 | Ad.shRNA MuRF1 | 6 hours stretch, 12 hours unloading |
| SA055281 | 46 | Ad.shRNA MuRF1 | 6 hours stretch, 1 hour unloading |
| SA055282 | 45 | Ad.shRNA MuRF1 | 6 hours stretch, 1 hour unloading |
| SA055283 | 44 | Ad.shRNA MuRF1 | 6 hours stretch, 1 hour unloading |
| SA055284 | 43 | Ad.shRNA MuRF1 | 6 hours stretch, 1 hour unloading |
| SA055285 | 48 | Ad.shRNA MuRF1 | 6 hours stretch, 1 hour unloading |
| SA055286 | 47 | Ad.shRNA MuRF1 | 6 hours stretch, 1 hour unloading |
| SA055293 | 69 | Ad.shRNA MuRF1 | 6 hours stretch, 3 hours unloading |
| SA055294 | 67 | Ad.shRNA MuRF1 | 6 hours stretch, 3 hours unloading |
| SA055295 | 70 | Ad.shRNA MuRF1 | 6 hours stretch, 3 hours unloading |
| SA055296 | 72 | Ad.shRNA MuRF1 | 6 hours stretch, 3 hours unloading |
| SA055297 | 71 | Ad.shRNA MuRF1 | 6 hours stretch, 3 hours unloading |
| SA055298 | 68 | Ad.shRNA MuRF1 | 6 hours stretch, 3 hours unloading |
| SA055299 | 96 | Ad.shRNA MuRF1 | 6 hours stretch, 6 hours unloading |
| SA055300 | 95 | Ad.shRNA MuRF1 | 6 hours stretch, 6 hours unloading |
| SA055301 | 91 | Ad.shRNA MuRF1 | 6 hours stretch, 6 hours unloading |
| SA055302 | 92 | Ad.shRNA MuRF1 | 6 hours stretch, 6 hours unloading |
| SA055303 | 93 | Ad.shRNA MuRF1 | 6 hours stretch, 6 hours unloading |
| SA055304 | 94 | Ad.shRNA MuRF1 | 6 hours stretch, 6 hours unloading |
| SA055305 | 15 | Ad.shRNA MuRF1 | Non-stretched |
| SA055306 | 13 | Ad.shRNA MuRF1 | Non-stretched |
| SA055307 | 14 | Ad.shRNA MuRF1 | Non-stretched |
| SA055308 | 16 | Ad.shRNA MuRF1 | Non-stretched |
| SA055309 | 17 | Ad.shRNA MuRF1 | Non-stretched |
| SA055310 | 18 | Ad.shRNA MuRF1 | Non-stretched |
| SA055311 | 10 | Ad.shRNA Scramble | 6 hours stretch |
| SA055312 | 9 | Ad.shRNA Scramble | 6 hours stretch |
| SA055313 | 11 | Ad.shRNA Scramble | 6 hours stretch |
| SA055314 | 8 | Ad.shRNA Scramble | 6 hours stretch |
| SA055315 | 7 | Ad.shRNA Scramble | 6 hours stretch |
| SA055316 | 12 | Ad.shRNA Scramble | 6 hours stretch |
| SA055323 | 103 | Ad.shRNA Scramble | 6 hours stretch, 12 hours unloading |
| SA055324 | 106 | Ad.shRNA Scramble | 6 hours stretch, 12 hours unloading |
| SA055325 | 107 | Ad.shRNA Scramble | 6 hours stretch, 12 hours unloading |
| SA055326 | 108 | Ad.shRNA Scramble | 6 hours stretch, 12 hours unloading |
| SA055327 | 104 | Ad.shRNA Scramble | 6 hours stretch, 12 hours unloading |
| SA055328 | 105 | Ad.shRNA Scramble | 6 hours stretch, 12 hours unloading |
| SA055317 | 34 | Ad.shRNA Scramble | 6 hours stretch, 1 hour unloading |
| SA055318 | 35 | Ad.shRNA Scramble | 6 hours stretch, 1 hour unloading |
| SA055319 | 33 | Ad.shRNA Scramble | 6 hours stretch, 1 hour unloading |
| SA055320 | 32 | Ad.shRNA Scramble | 6 hours stretch, 1 hour unloading |
| SA055321 | 31 | Ad.shRNA Scramble | 6 hours stretch, 1 hour unloading |
| SA055322 | 36 | Ad.shRNA Scramble | 6 hours stretch, 1 hour unloading |
| SA055329 | 55 | Ad.shRNA Scramble | 6 hours stretch, 3 hours unloading |
| SA055330 | 59 | Ad.shRNA Scramble | 6 hours stretch, 3 hours unloading |
| SA055331 | 57 | Ad.shRNA Scramble | 6 hours stretch, 3 hours unloading |
| SA055332 | 56 | Ad.shRNA Scramble | 6 hours stretch, 3 hours unloading |
| SA055333 | 60 | Ad.shRNA Scramble | 6 hours stretch, 3 hours unloading |
| SA055334 | 58 | Ad.shRNA Scramble | 6 hours stretch, 3 hours unloading |
| SA055335 | 82 | Ad.shRNA Scramble | 6 hours stretch, 6 hours unloading |
| SA055336 | 79 | Ad.shRNA Scramble | 6 hours stretch, 6 hours unloading |
| SA055337 | 84 | Ad.shRNA Scramble | 6 hours stretch, 6 hours unloading |
| SA055338 | 83 | Ad.shRNA Scramble | 6 hours stretch, 6 hours unloading |
| SA055339 | 81 | Ad.shRNA Scramble | 6 hours stretch, 6 hours unloading |
| SA055340 | 80 | Ad.shRNA Scramble | 6 hours stretch, 6 hours unloading |
| SA055341 | 3 | Ad.shRNA Scramble | Non-stretched |
| SA055342 | 2 | Ad.shRNA Scramble | Non-stretched |
| SA055343 | 4 | Ad.shRNA Scramble | Non-stretched |
| SA055344 | 6 | Ad.shRNA Scramble | Non-stretched |
| SA055345 | 1 | Ad.shRNA Scramble | Non-stretched |
| SA055346 | 5 | Ad.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 x 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 x 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 |
