Summary of Study ST002229

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 PR001419. The data can be accessed directly via it's Project DOI: 10.21228/M89D8V This work is supported by NIH grant, U2C- DK119886.

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This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST002229
Study TitleEstrogen receptor α deficiency in cardiac myocytes reprograms heart-derived extracellular vesicle proteome and induces obesity in female mice (Part 1)
Study SummaryDysregulation of ERα has been linked with increased metabolic and cardiovascular disease risk. Uncovering the impact of ERα deficiency in specific tissues has implications for understanding the role of ERα in normal physiology and disease, the increased disease risk in postmenopausal women, and the design of tissue-specific ERα-based therapies for a range of pathologies including cardiac disease and cancer. Cardiac myocyte-specific ER knockout mice (ERαHKO) were generated to assess the role of ERα in the heart. Female ERαHKO mice displayed a modest cardiac phenotype, but unexpectedly, the most striking phenotype was obesity in female ERαHKO but not male ERαHKO mice. In female ERαHKO mice we identified cardiac dysfunction, mild glucose and insulin intolerance, and reduced ERα gene expression in skeletal muscle and white adipose tissue (WAT). Gene expression, protein, lipidomic and metabolomic analyses showed evidence of contractile and/or metabolic dysregulation in heart, skeletal muscle and WAT. We also show that extracellular vesicles (EVs) collected from the perfusate of isolated hearts from female ERαHKO mice have a distinct proteome, and these EVs have the capacity to reprogram the proteome of a skeletal muscle cell including proteins linked with ERα, fatty acid regulation, lipid metabolism and mitochondrial function. This study uncovers a cardiac-initiated and sex-specific cardiometabolic phenotype that is regulated by ERα.
Institute
Baker Heart and Diabetes Institute
Last NameTham
First NameYow Keat
Address75 Commercial Rd, Melbourne, Victoria, 3004, Australia
Emailyowkeat.tham@baker.edu.au
Phone+65385321266
Submit Date2022-05-18
Num Groups4
Total Subjects25
Num Males10
Num Females15
Raw Data AvailableYes
Raw Data File Type(s)d
Analysis Type DetailLC-MS
Release Date2023-01-02
Release Version1
Yow Keat Tham Yow Keat Tham
https://dx.doi.org/10.21228/M89D8V
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR001419
Project DOI:doi: 10.21228/M89D8V
Project Title:Estrogen receptor α deficiency in cardiac myocytes reprograms heart-derived extracellular vesicle proteome and induces obesity in female mice
Project Summary:Tissues (ventricles, skeletal muscles-soleus, subcutaneous fat) from male and female ERalpha cardiac-specific knockout and floxed control aged mice (54-59 weeks old, male FC n=5, male KO n=5, female FC n=8, female KO n=7 ) were subjected to metabolomic profiling.
Institute:Baker Heart and Diabetes Institute
Department:Discovery and Preclinical Science
Laboratory:Cardiac Hypertrophy
Last Name:Tham
First Name:Yow Keat
Address:75 Commercial Rd, Melbourne, Victoria, 3004, Australia
Email:yowkeat.tham@baker.edu.au
Phone:0385321266
Publications:https://www.nature.com/articles/s44161-023-00223-z

Subject:

Subject ID:SU002315
Subject Type:Mammal
Subject Species:Mus musculus
Taxonomy ID:10090
Genotype Strain:C57BL6 and FVB mixed strain
Age Or Age Range:54-59 weeks old
Gender:Male and female
Animal Feed:Specialty Feeds Irradiated Rat and Mouse Standard Chow Diet

Factors:

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

mb_sample_id local_sample_id Sex Genotype
SA21243037899_LivFemale ERalpha-knockout
SA21243137915_SkFemale ERalpha-knockout
SA21243237910_SkFemale ERalpha-knockout
SA21243337906_LivFemale ERalpha-knockout
SA21243437914_SkFemale ERalpha-knockout
SA21243537917_LivFemale ERalpha-knockout
SA21243637914_HFemale ERalpha-knockout
SA21243737915_HFemale ERalpha-knockout
SA21243837910_LivFemale ERalpha-knockout
SA21243937917_SkFemale ERalpha-knockout
SA21244037899 _PFemale ERalpha-knockout
SA21244137914 _PFemale ERalpha-knockout
SA21244237915 _PFemale ERalpha-knockout
SA21244337905_SkFemale ERalpha-knockout
SA21244437899_SkFemale ERalpha-knockout
SA21244537910 _PFemale ERalpha-knockout
SA21244637917 _PFemale ERalpha-knockout
SA21244737910_HFemale ERalpha-knockout
SA21244837905 _PFemale ERalpha-knockout
SA21244937906 _PFemale ERalpha-knockout
SA21245037906_SkFemale ERalpha-knockout
SA21245137905_LivFemale ERalpha-knockout
SA21245237905_epiFemale ERalpha-knockout
SA21245337917_HFemale ERalpha-knockout
SA21245437906_epiFemale ERalpha-knockout
SA21245537910_epiFemale ERalpha-knockout
SA21245637915_epiFemale ERalpha-knockout
SA21245737899_epiFemale ERalpha-knockout
SA21245837915_LivFemale ERalpha-knockout
SA21245937905_HFemale ERalpha-knockout
SA21246037906_HFemale ERalpha-knockout
SA21246137899_HFemale ERalpha-knockout
SA21246237914_LivFemale ERalpha-knockout
SA21246337914_epiFemale ERalpha-knockout
SA21246437913_epiFemale Floxed Control
SA21246537916_SkFemale Floxed Control
SA21246637896_SkFemale Floxed Control
SA21246737891_SkFemale Floxed Control
SA21246837909_SkFemale Floxed Control
SA21246937912_epiFemale Floxed Control
SA21247037912_SkFemale Floxed Control
SA21247137913_SkFemale Floxed Control
SA21247237893_epiFemale Floxed Control
SA21247337909_LivFemale Floxed Control
SA21247437896_LivFemale Floxed Control
SA21247537916_LivFemale Floxed Control
SA21247637912_LivFemale Floxed Control
SA21247737890_SkFemale Floxed Control
SA21247837913_LivFemale Floxed Control
SA21247937893_LivFemale Floxed Control
SA21248037891_LivFemale Floxed Control
SA21248137896_epiFemale Floxed Control
SA21248237909_epiFemale Floxed Control
SA21248337891_epiFemale Floxed Control
SA21248437890_epiFemale Floxed Control
SA21248537890_LivFemale Floxed Control
SA21248637916_epiFemale Floxed Control
SA21248737893_SkFemale Floxed Control
SA21248837916_HFemale Floxed Control
SA21248937896 _PFemale Floxed Control
SA21249037916 _PFemale Floxed Control
SA21249137912 _PFemale Floxed Control
SA21249237912_HFemale Floxed Control
SA21249337913_HFemale Floxed Control
SA21249437890 _PFemale Floxed Control
SA21249537891 _PFemale Floxed Control
SA21249637893 _PFemale Floxed Control
SA21249737913 _PFemale Floxed Control
SA21249837909 _PFemale Floxed Control
SA21249937909_HFemale Floxed Control
SA21250037891_HFemale Floxed Control
SA21250137890_HFemale Floxed Control
SA21250237896_HFemale Floxed Control
SA21250337893_HFemale Floxed Control
SA21250437908_SkMale ERalpha-knockout
SA21250537898 _PMale ERalpha-knockout
SA21250637902_HMale ERalpha-knockout
SA21250737901_HMale ERalpha-knockout
SA21250837904_HMale ERalpha-knockout
SA21250937898_HMale ERalpha-knockout
SA21251037902 _PMale ERalpha-knockout
SA21251137901_epiMale ERalpha-knockout
SA21251237908_epiMale ERalpha-knockout
SA21251337904 _PMale ERalpha-knockout
SA21251437904_epiMale ERalpha-knockout
SA21251537902_epiMale ERalpha-knockout
SA21251637898_epiMale ERalpha-knockout
SA21251737908_HMale ERalpha-knockout
SA21251837901 _PMale ERalpha-knockout
SA21251937902_SkMale ERalpha-knockout
SA21252037902_LivMale ERalpha-knockout
SA21252137901_SkMale ERalpha-knockout
SA21252237898_SkMale ERalpha-knockout
SA21252337908 _PMale ERalpha-knockout
SA21252437898_LivMale ERalpha-knockout
SA21252537904_SkMale ERalpha-knockout
SA21252637901_LivMale ERalpha-knockout
SA21252737908_LivMale ERalpha-knockout
SA21252837904_LivMale ERalpha-knockout
SA21252937900_SkMale Floxed Control
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Collection:

Collection ID:CO002308
Collection Summary:Ventricles were dissected from mice such that each sample will include left and right ventricular tissue.Tissues were snap frozen in liquid nitrogen and stored in -80 freezer until tissues were processed for lipid extractions Blood was collected via cardiac puncture and stored in EDTA tubes on ice. EDTA tubes were spun at 3000g for 15 mins at 4 degrees. Plasma was then collected from these tubes (supernatant) and then stored at -80 degrees.
Sample Type:Ventricles, Plasma, Skeletal Muscles, Liver, Epididymal fat
Storage Conditions:-80℃
Collection Vials:1.5ml eppendorf tubes
Storage Vials:1.5ml eppendorf tubes

Treatment:

Treatment ID:TR002327
Treatment Summary:Mice did not undergo specific treatment, as this was a basal phenotyping study. Mice were fasted for 6 hours before dissections, and a lethal dose of anesthesia was delivered via intraperitoneal injection before tissue collection.
Animal Anesthesia:Pentobarbitone
Animal Fasting:6 hours

Sample Preparation:

Sampleprep ID:SP002321
Sampleprep Summary:tissues was homogenised in 1xPBS and then sonicated with a probe-sonicator for 15 seconds, 23 amplitude. BCA assays were then conducted to determine protein concentrations of these homogenates. Lipid extraction was conducted using 10ul of sample (ventricle, skeletal muscle homogenate at 5mg/ml, fat homogenate at 2mg/ml and liver homogenate at 2.5mg/ml) using the single phase chloroform methanol method. 10ul of internal standards and 200ul of chloroform:methanol (1:2) were added to samples before the mixture was vortexed. Samples were then placed on a rotary shaker for 10 mins at a speed of 90 before being transferred to a bath sonicator. Samples were then sonicated for 30 mins at water temperature below 28 degrees. Samples were then removed and rested at room temperature for 20 mins. Samples were then centrifuged at 13000rpm for 10 minutes. 200ul of the supernatant was then transferred to 0.5ml polypropylene 96 well plates, and spun dried using a speedvac vacuum concentrator. Lipids were reconstituted in 50ul water saturated butanol + 50ul of Ammonium Formate.
Sampleprep Protocol Filename:Agilent_appnote
Extract Storage:-80℃

Combined analysis:

Analysis ID AN003638
Analysis type MS
Chromatography type Reversed phase
Chromatography system Agilent 1290 Infinity II
Column Agilent Zorbax Eclipse Plus C18 (100 x 2.1mm, 1.8 um)
MS Type ESI
MS instrument type Triple quadrupole
MS instrument name Agilent 6490 QQQ
Ion Mode POSITIVE
Units pmol per mg (tissues) pmol per ml (plasma)

Chromatography:

Chromatography ID:CH002693
Chromatography Summary:The running solvent consisted of solvent A: 50% H2O / 30% acetonitrile / 20% isopropanol (v/v/v) containing 10mM ammonium formate and 5uM medronic acid, and solvent B: 1% H2O / 9% acetonitrile / 90% isopropanol (v/v/v) containing 10mM ammonium formate. We utilized a stepped linear gradient with a 16-minute cycle time per sample and a 1µL sample injection. To increase throughput, we used a dual column set up to equilibrate the second column while the first is running a sample. The sample analytical gradient was as follows: starting with a flow rate of 0.4mL/minute at 15% B and increasing to 50% B over 2.5 minutes, then to 57% over 0.1 minutes, to 70% over 6.4 minutes, to 93% over 0.1 minute, to 96% over 1.9 minutes and finally to 100% over 0.1 minute. The solvent was then held at 100% B for 0.9 minutes (total 12.0 minutes). Equilibration was started as follows: solvent was decreased from 100% B to 15% B over 0.2 minutes and held until a total of 16 minutes. The next sample is injected and the columns are switched.
Instrument Name:Agilent 1290 Infinity II
Column Name:Agilent Zorbax Eclipse Plus C18 (100 x 2.1mm, 1.8 um)
Flow Gradient:starting with a flow rate of 0.4mL/minute at 15% B and increasing to 50% B over 2.5 minutes, then to 57% over 0.1 minutes, to 70% over 6.4 minutes, to 93% over 0.1 minute, to 96% over 1.9 minutes and finally to 100% over 0.1 minute. The solvent was then held at 100% B for 0.9 minutes (total 12.0 minutes). Equilibration was started as follows: solvent was decreased from 100% B to 15% B over 0.2 minutes and held until a total of 16 minutes.
Flow Rate:0.4mL/min
Solvent A:50% water/30% acetonitrile/20% isopropanol; 10mM ammonium formate; 5uM medronic acid
Solvent B:1% water/9% acetonitrile/90% isopropanol; 10mM ammonium formate
Chromatography Type:Reversed phase

MS:

MS ID:MS003389
Analysis ID:AN003638
Instrument Name:Agilent 6490 QQQ
Instrument Type:Triple quadrupole
MS Type:ESI
MS Comments:Details previously published in https://doi.org/10.1016/j.chembiol.2018.10.008 Analysis of plasma extracts was performed on an Agilent 6490 QQQ mass spectrometer with an Agilent 1290 series HPLC system and a ZORBAX eclipse plus C18 column (2.1x100mm 1.8μm, Agilent) with the thermostat set at 60°C. Mass spectrometry analysis was performed in positive ion mode with dynamic scheduled multiple reaction monitoring (MRM). Mass spectrometry settings and MRM transitions for each lipid class, subclass and individual species are shown in Tables 1 and S1. The solvent system consisted of solvent A) 50% H2O / 30% acetonitrile / 20% isopropanol (v/v/v) containing 10mM ammonium formate and solvent B) 1% H2O / 9% acetonitrile / 90% isopropanol (v/v/v) containing 10mM ammonium formate. We utilized a stepped linear gradient with a 15-minute cycle time per sample and a 1μL sample injection. The gradient was as follows; starting with a flow rate of 0.4ml/minute at 10% B and increasing to 45% B over 2.7 minutes, then to 53% over 0.1 minutes, to 65% over 6.2 minutes, to 89% over 0.1 minute, to 92% over 1.9 minutes and finally to 100% over 0.1 minute. The solvent was then held at 100% B for 0.8 minutes (total 11.9 minutes). Equilibration was as follows, solvent was decreased from 100% B to 10% B over 0.1 minute and held for an additional 0.9 minutes. Flow rate was then switched to 0.6 ml/minute for 1 minute before returning to 0.4 ml/minute over 0.1 minutes. Solvent B was held at 10% B for a further 0.9 minutes at 0.4ml/minutes for a total cycle time of 15 minutes. The following mass spectrometer conditions were used; gas temperature, 150°C, gas flow rate 17L/min, nebulizer 20psi, Sheath gas temperature 200°C, capillary voltage 3500V and sheath gas flow 10L/min. Isolation widths for Q1 and Q3 were set to “unit” resolution (0.7 amu). PQC samples consisting of a pooled set of 6 healthy individuals were incorporated into the analysis at 1 PQC per 18 plasma samples. TQC consisted of PQC extracts which were pooled and split into individual vials to provide a measure of technical variation from the mass spectrometer only. These were included at a ratio of 1 TQC per 18 plasma samples. TQCs were monitored for changes in peak area, width and retention time to determine the performance of the LC-MS/MS analysis and were subsequently used to align for differential responses across the analytical batches.
Ion Mode:POSITIVE
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