Summary of study ST001246

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 PR000833. The data can be accessed directly via it's Project DOI: 10.21228/M8110J This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

Perform statistical analysis  |  Show all samples  |  Show named metabolites  |  Download named metabolite data  |  Download all metabolite data  |  Download mwTab file (text)   |  Download mwTab file(JSON)   |  Download data (Contains raw data)
Study IDST001246
Study TitleTFPa/HADHA is required for fatty acid beta-oxidation and cardiolipin re-modeling in human cardiomyocytes (part-I)
Study SummaryMitochondrial trifunctional protein deficiency, due to mutations in hydratase subunit A (HADHA), results in sudden infant death syndrome (SIDS) with no cure. To reveal the disease etiology, we generated stem cell-derived cardiomyocytes from HADHA-deficient hiPSCs and accelerated their maturation via a novel, engineered MicroRNA Maturation Cocktail (MiMaC) that upregulated the epigenetic regulator, HOPX. Fatty acid challenged MiMaC treated HADHA mutant cardiomyocytes manifested the disease phenotype: defective calcium dynamics and repolarization kinetics which resulted in a pro-arrhythmic state. Single cell RNA-seq revealed a novel cardiomyocyte developmental intermediate, based on metabolic gene expression. This intermediate gave rise to mature-like cardiomyocytes in control cells but, mutant cells transitioned to a pathological state with reduced fatty acid beta-oxidation (FAO), reduced mitochondrial proton gradient, disrupted cristae structure and defective cardiolipin remodeling. This study reveals that TFPa/HADHA, a MLCL-AT-like enzyme, is required for FAO and cardiolipin remodeling, essential for functional mitochondria in human cardiomyocytes.
Institute
University of California, Davis
Last NameShowalter
First NameMegan
AddressUC Davis Genome Center, room 1313, 451 Health Sci Drive
Emailmshowalter@ucdavis.edu
Phone5307529922
Submit Date2019-08-26
Raw Data AvailableYes
Raw Data File Type(s).raw
Analysis Type DetailLC-MS
Release Date2019-09-06
Release Version1
Megan Showalter Megan Showalter
https://dx.doi.org/10.21228/M8110J
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:


Project:

Project ID:PR000833
Project DOI:doi: 10.21228/M8110J
Project Title:TFPa/HADHA is required for fatty acid beta-oxidation and cardiolipin re-modeling in human cardiomyocytes
Project Summary:Mitochondrial trifunctional protein deficiency, due to mutations in hydratase subunit A (HADHA), results in sudden infant death syndrome (SIDS) with no cure. To reveal the disease etiology, we generated stem cell-derived cardiomyocytes from HADHA-deficient hiPSCs and accelerated their maturation via a novel, engineered MicroRNA Maturation Cocktail (MiMaC) that upregulated the epigenetic regulator, HOPX. Fatty acid challenged MiMaC treated HADHA mutant cardiomyocytes manifested the disease phenotype: defective calcium dynamics and repolarization kinetics which resulted in a pro-arrhythmic state. Single cell RNA-seq revealed a novel cardiomyocyte developmental intermediate, based on metabolic gene expression. This intermediate gave rise to mature-like cardiomyocytes in control cells but, mutant cells transitioned to a pathological state with reduced fatty acid beta-oxidation (FAO), reduced mitochondrial proton gradient, disrupted cristae structure and defective cardiolipin remodeling. This study reveals that TFPa/HADHA, a MLCL-AT-like enzyme, is required for FAO and cardiolipin remodeling, essential for functional mitochondria in human cardiomyocytes.
Institute:University of California, Davis
Last Name:Showalter
First Name:Megan
Address:GBSF 1313, 451 Health Sci Drive, Davis, Ca, 95616, USA
Email:mshowalter@ucdavis.edu
Phone:530-752-9922
Funding Source:R01GM097372, R01GM083867

Subject:

Subject ID:SU001314
Subject Type:Cultured cells
Subject Species:Homo sapiens
Taxonomy ID:9606

Factors:

Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id local_sample_id Genoytpe Treatment
SA090822KO_6DFA_01KO HADHA Control
SA090823KO_6DFA_03KO HADHA Control
SA090824KO_6DFA_02KO HADHA Control
SA090825MUT_6DFA_02_inj02MUT HADHA 10 Day Fatty Acid Supplementation
SA090826MUT_6DFA_02_inj03MUT HADHA 11 Day Fatty Acid Supplementation
SA090827MUT_12DFA_03MUT HADHA 12 Day Fatty Acid Supplementation
SA090828MUT_12DFA_01MUT HADHA 12 Day Fatty Acid Supplementation
SA090829MUT_12DFA_02MUT HADHA 12 Day Fatty Acid Supplementation
SA090830MUT_12DFA_04MUT HADHA 12 Day Fatty Acid Supplementation
SA090831MUT_6DFA_01_inj01MUT HADHA 6 Day Fatty Acid Supplementation
SA090832MUT_6DFA_01_inj02MUT HADHA 7 Day Fatty Acid Supplementation
SA090833MUT_6DFA_01_inj03MUT HADHA 8 Day Fatty Acid Supplementation
SA090834MUT_6DFA_02_inj01MUT HADHA 9 Day Fatty Acid Supplementation
SA090835WT_12DFA_04WT HADHA 12 Day Fatty Acid Supplementation
SA090836WT_12DFA_02WT HADHA 12 Day Fatty Acid Supplementation
SA090837WT_12DFA_01WT HADHA 12 Day Fatty Acid Supplementation
SA090838WT_12DFA_03WT HADHA 12 Day Fatty Acid Supplementation
Showing results 1 to 17 of 17

Collection:

Collection ID:CO001308
Collection Summary:hESC and hiPSC and cardiac differentiation: The hESC line RUES2 (NIHhESC-09-0013) and hiPSC line WTC #11, previously derived in the Conklin laboratory 83, were cultured on Matrigel growth factor-reduced basement membrane matrix (Corning) in mTeSR media (StemCell Technologies). A monolayer-based directed differentiation protocol was followed to generate hESC-CMs and hiPSC-CMs, as done previously 25. hiPSC-CM cardiolipin assay was done with a small molecule monolayer-based directed differentiation protocol, as done previously 84. 15 days after differentiation hPSC-CMs were enriched for the cardiomyocyte population using a lactate selection process 85. We generated cardiomyocyte populations ranging from 40-60% that were then enriched to 75-80% cardiomyocytes after 4 days of lactate enrichment. METABOLITE EXTRACTION: 1 million cells were extracted with 225 µl of methanol, after enzymatic lifting from cell culture plates.
Sample Type:Cultured cells

Treatment:

Treatment ID:TR001329
Treatment Summary:HADHA Line Creation: Using LentiCrisprV2 plasmid 91 (lentiCRISPRV2 was a gift from Feng Zhang (Addgene plasmid # 52961) two different gRNAs targeted to Exon 1 of HADHA were designed using CRISPRScan 92. Sequences for the gRNAs can be found in Supplemental Table S12. The gRNA and Cas9 expressing plasmids were transiently transfected into the WTC line using GeneJuice (EMD Millipore). 24 hours after transfection, WTCs were puromycin selected for two days and then clonally expanded. DNA of the clones was isolated, the region around the targeting guides was PCR amplified (see guides in Supplemental Table S12) and sequenced to determine the insertion and deletion errors generated by CRISPR-Cas9 system in exon 1 of HADHA. Western analysis was performed to determine the levels of HADHA protein in HADHA mutants. 31 clones were sent for sequencing from gRNA1 experiment, 6 clones (19%) had no mutations while 25 clones (81%) were found to have mutations. 24 clones were sent for sequencing from gRNA2, 1 clone had no mutations (4%) while 23 clones (96%) were found to have mutations. Two of the mutant lines were analyzed further in this study. Glucose and Fatty Acid Media: The base media which we are calling Glucose Media, is RPMI supplemented with B27 with insulin. The fatty acid media is the glucose media with oleic acid conjugated to BSA (Sigma O3008): 12.7μg/mL, linoleic acid conjugated to BSA (Sigma L9530): 7.05μg/mL, sodium palmitate (Sigma P9767) conjugated to BSA (Sigma A8806): 52.5μM and L-carnitine: 125μM. Fatty acid (FA) experiments used this B27 + insulin + the three FAs (oleic acid, linoleic acid and palmitatic acid), in RPMI media. This media was changed every other day during the 6-days or 12-days of treatment.

Sample Preparation:

Sampleprep ID:SP001322
Sampleprep Summary:1 million cells were extracted with 225 µl of methanol at −20°C containing an internal standard mixture of PE(17:0/17:0), PG(17:0/17:0), PC(17:0/0:0), C17 sphingosine, ceramide (d18:1/17:0), SM (d18:0/17:0), palmitic acid-d3, PC (12:0/13:0), cholesterol-d7, TG (17:0/17:1/17:0)-d5, DG (12:0/12:0/0:0), DG (18:1/2:0/0:0), MG (17:0/0:0/0:0), PE (17:1/0:0), LPC (17:0), LPE (17:1), and 750 µL of MTBE (methyl tertiary butyl ether) (Sigma Aldrich) at −20°C containing the internal standard cholesteryl ester 22:1. Cells were vortexed for 20 sec, sonicated for 5 min and shaken for 6 min at 4°C with an Orbital Mixing Chilling/Heating Plate (Torrey Pines Scientific Instruments). Then 188 µl of LC-MS grade water (Fisher) was added. Samples were vortexed, centrifuged at 14,000 rcf (Eppendorf 5415D). The upper (non-polar, organic) phase was collected in two 350 µL aliquots and evaporated to dryness.

Combined analysis:

Analysis ID AN002070
Analysis type MS
Chromatography type Reversed phase
Chromatography system Thermo Q Exactive HF Hybrid Quadrupole-Orbitrap
Column Waters Acquity CSH C18 (100 x 2.1mm, 1.7um)
MS Type ESI
MS instrument type Orbitrap
MS instrument name Thermo Q Exactive HF hybrid Orbitrap
Ion Mode UNSPECIFIED
Units ng

Chromatography:

Chromatography ID:CH001507
Chromatography Summary:Re-suspended samples were injected at 3 µL and 5 µL for ESI positive and negative modes respectively, onto a Waters Acquity UPLC CSH C18 (100 mm length × 2.1 mm id; 1.7 µm particle size) with an additional Waters Acquity VanGuard CSH C18 pre-column (5 mm × 2.1 mm id; 1.7 µm particle size) maintained at 65°C was coupled to a Vanquish UHPLC System. To improve lipid coverage, different mobile phase modifiers were used for positive and negative mode analysis 103. For positive mode 10 mM ammonium formate and 0.1% formic acid were used and 10 mM ammonium acetate (Sigma–Aldrich) was used for negative mode. Both positive and negative modes used the same mobile phase composition of (A) 60:40 v/v acetonitrile:water (LC-MS grade) and (B) 90:10 v/v isopropanol:acetonitrile. The gradient started at 0 min with 15% (B), 0–2 min 30% (B), 2–2.5 min 48% (B), 2.5–11 min 82% (B), 11–11.5 min 99% (B), 11.5–12 min 99% (B), 12–12.1 min 15% (B), and 12.1–15 min 15% (B). A flow rate of 0.6 mL/min was used.
Instrument Name:Thermo Q Exactive HF Hybrid Quadrupole-Orbitrap
Column Name:Waters Acquity CSH C18 (100 x 2.1mm, 1.7um)
Chromatography Type:Reversed phase

MS:

MS ID:MS001921
Analysis ID:AN002070
Instrument Name:Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:For data acquisition a Q-Exactive HF Hybrid Quadrupole-Orbitrap Mass Spectrometer was used with the following parameters: mass range, m/z 100-1200; MS1 resolution 60,000: data-dependent MS2 resolution 15,000; NCE 20, 30, 40; 4 targets/MS1 scan; gas temperature 369°C, sheath gas flow (nitrogen), 60 units, aux gas flow 25 units, sweep gas flow 2 units; spray voltage 3.59 kV. LC-MS data processing using MS-DIAL and statistics: Untargeted lipidomic data processing was performed using MS-DIAL 104 for deconvolution, peak picking, alignment, and identification. In house m/z and retention time libraries were used in addition to MS/MS spectra databases in msp format 105. Features were reported when present in at least 50% of samples in each group. Statistical analysis was done by first normalizing data using the sum of the knowns, or mTIC normalization, to scale each sample. Normalized peak heights were then submitted to R for statistical analysis. ANOVA analysis was performed with FDR correction and post hoc testing.
Ion Mode:UNSPECIFIED
  logo