Summary of study ST001246

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench,, 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.


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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.
University of California, Davis
Last NameShowalter
First NameMegan
AddressUC Davis Genome Center, room 1313, 451 Health Sci Drive
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 application/zip

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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.