Summary of Study ST002080

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 PR001320. The data can be accessed directly via it's Project DOI: 10.21228/M8399B This work is supported by NIH grant, U2C- DK119886.

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

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.

Study ID	ST002080
Study Title	Effects of Ferroptosis on the Metabolome in Cardiac Cells: The Role of Glutaminolysis
Study Type	GCMS
Study Summary	Ferroptosis is a novel iron-dependent regulated cell death mechanism that affects cell metabolism; however, a detailed metabolomic analysis of ferroptotic cells is not yet available. Here, we elucidated the metabolome of H9c2 cardioblasts by gas chromatography-mass spectrometry during ferroptosis induced by RSL3, a GPX4 inhibitor, in the presence of ferrostatin-1 (a ferroptosis inhibitor), XJB-5-131 (a mitochondrial-targeted ROS scavenger), or TSM-1005-44 (a newly developed cellular ROS scavenger). Results demonstrated that RSL3 decreased the levels of amino acids involved in glutathione synthesis more than two-fold. In contrast, saturated fatty acids levels were markedly increased in RSL3-challenged cells, with no effects on unsaturated fatty acids. RSL3 significantly altered the levels of mitochondrial tricarboxylic acid cycle intermediates; isocitrate and 2-oxoglutarate were found to increase, whereas succinate was significantly decreased in RSL3-challenged cells. Ferrostatin-1, XJB-5-131, and TSM-1005-44 prevented RSL3-induced cell death and conserved the metabolomic profile of the cells. Since 2-oxoglutarate is involved in the regulation of ferroptosis, particularly through glutamine metabolism, we further assessed the role of glutaminolysis in ferroptosis in H9c2 cardioblasts. Genetic silencing of GLS1, which encodes the K-type mitochondrial glutaminase (glutaminase C), protected against ferroptosis in the early stage. In conclusion, our study demonstrates that RSL3-induced ferroptosis impairs the metabolome of H9c2 cardioblasts.
Institute	University of Puerto Rico, School of Medicine
Department	Physiology
Laboratory	Cardiovascular Physiology, DR. Sabzali Javadov's Lab
Last Name	Rodriguez-Graciani
First Name	Keishla M
Address	Medical Sciences Campus, Main Building, 6th Floor, Department of Physiology, San Juan, Puerto Rico, 00936-5067, USA
Email	keishla.rodriguez20@upr.edu
Phone	7877582525x26888
Submit Date	2022-02-08
Num Groups	8
Publications	https://www.mdpi.com/1476010, Antioxidants 2022, 11(2), 278; https://doi.org/10.3390/antiox11020278
Raw Data Available	Yes
Raw Data File Type(s)	cdf
Analysis Type Detail	GC-MS
Release Date	2022-02-22
Release Version	1

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Project:

Project ID:	PR001320
Project DOI:	doi: 10.21228/M8399B
Project Title:	Effects of Ferroptosis on the Metabolome in Cardiac Cells: The Role of Glutaminolysis
Project Type:	GCMS
Project Summary:	Ferroptosis is a novel iron-dependent regulated cell death mechanism that affects cell metabolism; however, a detailed metabolomic analysis of ferroptotic cells is not yet available. Here, we elucidated the metabolome of H9c2 cardioblasts by gas chromatography-mass spectrometry during ferroptosis induced by RSL3, a GPX4 inhibitor, in the presence of ferrostatin-1 (a ferroptosis inhibitor), XJB-5-131 (a mitochondrial-targeted ROS scavenger), or TSM-1005-44 (a newly developed cellular ROS scavenger). Results demonstrated that RSL3 decreased the levels of amino acids involved in glutathione synthesis more than two-fold. In contrast, saturated fatty acids levels were markedly increased in RSL3-challenged cells, with no effects on unsaturated fatty acids. RSL3 significantly altered the levels of mitochondrial tricarboxylic acid cycle intermediates; isocitrate and 2-oxoglutarate were found to increase, whereas succinate was significantly decreased in RSL3-challenged cells. Ferrostatin-1, XJB-5-131, and TSM-1005-44 prevented RSL3-induced cell death and conserved the metabolomic profile of the cells. Since 2-oxoglutarate is involved in the regulation of ferroptosis, particularly through glutamine metabolism, we further assessed the role of glutaminolysis in ferroptosis in H9c2 cardioblasts. Genetic silencing of GLS1, which encodes the K-type mitochondrial glutaminase (glutaminase C), protected against ferroptosis in the early stage. In conclusion, our study demonstrates that RSL3-induced ferroptosis impairs the metabolome of H9c2 cardioblasts.
Institute:	University of Puerto Rico, School of Medicine
Department:	Physiology
Laboratory:	Cardiovascular Physiology, DR. Sabzali Javadov's Lab
Last Name:	Rodriguez-Graciani
First Name:	Keishla M
Address:	Medical Sciences Campus, Main Building, 6th Floor, Department of Physiology, San Juan, Puerto Rico, 00936-5067, USA
Email:	keishla.rodriguez20@upr.edu
Phone:	7877582525x26888
Funding Source:	This research was funded by the National Institutes of Health (Grants SC1GM128210, R25GM061838, U54MD007600, P20GM103475, HDTRA1-16-1-0041, and U19-AI1068021).
Publications:	https://www.mdpi.com/1476010, Antioxidants 2022, 11(2), 278; https://doi.org/10.3390/antiox11020278
Contributors:	Xavier R. Chapa-Dubocq, Esteban J. Ayala-Arroyo, Ivana Chaves-Negrón, Sehwan Jang, Nataliya Chorna, Taber S. Maskrey, Peter Wipf and Sabzali Javadov

Subject:

Subject ID:	SU002164
Subject Type:	Cultured cells
Subject Species:	Rattus norvegicus
Taxonomy ID:	10116
Cell Biosource Or Supplier:	American Type Culture Collection (ATCC)
Cell Strain Details:	H9c2(2-1) is a subclone of the original clonal cell line derived from embryonic BD1X rat heart tissue by B. Kimes and B. Brandt and exhibits many of the properties of skeletal muscle.
Cell Passage Number:	3-10
Cell Counts:	80-90% confluency

Factors:

Subject type: Cultured cells; Subject species: Rattus norvegicus (Factor headings shown in green)

mb_sample_id	local_sample_id	Treatment
SA196497	Control_ 2	Control
SA196498	Control_ 5	Control
SA196499	Control_ 1	Control
SA196500	Control_ 4	Control
SA196501	Control_ 3	Control
SA196502	Fer1-2	Ferrostatin-1
SA196503	Fer1-3	Ferrostatin-1
SA196504	Fer1-6	Ferrostatin-1
SA196505	Fer1-1	Ferrostatin-1
SA196506	Fer1-4	Ferrostatin-1
SA196507	Fer1-5	Ferrostatin-1
SA196508	RSL3_6	RSL3
SA196509	RSL3_1	RSL3
SA196510	RSL3_3	RSL3
SA196511	RSL3_2	RSL3
SA196512	RSL3_4	RSL3
SA196513	RSL3_5	RSL3
SA196514	RF_1	RSL3 + Ferrostatin-1
SA196515	RF_2	RSL3 + Ferrostatin-1
SA196516	RF_3	RSL3 + Ferrostatin-1
SA196517	RF_6	RSL3 + Ferrostatin-1
SA196518	RF_4	RSL3 + Ferrostatin-1
SA196519	RF_5	RSL3 + Ferrostatin-1
SA196520	RT_2	RSL3 + TSM-1005-44
SA196521	RT_4	RSL3 + TSM-1005-44
SA196522	RT_5	RSL3 + TSM-1005-44
SA196523	RT_3	RSL3 + TSM-1005-44
SA196524	RT_1	RSL3 + TSM-1005-44
SA196525	RX_2	RSL3 + XJB-5-131
SA196526	RX_1	RSL3 + XJB-5-131
SA196527	RX_3	RSL3 + XJB-5-131
SA196528	RX_6	RSL3 + XJB-5-131
SA196529	RX_5	RSL3 + XJB-5-131
SA196530	RX_4	RSL3 + XJB-5-131
SA196531	TSM_1	TSM-1005-44
SA196532	TSM_6	TSM-1005-44
SA196533	TSM_3	TSM-1005-44
SA196534	TSM_4	TSM-1005-44
SA196535	TSM_5	TSM-1005-44
SA196536	XJB_1	XJB-5-131
SA196537	XJB_2	XJB-5-131
SA196538	XJB_5	XJB-5-131
SA196539	XJB_4	XJB-5-131
SA196540	XJB_3	XJB-5-131

Showing results 1 to 44 of 44

Showing results 1 to 44 of 44

Collection:

Collection ID:	CO002157
Collection Summary:	H9c2 cardioblasts were cultured according to the manufacturer’s recommendations (ATCC, Manassas, VA, USA) with minor modifications. Briefly, the cells were incubated in DMEM based modified media containing 4 mM L-glutamine, 4.5 g/L glucose, 1 mM sodium pyruvate, and 1.5 g/L sodium bicarbonate, pH 7.4, and supplemented with 10% fetal bovine serum and 1% antibiotic solution (HyClone, Logan, UT, USA) in a CO2 incubator containing 95% air and 5% CO2 at 37 ◦C. Cells with 80–90% confluence from passages 3–10 were used for experiments. All chemicals were purchased from Sigma-Aldrich (St. Louis, MO). H9c2 cardioblasts demonstrate similar to the primary cardiomyocytes hypertrophic response as well as mitochondrial metabolism and morphology.
Sample Type:	Cultured cells

Treatment:

Treatment ID:	TR002176
Treatment Summary:	Cultured H9c2 cells reaching 80–90% confluence from passages 3–10 were used for experiments. Ferroptosis was induced by incubating the cells with 0.5 µM RSL3 for 3 h. In addition, the cells were exposed to RSL3 in the presence of 1 µM Fer-1, 0.2 µM XJB, and 0.6 µM TSM. Thus, experiments were performed in the following 8 groups: (i) control (n = 5), (ii) RSL3 (n = 6), (iii) Fer-1 (n = 6), (iv) XJB (n = 5), (v) TSM (n = 5), (vi) RSL3 + Fer-1 (n = 6), (vii) RSL3 + XJB (n = 6), and (viii) RSL3 + TSM (n = 5). Fer-1, XJB, or TSM were added to the culture medium simultaneously with RSL3. At the end of the incubation, the cells were harvested and processed for analysis by GC-MS.
Cell Pct Confluence:	80-90%

Sample Preparation:

Sampleprep ID:	SP002170
Sampleprep Summary:	Metabolites were extracted as previously described using 1 mL of cold MeOH/H2O (85:15). Samples were sonicated for 15 s (3×) on ice and centrifuged at 1400 rpm × 10 min at 4 ◦C (Rotor model: Eppendorf, FA-45-30-11). For protein quantification, the pellets were dried for 15 min in a rotary vacuum evaporator, then resuspended in 60 µL of denaturation buffer and sonicated for 1 min. All samples were centrifuged for 10 min at the maximum speed and the supernatant was used for protein quantification using the Bradford method. Supernatants were collected and evaporated to dryness using a SpeedVac (Savant AS160, Farmingdale, NY, USA). The metabolite samples were first derivatized through methoxyamination by adding 30 µL of 20 mg/mL solution of methoxyamine hydrochloride Antioxidants 2022, 11, 278 4 of 16 in pyridine (Sigma-Aldrich, St. Louis, MO, USA) and incubated at 37 ◦C for 2 h. Afterward, trimethylsilylation was performed by adding 30 µL of N-tert-butyldimethylsilyl-Nmethyltrifluoroacetamide (MTBSTFA + 1% TBDMSCl; Sigma-Aldrich, St. Louis, MO, USA) and incubated for 1 h at 65 ◦C. The reaction mixture was centrifuged at 1300 rpm × 10 min at RT. Supernatants were transferred to analytical vials. Then, 20 µL per sample was added to glass vials with inserts followed by the addition of 1 mM 2-fluobiphenyl (Sigma-Aldrich, St. Louis, MO, USA) as an internal standard. Samples were processed by GC-MS-QP2010 (Shimadzu Scientific Instruments, Inc., Columbia, MD) using analytical conditions as previously described.

Sampleprep ID:

SP002170

Sampleprep Summary:

Metabolites were extracted as previously described using 1 mL of cold MeOH/H2O (85:15). Samples were sonicated for 15 s (3×) on ice and centrifuged at 1400 rpm × 10 min at 4 ◦C (Rotor model: Eppendorf, FA-45-30-11). For protein quantification, the pellets were dried for 15 min in a rotary vacuum evaporator, then resuspended in 60 µL of denaturation buffer and sonicated for 1 min. All samples were centrifuged for 10 min at the maximum speed and the supernatant was used for protein quantification using the Bradford method. Supernatants were collected and evaporated to dryness using a SpeedVac (Savant AS160, Farmingdale, NY, USA). The metabolite samples were first derivatized through methoxyamination by adding 30 µL of 20 mg/mL solution of methoxyamine hydrochloride Antioxidants 2022, 11, 278 4 of 16 in pyridine (Sigma-Aldrich, St. Louis, MO, USA) and incubated at 37 ◦C for 2 h. Afterward, trimethylsilylation was performed by adding 30 µL of N-tert-butyldimethylsilyl-Nmethyltrifluoroacetamide (MTBSTFA + 1% TBDMSCl; Sigma-Aldrich, St. Louis, MO, USA) and incubated for 1 h at 65 ◦C. The reaction mixture was centrifuged at 1300 rpm × 10 min at RT. Supernatants were transferred to analytical vials. Then, 20 µL per sample was added to glass vials with inserts followed by the addition of 1 mM 2-fluobiphenyl (Sigma-Aldrich, St. Louis, MO, USA) as an internal standard. Samples were processed by GC-MS-QP2010 (Shimadzu Scientific Instruments, Inc., Columbia, MD) using analytical conditions as previously described.

Combined analysis:

Analysis ID	AN003394
Analysis type	MS
Chromatography type	GC
Chromatography system	Shimadzu GCMS-QP2010 ultra
Column	Shimadzu SH-RXI (30m x 0.25mm,0.25um)
MS Type	EI
MS instrument type	Single quadrupole
MS instrument name	Shimadzu QP2010 Ultra
Ion Mode	POSITIVE
Units	mM

Chromatography:

Chromatography ID:	CH002509
Instrument Name:	Shimadzu GCMS-QP2010 ultra
Column Name:	Shimadzu SH-RXI (30m x 0.25mm,0.25um)
Chromatography Type:	GC

MS:

MS ID:	MS003161
Analysis ID:	AN003394
Instrument Name:	Shimadzu QP2010 Ultra
Instrument Type:	Single quadrupole
MS Type:	EI
MS Comments:	Raw chromatography data were obtained and processed in GC-MS Labsolution Postrun Analysis software (Shimadzu Scientific Instruments, Inc., Columbia, MD) for identification of metabolites from their electron impact mass spectra by comparison to the database (NIST14/2014/EPA/NIH) [26]. Peak integration for all metabolites and multiple searches in the mass spectral library database resulted in a final database of 52 metabolic features chosen for this analysis. To assess analytical accuracy and precision, an external quality evaluation was performed using 2-fluorobiphenyl spiked into derivatization blank samples before running on the GC-MS. The quantitative analysis corresponding to the metabolite concentrations in each sample was calculated based on the internal standard in mM. A table in the format of comma-delimited (* csv) was created and uploaded to MetaboAnalyst.ca.
Ion Mode:	POSITIVE