MAGE-TAB Version	1.1
Comment[MetaboBank accession]	MTBKS202
Study Title	Lysophosphatidylserine induces necrotic cell death in pressure-overloaded hearts via G protein-coupled receptor 34
Study Description	Heart failure is a leading cause of mortality in developed countries. Cell death is a key player in the development of heart failure. Calcium-independent phospholipase A2β (iPLA2β) produces lipid mediators by catalyzing lipids, and induces nuclear shrinkage in caspase-independent cell death. Here, we show that lysophosphatidylserine generated by iPLA2β induces necrotic cardiomyocyte death, as well as contractile dysfunction mediated though its receptor, G protein-coupled receptor 34 (GRP34). Cardiomyocyte-specific iPLA2β-deficient mice were subjected to pressure overload. While control mice showed left ventricular systolic dysfunction with necrotic cardiomyocyte death, iPLA2β-deficient mice preserved cardiac function. Lipidomic analysis revealed a reduction of 18:0 lysophosphatidylserine in iPLA2β-deficient hearts. Knockdown of Grp34 attenuated 18:0 lysophosphatidylserine-induced necrosis in neonatal rat cardiomyocytes, while the ablation of Grp34 reduced the development of pressure overload-induced cardiac remodeling. Thus, the iPLA2β-lysophosphatidylserine-G protein-coupled receptor 34-necrosis signaling axis plays a detrimental role in the heart in response to pressure overload.

Experimental Design	genetic modification design	injury design
Experimental Factor Name	genetic_modification	treatment
Experimental Factor Type	genetic_modification	treatment

Person Last Name	Ikeda	Taneike
Person First Name	Kazutaka	Manabu
Person Mid Initials
Person Affiliation	Laboratory for Metabolomics, RIKEN Center for Integrative Medical Sciences (IMS); Cellular and Molecular Epigenetics Laboratory, Graduate School of Medical Life Science, Yokohama-City University	Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine
Person Roles	submitter	submitter

PubMed ID
Publication DOI

Protocol Name	Sample collection	Extraction	Chromatography	Mass spectrometry	Data processing	Metabolite identification
Protocol Type	Sample collection	Extraction	Chromatography	Mass spectrometry	Data processing	Metabolite identification
Protocol Description	Wild-type C57BL/6 (C57BL/6JJmsSlc) mice were purchased from SLC, Japan. All mice were housed in a temperature-controlled (23 ± 1 ℃) room with a 12-hr light-dark cycle (lights on from 8:00 to 20:00); they were acclimated to the environment at least 7 days before the experiments. The Pla2g6 gene-targeting vector was constructed using C57BL/6J mouse genomic DNA. The targeting construct was developed by inserting the loxP-flippase (FLP) recombinase target (FRT)-neomycin-FRT resistance cassette, a loxP site encompassing exon 10 of the Pla2g6 gene, and the diptheria toxin A subunit (DTA) gene. The targeting vector was electroporated into ES cells (F1; SVJ129 and C57BL/6J); the transfected ES clones were selected for diphtheria toxin and neomycin resistance according to standard protocols. Circular pCAG-Flpe plasmid was electroporated into the selected ES clones. The neomycin cassette-excised ES clones were screened by PCR. Southern blotting and karyotyping analyses were performed to obtain ES clones exhibiting the desired homologous recombination and normal karyotype. These targeted ES clones were injected into C57BL/6J mouse blastocyst to generate chimeric mice. The chimeric mice were crossed with C57BL/6J mice to validate germ line transmission. We generated mice with the floxed Pla2g6 allele and crossed them with transgenic mice expressing α-myosin heavy chain promoter-driven Cre recombinase (Myh6-Cre+), in order to obtain cardiomyocyte-specific iPLA2β-deficient mice (Pla2g6flox/flox; Myh6-Cre+, Pla2g6-/-). Their Pla2g6flox/flox; Myh6-Cre- (Pla2g6+/+) littermates were used as controls. GPR34-deficient mice were backcrossed to the C57BL/6 background for 12 generations. Only male mice were used in this study. The mice were given food and water ad libitum. We performed TAC surgery on male mice aged 10-14 weeks using 27-gauge needles. Sham-operated animals underwent the same operation without aortic constriction. Non-invasive measurements of blood pressure were performed on mice anaesthetized with 2.5% avertin, using a blood pressure monitor for mice (Model MK-2000, Muromachi Kikai) according to the manufacturer's instructions, as previously described30. The pressure gradient across TAC was estimated based on the difference in blood pressure between the two arms. Adult cardiomyocytes were isolated from 10-week-old male mice using a Langendorff system, as previously reported (Oka, T et al., (2012)). In brief, after the mice were deeply anesthetized, the heart was quickly excised, cannulated via the aorta, and perfused at constant flow. Hearts were first perfused for 1 minute at 37°C with a perfusion buffer containing 120 mM NaCl (31319, Nakalai Tesque), 5.4 mM KCl (163-03545, FUJIFILM Wako Pure Chemical Corporation), 1.6 mM MgCl2 (135-00165, FUJIFILM Wako Pure Chemical Corporation), 1.2 mM NaH2PO4 (197-02865, FUJIFILM Wako Pure Chemical Corporation), 5.6 mM glucose (G8270, Sigma-Aldrich), 20 mM NaHCO3 (191-01305, FUJIFILM Wako Pure Chemical Corporation), and 5 mM taurine (T0625, Sigma-Aldrich), followed by collagenase buffer containing 1.2 mg/ml collagenase type 2 (CLS-2, Worthington Biochemical Corporation) and 0.020 mg/ml protease type XIV (P-5147, Sigma-Aldrich). After collagenase and protease digestion, the supernatant containing the dispersed myocytes was filtered into a sterilized tube and gently centrifuged at 20x g for 3 minutes. The cell pellet was promptly resuspended in perfusion buffer containing 200 μM Ca2+. The cardiomyocytes were pelleted by gravity for 10 minutes; the supernatant was aspirated and the suspension of rod-shaped cardiomyocytes was then used.	"Untargeted lipidomics: Untargeted lipidomics was performed as described(Tsugawa H et al., (2020)). In brief, 200 μl of suspension from each heart tissue in methanol containing internal standards was mixed with 100 μl chloroform and incubated for 1 hour at room temperature. Subsequently, 20 μl of water was added, before the samples were incubated for 10 minutes and then centrifuged at 2000×g for 10 minutes. The supernatants were then collected.; Targeted lipidomics: Targeted lipidomics was performed as described(Naoe S et al., (2019)). 
In brief, samples were prepared by solid-phase extraction with a Sep-Pak C18 cartridge (Waters) using deuterium-labeled internal standards."	Untargeted lipidomics: LC separation was performed using a reverse-phase column (Acquity UPLC BEH peptide C18; 2.1 × 50 mm, 1.7 µm particle size; Waters, Milford, MA, USA) with a gradient elution of mobile phase A (methanol: acetonitrile: water = 1:1:3, v/v/v for volume ratio containing 5 mM ammonium acetate and 10 nM EDTA) and mobile phase B (100% isopropanol containing 5 mM ammonium acetate and 10 nM EDTA), and the composition was produced by mixing those solvents. LC gradient consisted of holding solvent (A/B: 100/0) for 1 min, then linearly converting to solvent (A/B: 60/40) for 4 min, linearly converting to solvent (A/B:36/64) for 2.5 min and holding for 4.5 min, then linearly converting to solvent (A/B: 17.5/82.5) for 0.5 min, linearly converting to solvent (A/B: 15/85) for 8.5 min, and linearly converting to solvent (A/B: 5/95) for 1 min followed by returning to solvent (A/B: 100/0) and holding for 5 min for re-equilibration. The flow rate and column temperature were set to 0.3 mL/min and 45 °C, respectively. ; Targeted lipidomics: The targeted analysis was performed using a UPLC system (Waters UPLC, Waters, Milford, MA, USA) with a triple quadruple linear ion trap mass spectrometer (QTRAP 5500; SCIEX, Framingham, MA, USA), equipped with Acquity UPLC BEH C18 column (1.0 × 150 mm, 1.7 µm particle size; Waters, Milford, MA, USA). Samples were eluted with a mobile phase composed of water/acetate (100:0.1, v/v) and acetonitrile/methanol (4:1, v/v) (73:27) for 5 min, and ramped to 30:70 over 15 min, to 20:80 over 25 min and held for 8 min, ramped to 0:100 over 35 min, and held for 10 min with flow rates of 70 µL/min (0-30 min), 80 µL/min (30-33 min), and 100 µL/min (33-45 min).	Untargeted lipidomics: The supernatants were then collected and analyzed using an ACQUITY UPLC system (Waters, Milford, MA) coupled with a QTOF-MS (TripleTOF 6600; Sciex, Framingham, MA). The information-dependent acquisition (IDA) mode was applied to confirm each of the lipid profiles and structures in negative-ion and positive-ion mode. ; Targeted lipidomics: Targeted lipidomics for PUFA metabolites were performed using an ACQUITY UPLC system (Waters) with a linear ion-trap quadrupole mass spectrometer (QTRAP5500; Sciex). MS/MS analyses were conducted in negative-ion mode, and fatty acid metabolites were identified and quantified by multiple reaction monitoring (MRM).	The software used is as follows. Untargeted lipidomics: Analyst TF ver.1.8.1 (SCIEX), SCIEX MS converter Ver. Beta 1.3 (SCIEX), 2DICAL ver. 0.91 (Mitsui Knowledge Industry) and Microsoft Excel 2019; Targeted lipidomics: MultiQuant software ver.2.0 (SCIEX). ;Targeted lipidomics: The targeted analysis data was analyzed using MultiQuant software (SCIEX). The chromatogram peaks were manually curated.	Untargeted lipidomics: The untargeted analysis data was analyzed using manual identification with reference samples or MS-DIAL software version 4 (http://prime.psc.riken.jp/);
Protocol Parameters		Post extraction;Derivatization	Chromatography instrument;Autosampler model;Column model;Column type;Guard column	Scan polarity;Scan m/z range;Instrument;Ion source;Mass analyzer
Protocol Hardware
Protocol Software

Public Release Date	2023-07-06

Term Source Name
Term Source File
Term Source Version

SDRF File	MTBKS202.sdrf.txt

Comment[Study type]	untargeted metabolite profiling	targeted metabolite profiling
Comment[Experiment type]	liquid chromatography-mass spectrometry	time-of-flight mass spectrometry	quadrupole mass spectrometer
Comment[Submission type]	LC-MS
Comment[BioProject]	PRJDB12651
Comment[Submission Date]	2021-11-25
Comment[Last Update Date]	2023-07-06