{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST003246","ANALYSIS_ID":"AN005316","PROJECT_ID":"PR002016","VERSION":"1","CREATED_ON":"June 5, 2024, 7:06 am"},
"PROJECT":{"PROJECT_TITLE":"Effects of mitoregulin loss on cardiac and mitochondrial lipids in mice","PROJECT_SUMMARY":"We and others discovered a highly-conserved mitochondrial transmembrane microprotein, named Mitoregulin (Mtln), that supports lipid metabolism. We reported that Mtln strongly binds cardiolipin (CL), increases mitochondrial respiration and Ca2+ retention capacities, and reduces reactive oxygen species (ROS). Here we extend our observation of Mtln-CL binding and examine Mtln influence on cristae structure and mitochondrial membrane integrity during stress. We demonstrate that mitochondria from constitutive- and inducible Mtln-knockout (KO) mice are susceptible to membrane freeze-damage and that this can be rescued by acute Mtln re-expression. In mitochondrial-simulated lipid monolayers, we show that synthetic Mtln decreases lipid packing and monolayer elasticity. Lipidomics revealed that Mtln-KO heart tissues show broad decreases in 22:6-containing lipids and increased cardiolipin damage/remodeling. Lastly, we demonstrate that Mtln-KO mice suffer worse myocardial ischemia-reperfusion injury, hinting at a translationally-relevant role for Mtln in cardioprotection. Our work supports a model in which Mtln binds cardiolipin and stabilizes mitochondrial membranes to broadly influence diverse mitochondrial functions, including lipid metabolism, while also protecting against stress.","INSTITUTE":"University of Iowa","LAST_NAME":"Boudreau","FIRST_NAME":"Ryan","ADDRESS":"4334 PBDB, 169 Newton Rd, Iowa City, IA 52242","EMAIL":"ryan-boudreau@uiowa.edu","PHONE":"3193535573"},
"STUDY":{"STUDY_TITLE":"Effects of mitoregulin loss on cardiac and mitochondrial lipids in mice","STUDY_SUMMARY":"Cardiac lipidome analysis in aged (21 to 23-months old) male wildtype and mtln knockout mice","INSTITUTE":"University of Iowa","LAST_NAME":"Boudreau","FIRST_NAME":"Ryan","ADDRESS":"405 Galway Dr, Iowa City, Iowa, 52246, USA","EMAIL":"ryan-boudreau@uiowa.edu","PHONE":"3193535573"},
"SUBJECT":{"SUBJECT_TYPE":"Mammal","SUBJECT_SPECIES":"Mus musculus","TAXONOMY_ID":"10090"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"1102-3",
"Factors":{"Genotype":"WT","Sample source":"heart tissue"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name POS mode)":"6545_20190605_08_+_1102-3.mzML","RAW_FILE_NAME(Raw file name NEG mode)":"6545_20190605_110_-_1102-3.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"1116-1",
"Factors":{"Genotype":"WT","Sample source":"heart tissue"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name POS mode)":"6545_20190605_16_+_1116-1.mzML","RAW_FILE_NAME(Raw file name NEG mode)":"6545_20190605_106_-_1116-1.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"1116-4",
"Factors":{"Genotype":"WT","Sample source":"heart tissue"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name POS mode)":"6545_20190605_06_+_1116-4.mzML","RAW_FILE_NAME(Raw file name NEG mode)":"6545_20190605_105_-_1116-4.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"1134-1",
"Factors":{"Genotype":"WT","Sample source":"heart tissue"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name POS mode)":"6545_20190605_17_+_1134-1.mzML","RAW_FILE_NAME(Raw file name NEG mode)":"6545_20190605_117_-_1134-1.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"1134-2",
"Factors":{"Genotype":"KO","Sample source":"heart tissue"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name POS mode)":"6545_20190605_15_+_1134-2.mzML","RAW_FILE_NAME(Raw file name NEG mode)":"6545_20190605_120_-_1134-2.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"1134-3",
"Factors":{"Genotype":"KO","Sample source":"heart tissue"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name POS mode)":"6545_20190605_19_+_1134-3.mzML","RAW_FILE_NAME(Raw file name NEG mode)":"6545_20190605_108_-_1134-3.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"1134-4",
"Factors":{"Genotype":"KO","Sample source":"heart tissue"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name POS mode)":"6545_20190605_07_+_1134-4.mzML","RAW_FILE_NAME(Raw file name NEG mode)":"6545_20190605_109_-_1134-4.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"1134-5",
"Factors":{"Genotype":"KO","Sample source":"heart tissue"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name POS mode)":"6545_20190605_10_+_1134-5.mzML","RAW_FILE_NAME(Raw file name NEG mode)":"6545_20190605_116_-_1134-5.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"1153-1",
"Factors":{"Genotype":"KO","Sample source":"heart tissue"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name POS mode)":"6545_20190605_21_+_1153-1.mzML","RAW_FILE_NAME(Raw file name NEG mode)":"6545_20190605_114_-_1153-1.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"2191-1",
"Factors":{"Genotype":"WT","Sample source":"heart tissue"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name POS mode)":"6545_20190605_11_+_2191-1.mzML","RAW_FILE_NAME(Raw file name NEG mode)":"6545_20190605_118_-_2191-1.mzML"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"Mtln-KO and WT mice were fasted for 4-6 h, and while under deep ketamine/xylazine anesthesia, the heart apex was snipped off and immediately snap-frozen by LN2 immersion.","SAMPLE_TYPE":"Heart"},
"TREATMENT":{"TREATMENT_SUMMARY":"No treatment."},
"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Lipid extraction. Lipid extraction, based on Matyash et al.72, was performed as follows. All solutions were pre-chilled on ice. Tissues or mitochondrial pellets were transferred to labeled bead-mill tubes (1.4 mm, MoBio Cat# 13113-50) where lipids were extracted in a solution of 250 µL PBS, 225 µL MeOH containing internal standards, and 750 µL MTBE (methyl tert-butyl ether). Internal standards were Avanti SPLASH LipidoMix (Lot#12) at 10 µL per sample and Cambridge Isotope laboratories NSK-B and NSK-B-G1 (deuterated carnitines) at 10 µL per sample. The samples were homogenized in one 30 s cycle using the Omni Bead Ruptor followed by a rest on ice for 1 h. An addition of 188 µL PBS was made to induce phase separation. After centrifugation at 16,000 g for 5 minutes at 4 °C, the upper phases were collected and evaporated to dryness under a gentle nitrogen stream at room temperature. Lipid samples were reconstituted in 500 µL IPA (isopropyl alcohol) and transferred to an LC-MS vial with insert (Agilent 5182-0554 and 5183-2086) for analysis. Concurrently, a process blank sample and pooled quality control (QC) sample was prepared by taking equal volumes (~50 µL) from each sample after final resuspension."},
"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"Positive Mode RP LCMS","CHROMATOGRAPHY_TYPE":"Reversed phase","INSTRUMENT_NAME":"Agilent 1290 Infinity","COLUMN_NAME":"Waters ACQUITY UPLC CSH C18 (100 x 2.1mm,1.7um)","SOLVENT_A":"40% water/60% acetonitrile; 0.1% formic acid; 10 mM ammonium formate","SOLVENT_B":"90% isopropanol/9% acetonitrile/1% water; 0.1% formic acid; 10 mM ammonium formate","FLOW_GRADIENT":"The chromatography gradient for both positive and negative modes started at 15% mobile phase B then increased to 30% B over 2.4 min. It sequentially increased to 48% B from 2.4 – 3.0 min, 82% B from 3 – 13.2 min, and 99% B from 13.2 – 13.8 min where it’s held until 16.7 min and returned to the initial conditions and equilibrated for 5 min.","FLOW_RATE":"0.4 mL min","COLUMN_TEMPERATURE":"65"},
"ANALYSIS":{"ANALYSIS_TYPE":"MS"},
"MS":{"INSTRUMENT_NAME":"Agilent 6545 QTOF","INSTRUMENT_TYPE":"QTOF","MS_TYPE":"ESI","ION_MODE":"POSITIVE","MS_COMMENTS":"For positive mode, the source gas temperature was set to 225 °C, with a drying gas flow of 11 L/minute, nebulizer pressure of 40 psig, sheath gas temp of 350 °C and sheath gas flow of 11 L/minute. VCap voltage is set at 3500 V, nozzle voltage 500V, fragmentor at 110 V, skimmer at 85 V and octopole RF peak at 750 V. For data processing, Agilent MassHunter (MH) Workstation and software packages MH Qualitiative and MH Quantitative were used. The pooled QC (n=8) and process blank (n=4) were injected throughout the sample queue to ensure the reliability of acquired lipidomics data. For lipid annotation, accurate mass and MS/MS matching was used with the Agilent Lipid Annotator library. Results from the positive and negative ionization modes from Lipid Annotator were merged based on the class of lipid identified. Data exported from MH Quantitative was evaluated using Excel where initial lipid targets are parsed based on the following criteria. Only lipids with relative standard deviations (RSD) less than 30% in QC samples are used for data analysis. Additionally, only lipids with background AUC counts in process blanks that are less than 30% of QC are used for data analysis. The parsed excel data tables are normalized based on the ratio to class-specific internal standards, then to sum prior to statistical analysis."},
"MS_METABOLITE_DATA":{
"Units":"counts normalized to total lipid signal",
"Data":[{"Metabolite":"SM 42:3;O2","1134-2":"3452825.537","1134-3":"3281076.848","1134-4":"3470575.475","1134-5":"3888974.41","1153-1":"2821116.25","1102-3":"4038182.846","1116-1":"4130440.57","1116-4":"4242181.79","1134-1":"3921084.263","2191-1":"4439050.664"},{"Metabolite":"PC 18:2_18:3","1134-2":"719316.431","1134-3":"1108763.586","1134-4":"674529.0284","1134-5":"815292.9298","1153-1":"1160157.057","1102-3":"1512371.504","1116-1":"1135003.622","1116-4":"1357180.634","1134-1":"1033969.047","2191-1":"1317429.74"},{"Metabolite":"TG 18:0_18:1_22:0","1134-2":"509389.0598","1134-3":"516827.529","1134-4":"597849.5048","1134-5":"633781.8933","1153-1":"593519.1987","1102-3":"459477.1883","1116-1":"438997.7659","1116-4":"572592.8452","1134-1":"348490.2679","2191-1":"418720.1415"},{"Metabolite":"TG 18:1_20:1_22:1","1134-2":"1579032.314","1134-3":"1816398.595","1134-4":"2160702.921","1134-5":"2459522.524","1153-1":"2124986.164","1102-3":"1493617.224","1116-1":"1628526.412","1116-4":"1879613.883","1134-1":"1090285.533","2191-1":"1052426.509"},{"Metabolite":"TG 16:0_17:0_18:1","1134-2":"1275062.229","1134-3":"1350705.094","1134-4":"1480157.066","1134-5":"1364862.4","1153-1":"1074315.077","1102-3":"1051641.745","1116-1":"1010723.374","1116-4":"1046435.731","1134-1":"1269707.369","2191-1":"777441.4702"},{"Metabolite":"TG 16:0_16:0_18:1","1134-2":"22125423.77","1134-3":"20953202.47","1134-4":"26391261.34","1134-5":"24559626.73","1153-1":"21606551.23","1102-3":"20531726.83","1116-1":"17692455.97","1116-4":"19609390.41","1134-1":"22178263.67","2191-1":"17174537.96"},{"Metabolite":"TG 58:2","1134-2":"1825870.673","1134-3":"2037205.658","1134-4":"2602604.062","1134-5":"2737786.83","1153-1":"2435124.517","1102-3":"1672315.516","1116-1":"1881604.15","1116-4":"2185350.565","1134-1":"1471905.478","2191-1":"1231317.704"},{"Metabolite":"PC O-36:6","1134-2":"456968.6164","1134-3":"372381.8669","1134-4":"395843.3981","1134-5":"480768.6604","1153-1":"409740.5498","1102-3":"583281.1808","1116-1":"561954.315","1116-4":"504911.8618","1134-1":"417800.7191","2191-1":"514267.7347"},{"Metabolite":"TG 18:1_20:1_22:0","1134-2":"707057.7415","1134-3":"771591.2534","1134-4":"959280.3941","1134-5":"1140796.201","1153-1":"914536.8461","1102-3":"665816.6283","1116-1":"709210.7514","1116-4":"817898.3896","1134-1":"439097.0708","2191-1":"505833.7125"},{"Metabolite":"PC 14:0_18:2","1134-2":"629484.7315","1134-3":"742505.6272","1134-4":"458504.2763","1134-5":"794195.1082","1153-1":"900147.222","1102-3":"1128271.951","1116-1":"947928.2044","1116-4":"1037874.515","1134-1":"706922.4761","2191-1":"1123171.695"},{"Metabolite":"TG 58:3","1134-2":"3778717.744","1134-3":"4273602.702","1134-4":"5875278.668","1134-5":"6152974.578","1153-1":"5208685.805","1102-3":"3557720.117","1116-1":"4082232.398","1116-4":"4313850","1134-1":"3661654.585","2191-1":"2503038.13"},{"Metabolite":"TG 18:1_18:1_20:1","1134-2":"12722260.01","1134-3":"12230502.26","1134-4":"18770008.31","1134-5":"18018773.97","1153-1":"14640566.03","1102-3":"10333822.25","1116-1":"11789382.03","1116-4":"12376296.75","1134-1":"13056637.38","2191-1":"6400516.807"},{"Metabolite":"TG 56:2","1134-2":"3653119.808","1134-3":"3941411.441","1134-4":"5857966.544","1134-5":"5862324.451","1153-1":"4871535.622","1102-3":"3590753.085","1116-1":"3828106.382","1116-4":"4058388.743","1134-1":"3527174.142","2191-1":"2530996.74"},{"Metabolite":"PC 33:1","1134-2":"2270727.109","1134-3":"2358726.469","1134-4":"1735929.076","1134-5":"1910678.591","1153-1":"2258750.975","1102-3":"3015592.743","1116-1":"2250451.467","1116-4":"2815609.651","1134-1":"2171745.167","2191-1":"2819458.306"},{"Metabolite":"PC 39:5","1134-2":"234892.1336","1134-3":"175843.7221","1134-4":"184600.6409","1134-5":"230578.9399","1153-1":"174649.0744","1102-3":"253616.257","1116-1":"240936.8185","1116-4":"219325.1145","1134-1":"222668.019","2191-1":"247624.66"},{"Metabolite":"TG 18:0_22:6_22:6","1134-2":"674525.01","1134-3":"806795.1177","1134-4":"573482.3367","1134-5":"1092820.468","1153-1":"973814.6998","1102-3":"1313444.985","1116-1":"2443190.086","1116-4":"1645816.872","1134-1":"701325.1682","2191-1":"1645641.555"},{"Metabolite":"TG 18:1_18:2_20:1","1134-2":"17977171.37","1134-3":"20130920.78","1134-4":"22268027.5","1134-5":"20937749.18","1153-1":"22154354.45","1102-3":"16216773.58","1116-1":"18103071.7","1116-4":"17830880.76","1134-1":"19736627.6","2191-1":"10909859.24"},{"Metabolite":"PC 14:0_22:6","1134-2":"1210456.106","1134-3":"937915.7491","1134-4":"533621.5587","1134-5":"1354294.901","1153-1":"1099419.862","1102-3":"1481265.368","1116-1":"1917015.713","1116-4":"1492305.762","1134-1":"1131118.277","2191-1":"1399553.763"},{"Metabolite":"PC 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