#METABOLOMICS WORKBENCH Codreags00_20230929_134531 DATATRACK_ID:4349 STUDY_ID:ST002891 ANALYSIS_ID:AN004750 PROJECT_ID:PR001804 VERSION 1 CREATED_ON October 2, 2023, 8:16 am #PROJECT PR:PROJECT_TITLE Glutamine metabolism improves left ventricular function but not PR:PROJECT_TITLE macrophage-mediated inflammation following myocardial infarction PR:PROJECT_TYPE Untargeted Metabolomics analysis PR:PROJECT_SUMMARY Glutamine is a critical amino acid that serves as an energy source, building PR:PROJECT_SUMMARY block, and signaling molecule for both the heart tissue and the immune system. PR:PROJECT_SUMMARY However, the role of glutamine metabolism in regulating cardiac remodeling PR:PROJECT_SUMMARY following myocardial infarction (MI) is unknown. In this study, we show that PR:PROJECT_SUMMARY glutamine metabolism is altered both in the remote (contractile) area and in PR:PROJECT_SUMMARY infiltrating macrophages in the infarct area after MI in adult male mice by PR:PROJECT_SUMMARY permanent left anterior descending artery occlusion. Using untargeted PR:PROJECT_SUMMARY metabolomics in extracted LV macrophages, we found that metabolites related to PR:PROJECT_SUMMARY glutamine metabolism were differentially altered at days 1, 3, and 7 after MI. PR:PROJECT_SUMMARY Glutamine metabolism in live cells was found to be increased after MI relative PR:PROJECT_SUMMARY to no MI controls. Gene expression in the remote area of the heart indicated a PR:PROJECT_SUMMARY loss of glutamine metabolism. Glutamine administration improved LV function at PR:PROJECT_SUMMARY days 1, 3, and 7, which was associated with improved contractile and metabolic PR:PROJECT_SUMMARY gene expression. PR:INSTITUTE Vanderbilt University PR:DEPARTMENT Chemistry PR:LABORATORY Center for Innovative Technology PR:LAST_NAME Codreanu PR:FIRST_NAME Simona PR:ADDRESS 1234 STEVENSON CENTER LANE, NASHVILLE, TN, 37235, USA PR:EMAIL SIMONA.CODREANU@VANDERBILT.EDU PR:PHONE 6158758422 #STUDY ST:STUDY_TITLE Glutamine metabolism improves left ventricular function but not ST:STUDY_TITLE macrophage-mediated inflammation following myocardial infarction ST:STUDY_SUMMARY Glutamine is a critical amino acid that serves as an energy source, building ST:STUDY_SUMMARY block, and signaling molecule for both the heart tissue and the immune system. ST:STUDY_SUMMARY However, the role of glutamine metabolism in regulating cardiac remodeling ST:STUDY_SUMMARY following myocardial infarction (MI) is unknown. In this study, we show that ST:STUDY_SUMMARY glutamine metabolism is altered both in the remote (contractile) area and in ST:STUDY_SUMMARY infiltrating macrophages in the infarct area after MI in adult male mice by ST:STUDY_SUMMARY permanent left anterior descending artery occlusion. Using untargeted ST:STUDY_SUMMARY metabolomics in extracted LV macrophages, we found that metabolites related to ST:STUDY_SUMMARY glutamine metabolism were differentially altered at days 1, 3, and 7 after MI. ST:STUDY_SUMMARY Glutamine metabolism in live cells was found to be increased after MI relative ST:STUDY_SUMMARY to no MI controls. Gene expression in the remote area of the heart indicated a ST:STUDY_SUMMARY loss of glutamine metabolism. Glutamine administration improved LV function at ST:STUDY_SUMMARY days 1, 3, and 7, which was associated with improved contractile and metabolic ST:STUDY_SUMMARY gene expression. ST:INSTITUTE Vanderbilt University ST:DEPARTMENT Chemistry ST:LABORATORY Center for Innovative Technology ST:LAST_NAME Codreanu ST:FIRST_NAME Simona ST:ADDRESS 1234 STEVENSON CENTER LANE, NASHVILLE, TN, 37235, USA ST:EMAIL SIMONA.CODREANU@VANDERBILT.EDU ST:STUDY_TYPE untargeted metabolomics analysis ST:PHONE 6158758422 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:AGE_OR_AGE_RANGE 15-20 week old SU:GENDER Male SU:SPECIES_GROUP C57BL/6J #FACTORS #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - D1_1 Gln Treatment:Day 1 RAW_FILE_NAME=SC_20211103_HILICn_FMS_Mouton_D1_1 SUBJECT_SAMPLE_FACTORS - D1_2 Gln Treatment:Day 1 RAW_FILE_NAME=SC_20211103_HILICn_FMS_Mouton_D1_2 SUBJECT_SAMPLE_FACTORS - D1_3 Gln Treatment:Day 1 RAW_FILE_NAME=SC_20211103_HILICn_FMS_Mouton_D1_3 SUBJECT_SAMPLE_FACTORS - D1_4 Gln Treatment:Day 1 RAW_FILE_NAME=SC_20211103_HILICn_FMS_Mouton_D1_4 SUBJECT_SAMPLE_FACTORS - D1_5 Gln Treatment:Day 1 RAW_FILE_NAME=SC_20211103_HILICn_FMS_Mouton_D1_5 SUBJECT_SAMPLE_FACTORS - D1_6 Gln Treatment:Day 1 RAW_FILE_NAME=SC_20211103_HILICn_FMS_Mouton_D1_6 SUBJECT_SAMPLE_FACTORS - D3_1 Gln Treatment:Day 3 RAW_FILE_NAME=SC_20211103_HILICn_FMS_Mouton_D3_1 SUBJECT_SAMPLE_FACTORS - D3_2 Gln Treatment:Day 3 RAW_FILE_NAME=SC_20211103_HILICn_FMS_Mouton_D3_2 SUBJECT_SAMPLE_FACTORS - D3_3 Gln Treatment:Day 3 RAW_FILE_NAME=SC_20211103_HILICn_FMS_Mouton_D3_3 SUBJECT_SAMPLE_FACTORS - D3_4 Gln Treatment:Day 3 RAW_FILE_NAME=SC_20211103_HILICn_FMS_Mouton_D3_4 SUBJECT_SAMPLE_FACTORS - D3_5 Gln Treatment:Day 3 RAW_FILE_NAME=SC_20211103_HILICn_FMS_Mouton_D3_5 SUBJECT_SAMPLE_FACTORS - D3_6 Gln Treatment:Day 3 RAW_FILE_NAME=SC_20211103_HILICn_FMS_Mouton_D3_6 SUBJECT_SAMPLE_FACTORS - D7_1 Gln Treatment:Day 7 RAW_FILE_NAME=SC_20211103_HILICn_FMS_Mouton_D7_1 SUBJECT_SAMPLE_FACTORS - D7_2 Gln Treatment:Day 7 RAW_FILE_NAME=SC_20211103_HILICn_FMS_Mouton_D7_2 SUBJECT_SAMPLE_FACTORS - D7_3 Gln Treatment:Day 7 RAW_FILE_NAME=SC_20211103_HILICn_FMS_Mouton_D7_3 SUBJECT_SAMPLE_FACTORS - D7_4 Gln Treatment:Day 7 RAW_FILE_NAME=SC_20211103_HILICn_FMS_Mouton_D7_4 SUBJECT_SAMPLE_FACTORS - D7_5 Gln Treatment:Day 7 RAW_FILE_NAME=SC_20211103_HILICn_FMS_Mouton_D7_5 #COLLECTION CO:COLLECTION_SUMMARY MI was induced in adult (15-20 week old) male C57BL/6J mice as previously CO:COLLECTION_SUMMARY described.2,4,6 Mice were anesthetized (2% isoflurane) and laid supine on a CO:COLLECTION_SUMMARY heated stage (37°C), intubated and connected to a mouse ventilator (Harvard CO:COLLECTION_SUMMARY Apparatus; 300µL stroke volume, 300 breaths/min). The chest was incised to CO:COLLECTION_SUMMARY expose the ribs, and the heart exposed between the 3rd and 4th ribs. The CO:COLLECTION_SUMMARY pericardial layer was gently removed, and the left coronary artery was ligated CO:COLLECTION_SUMMARY ~1mm below the left atrium using 8-0 suture. Ischemia was confirmed by blanching CO:COLLECTION_SUMMARY of the LV. For glutamine administration, mice were injected intraperitoneally CO:COLLECTION_SUMMARY with glutamine (0.75-1g/kg) or saline vehicle beginning at either day 1 post-MI CO:COLLECTION_SUMMARY (for day 3 time-point) or beginning at day 3 (for day 7 time-point). A separate CO:COLLECTION_SUMMARY cohort of mice was injected 2 hr after MI and studied 24 hr later (day 1). To CO:COLLECTION_SUMMARY inhibit glutamine metabolism, mice were administered BPTES (12.5mg/kg; 10% in CO:COLLECTION_SUMMARY DMSO/corn oil), a glutaminase-1 inhibitor, beginning at either day 1 or day 3. CO:SAMPLE_TYPE Macrophages #TREATMENT TR:TREATMENT_SUMMARY For glutamine administration, mice were injected intraperitoneally with TR:TREATMENT_SUMMARY glutamine (0.75-1g/kg) or saline vehicle beginning at either day 1 post-MI (for TR:TREATMENT_SUMMARY day 3 time-point) or beginning at day 3 (for day 7 time-point). A separate TR:TREATMENT_SUMMARY cohort of mice was injected 2 hr after MI and studied 24 hr later (day 1). To TR:TREATMENT_SUMMARY inhibit glutamine metabolism, mice were administered BPTES (12.5mg/kg; 10% in TR:TREATMENT_SUMMARY DMSO/corn oil), a glutaminase-1 inhibitor, beginning at either day 1 or day 3. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Frozen samples were stored at -80°C until analyzed by LC-MS-based metabolomics SP:SAMPLEPREP_SUMMARY in the Vanderbilt Center for Innovative Technology (CIT). Isotopically labeled SP:SAMPLEPREP_SUMMARY phenylalanine-D8 and biotin-D2 were added to 200 μL of culture supernatant per SP:SAMPLEPREP_SUMMARY sample, and protein was precipitated by addition of 800 μL of ice-cold methanol SP:SAMPLEPREP_SUMMARY followed by overnight incubation at −80°C. Precipitated proteins were SP:SAMPLEPREP_SUMMARY pelleted by centrifugation (15,000 rpm, 15 min), and extracted metabolites SP:SAMPLEPREP_SUMMARY were dried down in vacuo and stored at −80°C. Individual samples were SP:SAMPLEPREP_SUMMARY reconstituted in 100 μL of reconstitution buffer (acetonitrile/water, 90:10, SP:SAMPLEPREP_SUMMARY vol/vol) containing tryptophan-D3 and inosine-4N15. Equal volumes of individual SP:SAMPLEPREP_SUMMARY samples were pooled to create a quality control (QC) pooled sample used for SP:SAMPLEPREP_SUMMARY column conditioning, retention time alignment, to assess instrument SP:SAMPLEPREP_SUMMARY reproducibility, and for batch acceptance. SP:PROCESSING_STORAGE_CONDITIONS -80℃ SP:EXTRACT_STORAGE -80℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Metabolite extracts were separated on ACQUITY UPLC BEH Amide HILIC 1.7 μm, CH:CHROMATOGRAPHY_SUMMARY 2.1 × 100 mm column (Waters Corporation, Milford, MA) held at 30°C. CH:CHROMATOGRAPHY_SUMMARY Liquid chromatography was performed at a 200 μL min using solvent A (5 mM CH:CHROMATOGRAPHY_SUMMARY Ammonium formate in 90% water, 10% acetonitrile, and 0.1% formic acid) and CH:CHROMATOGRAPHY_SUMMARY solvent B (5 mM Ammonium formate in 90% acetonitrile, 10% water, and 0.1% CH:CHROMATOGRAPHY_SUMMARY formic acid) with a gradient length of 30 min. CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Vanquish UHPLC CH:COLUMN_NAME ACQUITY UPLC BEH Amide HILIC CH:SOLVENT_A 90% water/10% acetonitrile; 5mM ammonium formate; 0.1% formic acid CH:SOLVENT_B 10% water/90% acetonitrile; 5mM ammonium formate; 0.1% formic acid CH:FLOW_GRADIENT Linear gradient of 30 min CH:FLOW_RATE 0.20mL/min CH:COLUMN_TEMPERATURE 30 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive HF hybrid Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS The acquired raw data were imported, processed, normalized and reviewed using MS:MS_COMMENTS Progenesis QI v.3.0 (Non-linear Dynamics, Newcastle, UK). All MS and MS/MS MS:MS_COMMENTS sample runs were aligned against a QC pool reference run, and unique ions MS:MS_COMMENTS (retention time and m/z pairs) were deadducted and deisotoped to generate unique MS:MS_COMMENTS "features" (retention time and m/z pairs). Data were normalized to all features MS:MS_COMMENTS using Progenesis QI. Experimental data annotations were assigned based on MS:MS_COMMENTS consistent retention time and MS2 fragmentation pattern matches with reference MS:MS_COMMENTS standards. MS:MS_RESULTS_FILE ST002891_AN004750_Results.txt UNITS:time_m/z Has m/z:Yes Has RT:Yes RT units:Minutes #END