#METABOLOMICS WORKBENCH Euphemia2023_20231218_070349 DATATRACK_ID:4534 STUDY_ID:ST003030 ANALYSIS_ID:AN004967 VERSION 1 CREATED_ON 01-11-2024 #PROJECT PR:PROJECT_TITLE A small molecule macrophage migration inhibitory factor agonist ameliorates PR:PROJECT_TITLE age-related myocardial intolerance to ischemia-reperfusion insults via metabolic PR:PROJECT_TITLE regulation PR:PROJECT_SUMMARY Macrophage migration inhibitory factor (MIF) is an innate cytokine that PR:PROJECT_SUMMARY regulates both inflammatory and homeostatic responses. MIF is expressed by PR:PROJECT_SUMMARY cardiomyocytes, where it exerts a protective action against ischemia-reperfusion PR:PROJECT_SUMMARY (I/R) injury by activating AMP-activated protein kinase (AMPK). This effect is PR:PROJECT_SUMMARY attenuated in the senescent heart due to an intrinsic, age-related reduction in PR:PROJECT_SUMMARY MIF expression. We hypothesized that treating the aged heart with the small PR:PROJECT_SUMMARY molecule MIF agonist (MIF20) can reinforce protective MIF signaling in PR:PROJECT_SUMMARY cardiomyocytes, leading to a beneficial effect against I/R stress. The PR:PROJECT_SUMMARY administration of MIF20 at the onset of reperfusion was found to not only PR:PROJECT_SUMMARY decrease myocardial infarct size but also preserves systolic function in the PR:PROJECT_SUMMARY aged heart. Protection from I/R injury was reduced in mice with PR:PROJECT_SUMMARY cardiomyocyte-specific Mif deletion, consistent with the mechanism of action of PR:PROJECT_SUMMARY MIF20 to allosterically increase MIF affinity for its cognate receptor CD74. We PR:PROJECT_SUMMARY further found MIF20 to contribute to the maintenance of mitochondrial fitness PR:PROJECT_SUMMARY and to preserve the contractile properties of aged cardiomyocytes under PR:PROJECT_SUMMARY hypoxia/reoxygenation. MIF20 augments protective metabolic responses by reducing PR:PROJECT_SUMMARY the NADH/NAD ratio, leading to a decrease in the accumulation of reactive oxygen PR:PROJECT_SUMMARY species (ROS) in the aged myocardium under I/R stress. We also identify PR:PROJECT_SUMMARY alterations in the expression levels of the downstream effectors PDK4 and LCAD, PR:PROJECT_SUMMARY which participate in the remodeling of the cardiac metabolic profile. Data from PR:PROJECT_SUMMARY this study demonstrates that pharmacologic augmentation of MIF signaling PR:PROJECT_SUMMARY provides beneficial homeostatic actions on senescent myocardium under I/R PR:PROJECT_SUMMARY stress. PR:INSTITUTE University of Mississippi Medical Center PR:LAST_NAME Li PR:FIRST_NAME Ji PR:ADDRESS 2500 N State St, Jackson, MS 39216-4505 PR:EMAIL jli3@umc.edu PR:PHONE 6018158995 PR:DOI http://dx.doi.org/10.21228/M89X4H #STUDY ST:STUDY_TITLE A small molecule macrophage migration inhibitory factor agonist ameliorates ST:STUDY_TITLE age-related myocardial intolerance to ischemia-reperfusion insults via metabolic ST:STUDY_TITLE regulation - Part 2 ST:STUDY_SUMMARY Macrophage migration inhibitory factor (MIF) is an innate cytokine that ST:STUDY_SUMMARY regulates both inflammatory and homeostatic responses. MIF is expressed by ST:STUDY_SUMMARY cardiomyocytes, where it exerts a protective action against ischemia-reperfusion ST:STUDY_SUMMARY (I/R) injury by activating AMP-activated protein kinase (AMPK). This effect is ST:STUDY_SUMMARY attenuated in the senescent heart due to an intrinsic, age-related reduction in ST:STUDY_SUMMARY MIF expression. We hypothesized that treating the aged heart with the small ST:STUDY_SUMMARY molecule MIF agonist (MIF20) can reinforce protective MIF signaling in ST:STUDY_SUMMARY cardiomyocytes, leading to a beneficial effect against I/R stress. The ST:STUDY_SUMMARY administration of MIF20 at the onset of reperfusion was found to not only ST:STUDY_SUMMARY decrease myocardial infarct size but also preserves systolic function in the ST:STUDY_SUMMARY aged heart. Protection from I/R injury was reduced in mice with ST:STUDY_SUMMARY cardiomyocyte-specific Mif deletion, consistent with the mechanism of action of ST:STUDY_SUMMARY MIF20 to allosterically increase MIF affinity for its cognate receptor CD74. We ST:STUDY_SUMMARY further found MIF20 to contribute to the maintenance of mitochondrial fitness ST:STUDY_SUMMARY and to preserve the contractile properties of aged cardiomyocytes under ST:STUDY_SUMMARY hypoxia/reoxygenation. MIF20 augments protective metabolic responses by reducing ST:STUDY_SUMMARY the NADH/NAD ratio, leading to a decrease in the accumulation of reactive oxygen ST:STUDY_SUMMARY species (ROS) in the aged myocardium under I/R stress. We also identify ST:STUDY_SUMMARY alterations in the expression levels of the downstream effectors PDK4 and LCAD, ST:STUDY_SUMMARY which participate in the remodeling of the cardiac metabolic profile. Data from ST:STUDY_SUMMARY this study demonstrates that pharmacologic augmentation of MIF signaling ST:STUDY_SUMMARY provides beneficial homeostatic actions on senescent myocardium under I/R ST:STUDY_SUMMARY stress. ST:INSTITUTE University of Mississippi Medical Center ST:LAST_NAME Li ST:FIRST_NAME Ji ST:ADDRESS 2500 N State St, Jackson, MS 39216-4505 ST:EMAIL jli3@umc.edu ST:PHONE 6018158995 ST:SUBMIT_DATE 2023-12-18 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - 7_19 Genotype:Aged-wildtype | Treatment:Aged I/R Weight=78 SUBJECT_SAMPLE_FACTORS - 7_20 Genotype:Aged-wildtype | Treatment:Aged I/R Weight=46 SUBJECT_SAMPLE_FACTORS - 7_21 Genotype:Aged-wildtype | Treatment:Aged I/R Weight=68 SUBJECT_SAMPLE_FACTORS - 8_22 Genotype:Aged-wildtype | Treatment:Aged I/R +MIF20 Weight=73 SUBJECT_SAMPLE_FACTORS - 8_23 Genotype:Aged-wildtype | Treatment:Aged I/R +MIF20 Weight=62 SUBJECT_SAMPLE_FACTORS - 8_24 Genotype:Aged-wildtype | Treatment:Aged I/R +MIF20 Weight=78 SUBJECT_SAMPLE_FACTORS - 5_13 Genotype:Aged-wildtype | Treatment:Sham aged Weight=80 SUBJECT_SAMPLE_FACTORS - 5_14 Genotype:Aged-wildtype | Treatment:Sham aged Weight=64 SUBJECT_SAMPLE_FACTORS - 5_15 Genotype:Aged-wildtype | Treatment:Sham aged Weight=58 SUBJECT_SAMPLE_FACTORS - 6_16 Genotype:Aged-wildtype | Treatment:Sham aged+MIF20 Weight=54 SUBJECT_SAMPLE_FACTORS - 6_17 Genotype:Aged-wildtype | Treatment:Sham aged+MIF20 Weight=65 SUBJECT_SAMPLE_FACTORS - 6_18 Genotype:Aged-wildtype | Treatment:Sham aged+MIF20 Weight=79 SUBJECT_SAMPLE_FACTORS - 1_1 Genotype:Young-wildtype | Treatment:Sham young Weight=77 SUBJECT_SAMPLE_FACTORS - 1_2 Genotype:Young-wildtype | Treatment:Sham young Weight=66 SUBJECT_SAMPLE_FACTORS - 1_3 Genotype:Young-wildtype | Treatment:Sham young Weight=80 SUBJECT_SAMPLE_FACTORS - 2_4 Genotype:Young-wildtype | Treatment:Sham young+MIF20 Weight=72 SUBJECT_SAMPLE_FACTORS - 2_5 Genotype:Young-wildtype | Treatment:Sham young+MIF20 Weight=45 SUBJECT_SAMPLE_FACTORS - 2_6 Genotype:Young-wildtype | Treatment:Sham young+MIF20 Weight=60 SUBJECT_SAMPLE_FACTORS - 3_7 Genotype:Young-wildtype | Treatment:Young I/R Weight=71 SUBJECT_SAMPLE_FACTORS - 3_8 Genotype:Young-wildtype | Treatment:Young I/R Weight=65 SUBJECT_SAMPLE_FACTORS - 3_9 Genotype:Young-wildtype | Treatment:Young I/R Weight=80 SUBJECT_SAMPLE_FACTORS - 4_10 Genotype:Young-wildtype | Treatment:Young I/R+MIF20 Weight=80 SUBJECT_SAMPLE_FACTORS - 4_11 Genotype:Young-wildtype | Treatment:Young I/R+MIF20 Weight=66 SUBJECT_SAMPLE_FACTORS - 4_12 Genotype:Young-wildtype | Treatment:Young I/R+MIF20 Weight=57 #COLLECTION CO:COLLECTION_SUMMARY The sample collection method was as follows: All mouse groups underwent CO:COLLECTION_SUMMARY anesthesia with 2%-3% isoflurane and 100% O2. The mouse hearts were excised and CO:COLLECTION_SUMMARY rinsed in ice-cold PBS. Both atriums were removed; only the left ventricular CO:COLLECTION_SUMMARY myocardium, including the infarct area, was collected and was quickly frozen by CO:COLLECTION_SUMMARY using liquid nitrogen. Samples were preserved below -80 °C and were sent out CO:COLLECTION_SUMMARY for metabolomic analysis. CO:SAMPLE_TYPE Heart #TREATMENT TR:TREATMENT_SUMMARY Sham young;Sham young+MIF20;Young I/R;Young I/R+MIF20;Sham aged;Sham TR:TREATMENT_SUMMARY aged+MIF20;Aged I/R;Aged I/R +MIF20 #SAMPLEPREP SP:SAMPLEPREP_SUMMARY The metabolomic sample preparation method was: The tissue samples were ground SP:SAMPLEPREP_SUMMARY into a homogenous powder with a Retsch Cryomill (Retsch-Allee, Haan, Germany). SP:SAMPLEPREP_SUMMARY 45 – 80 mg of the pulverized sample was weighed and stored in the -80 oC SP:SAMPLEPREP_SUMMARY freezer prior to extraction. Samples were allowed to thaw at 4 °C for 30 min SP:SAMPLEPREP_SUMMARY before adding 1.5 mL of 20:40:40 water/methanol/acetonitrile with 0.1 M formic SP:SAMPLEPREP_SUMMARY acid for metabolites extraction. The samples were vortexed and centrifuged and SP:SAMPLEPREP_SUMMARY the supernatants were isolated and dried completely under nitrogen. The dried SP:SAMPLEPREP_SUMMARY samples were resuspended in 300 µL water and an aliquot were transferred to SP:SAMPLEPREP_SUMMARY autosampler vials for UHPLC-HRMS analysis. Mass analysis was carried out at the SP:SAMPLEPREP_SUMMARY University of Tennessee Biological and Small Molecule Mass Spectrometry Core SP:SAMPLEPREP_SUMMARY (RRID: SCR_021368) using an UltiMate 3000 liquid chromatography system (Dionex, SP:SAMPLEPREP_SUMMARY Sunnyvale, CA, USA) coupled to an Exactive Plus Orbitrap mass spectrometer SP:SAMPLEPREP_SUMMARY (Thermo Fisher Scientific, Waltham, MA). #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Mass analysis was carried out at the University of Tennessee Biological and CH:CHROMATOGRAPHY_SUMMARY Small Molecule Mass Spectrometry Core (RRID: SCR_021368) using an UltiMate 3000 CH:CHROMATOGRAPHY_SUMMARY liquid chromatography system (Dionex, Sunnyvale, CA, USA) coupled to an Exactive CH:CHROMATOGRAPHY_SUMMARY Plus Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA). CH:CHROMATOGRAPHY_SUMMARY Metabolites were separated using a Synergi Hydro RP column (2.5 μm, 100 × 2.0 CH:CHROMATOGRAPHY_SUMMARY mm; Phenomenex, Torrance, CA, USA) using a reversed phase ion pairing CH:CHROMATOGRAPHY_SUMMARY chromatographic method. This method uses a tributylamine ion pairing reagent CH:CHROMATOGRAPHY_SUMMARY with a water: methanol solvent gradient elution within a 25-minute duration as CH:CHROMATOGRAPHY_SUMMARY reported previously (Bazurto et al, 2018: Bazurto JV, Dearth SP, Tague ED, CH:CHROMATOGRAPHY_SUMMARY Campagna SR, Downs DM. Untargeted metabolomics confirms and extends the CH:CHROMATOGRAPHY_SUMMARY understanding of the impact of aminoimidazole carboxamide ribotide (AICAR) in CH:CHROMATOGRAPHY_SUMMARY the metabolic network of Salmonella enterica. Microb Cell. 2017 Nov CH:CHROMATOGRAPHY_SUMMARY 22;5(2):74-87. doi: 10.15698/mic2018.02.613. PMID: 29417056; PMCID: CH:CHROMATOGRAPHY_SUMMARY PMC5798407.). CH:INSTRUMENT_NAME Thermo Dionex Ultimate 3000 CH:COLUMN_NAME Phenomenex Synergi Hydro-RP (100 x 2mm,2.5um) CH:COLUMN_TEMPERATURE 25 CH:FLOW_GRADIENT 0 to 5 min 0% B, from 5 to 13 min 20% B, from 13 to 15.5 min 55% B, from 15.5 to CH:FLOW_GRADIENT 19 min 95% B, and from 19 to 25 min 0% B CH:FLOW_RATE 200 µL/min CH:SOLVENT_A 97:3 LCMS grade water : methanol, 11 mM tributylamine, 15 mM acetic acid. CH:SOLVENT_B LCMS grade methanol CH:CHROMATOGRAPHY_TYPE Reversed phase #ANALYSIS AN:LABORATORY_NAME Biological Small Molecule Mass Spectrometry Core, Univ of Tenn, Knoxville AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Exactive Plus Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:MS_COMMENTS Mass analysis was carried out at the University of Tennessee Biological and MS:MS_COMMENTS Small Molecule Mass Spectrometry Core (RRID: SCR_021368) using an UltiMate 3000 MS:MS_COMMENTS liquid chromatography system (Dionex, Sunnyvale, CA, USA) coupled to an Exactive MS:MS_COMMENTS Plus Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA). MS:MS_COMMENTS Metabolites were separated using a Synergi Hydro RP column (2.5 μm, 100 × 2.0 MS:MS_COMMENTS mm; Phenomenex, Torrance, CA, USA) using a reversed phase ion pairing MS:MS_COMMENTS chromatographic method. This method uses a tributylamine ion pairing reagent MS:MS_COMMENTS with a water: methanol solvent gradient elution within a 25-minute duration as MS:MS_COMMENTS reported previously (Bazurto et al., 2018). All solvents used were LC-MS grade. MS:MS_COMMENTS The chromatographic gradient was from 0 to 5 min 0% B, from 5 to 13 min 20% B, MS:MS_COMMENTS from 13 to 15.5 min 55% B, from 15.5 to 19 min 95% B, and from 19 to 25 min 0% MS:MS_COMMENTS B. (ref to paper: (Bazurto et al, 2018)) The separated metabolites were ionized MS:MS_COMMENTS using electrospray ionization with negative polarity and the full scan mass MS:MS_COMMENTS analysis was performed with a resolution of 140,000 as previously reported. The MS:MS_COMMENTS Xcalibur (RAW) files generated from the UPLC-HRMS analysis were converted to the MS:MS_COMMENTS mzML format using the msconvert software to enable data centroiding. Metabolomic MS:MS_COMMENTS Analysis and Visualization Engine (MAVEN) was used to integrate the peak areas MS:MS_COMMENTS from the extracted ion chromatograms (EIC). Prior to identification and MS:MS_COMMENTS integration, MAVEN was used to perform a nonlinear retention time correction MS:MS_COMMENTS across all samples. Metabolites were identified by comparing chromatographic MS:MS_COMMENTS retention time and exact masses within ± 5 ppm mass tolerance to an in-house MS:MS_COMMENTS standard library. Identifications were validated using the natural abundance of MS:MS_COMMENTS isotopes in the compound. The integrated peak areas were used for further MS:MS_COMMENTS statistical and biological analysis. Unidentified spectral features were MS:MS_COMMENTS annotated and analyzed using online LC-MS raw data spectra processing features MS:MS_COMMENTS in MetaboAnalyst 5.0. MS:ION_MODE NEGATIVE MS:MS_RESULTS_FILE ST003030_AN004967_Results.txt UNITS:Peak area Has m/z:Yes Has RT:Yes RT units:Seconds #END