#METABOLOMICS WORKBENCH mikagm_20230516_132600 DATATRACK_ID:4029 STUDY_ID:ST002706 ANALYSIS_ID:AN004387 PROJECT_ID:PR001677 VERSION 1 CREATED_ON May 18, 2023, 11:24 am #PROJECT PR:PROJECT_TITLE Changes in the plasma metabolomics en in the muscle-specific rescue of Bmal1 PR:PROJECT_SUMMARY This study examines the systemic effects of muscle-specific Bmal1 expression in PR:PROJECT_SUMMARY the Bmal1-KO mouse model. We used an adeno-associated virus to rescue the PR:PROJECT_SUMMARY expression of the clock gene Bmal1 in skeletal muscle of the Bmal1 KO. PR:INSTITUTE University of Florida PR:DEPARTMENT Department of Physiology and Aging PR:LAST_NAME Esser PR:FIRST_NAME Karyn PR:ADDRESS 1345 Center Drive, M552, Gainesville, Florida, 32610, USA PR:EMAIL kaesser@ufl.edu PR:PHONE 352-273-5728 #STUDY ST:STUDY_TITLE Plasma metabolomics of Bmal1-KO and Bmal1-KO+AAV ST:STUDY_SUMMARY Disruption of the circadian clock in skeletal muscle worsens local and systemic ST:STUDY_SUMMARY health, leading to decreased muscle strength, metabolic dysfunction, and ST:STUDY_SUMMARY aging-like phenotypes. Whole-body knockout mice that lack Bmal1, a key component ST:STUDY_SUMMARY of the molecular clock, display premature aging. Here, by using adeno-associated ST:STUDY_SUMMARY viruses, we rescued Bmal1 expression specifically in the skeletal muscle of ST:STUDY_SUMMARY Bmal1-KO mice and found that this improves their healthspan and lifespan. Plasma ST:STUDY_SUMMARY samples from 40-week-old KO and KO+AAV male mice were collected at ZT1 to ST:STUDY_SUMMARY characterize the systemic effects of muscle-specific expression of Bmal1. ST:STUDY_SUMMARY Overall, our findings highlight the critical role of skeletal muscle in systemic ST:STUDY_SUMMARY health. ST:INSTITUTE University of Florida ST:LAST_NAME Esser ST:FIRST_NAME Karyn ST:ADDRESS 1345 Center Drive, M552, Gainesville, Florida, 32610, USA ST:EMAIL kaesser@ufl.edu ST:PHONE 352-273-5728 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN B6.129-Arntltm1Bra/J SU:AGE_OR_AGE_RANGE 40 weeks SU:GENDER Male SU:ANIMAL_FEED Ad libitum SU:ANIMAL_WATER Ad libitum #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 26 PL5 Genotype:Bmal1-KO | Treatment:Control RAW_FILE_NAME=QE2_jdg_341_Esser_29[PL5]p.mzXML RAW_FILE_NAME=QE2_jdg_341_Esser_29[PL5]n.mzXML SUBJECT_SAMPLE_FACTORS 238 PL6 Genotype:Bmal1-KO | Treatment:Control RAW_FILE_NAME=QE2_jdg_341_Esser_30[PL6]p.mzXML RAW_FILE_NAME=QE2_jdg_341_Esser_30[PL6]n.mzXML SUBJECT_SAMPLE_FACTORS 205 PL7 Genotype:Bmal1-KO | Treatment:Control RAW_FILE_NAME=QE2_jdg_341_Esser_31[PL7]p.mzXML RAW_FILE_NAME=QE2_jdg_341_Esser_31[PL7]n.mzXML SUBJECT_SAMPLE_FACTORS 247 PL8 Genotype:Bmal1-KO | Treatment:Control RAW_FILE_NAME=QE2_jdg_341_Esser_32[PL8]p.mzXML RAW_FILE_NAME=QE2_jdg_341_Esser_32[PL8]n.mzXML SUBJECT_SAMPLE_FACTORS 82 PL9 Genotype:Bmal1-KO | Treatment:AAV RAW_FILE_NAME=QE2_jdg_341_Esser_33[PL9]p.mzXML RAW_FILE_NAME=QE2_jdg_341_Esser_33[PL9]n.mzXML SUBJECT_SAMPLE_FACTORS 143 PL10 Genotype:Bmal1-KO | Treatment:AAV RAW_FILE_NAME=QE2_jdg_341_Esser_34[PL10]p.mzXML RAW_FILE_NAME=QE2_jdg_341_Esser_34[PL10]n.mzXML SUBJECT_SAMPLE_FACTORS 144 PL11 Genotype:Bmal1-KO | Treatment:AAV RAW_FILE_NAME=QE2_jdg_341_Esser_35[PL11]p.mzXML RAW_FILE_NAME=QE2_jdg_341_Esser_35[PL11]n.mzXML SUBJECT_SAMPLE_FACTORS 146 PL12 Genotype:Bmal1-KO | Treatment:AAV RAW_FILE_NAME=QE2_jdg_341_Esser_36[PL12]p.mzXML RAW_FILE_NAME=QE2_jdg_341_Esser_36[PL12]n.mzXML SUBJECT_SAMPLE_FACTORS Pool1 Pool1 Genotype:Bmal1-KO | Treatment:Control RAW_FILE_NAME=QE2_jdg_341_Esser_1[Pool_8]p.mzXML RAW_FILE_NAME=QE2_jdg_341_Esser_1[Pool_8]n.mzXML SUBJECT_SAMPLE_FACTORS Pool2 Pool2 Genotype:Bmal1-KO | Treatment:AAV RAW_FILE_NAME=QE2_jdg_341_Esser_1[Pool_9]p.mzXML RAW_FILE_NAME=QE2_jdg_341_Esser_1[Pool_9]n.mzXML #COLLECTION CO:COLLECTION_SUMMARY Mice were anesthetized using isofluorane. Blood was collected by intracardiac CO:COLLECTION_SUMMARY puncture. EDTA was used as anticoagulant. Blood was centrifuged at 2,000xg for CO:COLLECTION_SUMMARY 15 mins and plasma was collected and subsequently frozen in liquid nitrogen. CO:COLLECTION_SUMMARY Samples are stored at -80°C. CO:SAMPLE_TYPE Blood (plasma) CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY Bmal1-KO+AAV mice were systemically infected in the subxiphoid region with 20μl TR:TREATMENT_SUMMARY containing 2 × 10^11 genome copies of AAV9 on postnatal day 5. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY All provided samples were extracted following our cellular extraction procedure SP:SAMPLEPREP_SUMMARY with pre-normalization to the sample protein content. Each extract was spiked SP:SAMPLEPREP_SUMMARY with 1 ul of standard mixture consisting of deuterium labeled carnitine, SP:SAMPLEPREP_SUMMARY acetylcarnitine, propionylcarnitine, butyrylcarnitine, isovalerylcarnitine, SP:SAMPLEPREP_SUMMARY octanoylcarnitine, myristoylcarnitine and palmitoylcarnitine to targeted SP:SAMPLEPREP_SUMMARY identification of carnitines in the sample. Metabolite extraction was done by SP:SAMPLEPREP_SUMMARY protein precipitation using 200 µl of 8:1:1 Acetonitrile: Methanol: Acetone SP:SAMPLEPREP_SUMMARY with 0.1% formic acid. Further protein precipitation was allowed by incubating SP:SAMPLEPREP_SUMMARY the samples at 4°C for 20 min. Samples were placed in an ultrasonic bath for 10 SP:SAMPLEPREP_SUMMARY min and then centrifuged at 20 000 xg for 5min at 4°C to pellet the protein. SP:SAMPLEPREP_SUMMARY 190 µl supernatant was transferred from each sample into clean tube and dried SP:SAMPLEPREP_SUMMARY under a gentle stream of nitrogen at 30°C. The dried extracts were re-suspended SP:SAMPLEPREP_SUMMARY with 25 µL water with 0.1% formic acid. Resuspension was allowed at 4°C for 10 SP:SAMPLEPREP_SUMMARY -15 min then samples were centrifuged at 20000 xg for 5 min at 4°C. SP:SAMPLEPREP_SUMMARY Supernatants were transferred into clean LC-vials for targeted LC-MS SP:SAMPLEPREP_SUMMARY quantitation on a Thermo Q-Exactive Oribtrap mass spectrometer with Dionex UHPLC SP:SAMPLEPREP_SUMMARY and autosampler. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Global metabolomics profiling was performed on a Thermo Q-Exactive Oribtrap mass CH:CHROMATOGRAPHY_SUMMARY spectrometer with Dionex UHPLC and autosampler. Each extract was spiked with 1 CH:CHROMATOGRAPHY_SUMMARY ul of standard mixture consisting of deuterium labeled carnitine, CH:CHROMATOGRAPHY_SUMMARY acetylcarnitine, propionylcarnitine, butyrylcarnitine, isovalerylcarnitine, CH:CHROMATOGRAPHY_SUMMARY octanoylcarnitine, myristoylcarnitine and palmitoylcarnitine to targeted CH:CHROMATOGRAPHY_SUMMARY identification of carnitines in the sample. All samples were analyzed in CH:CHROMATOGRAPHY_SUMMARY positive and negative heated electrospray ionization with a mass resolution of CH:CHROMATOGRAPHY_SUMMARY 35,000 at m/z 200 as separate injections. Separation was achieved on an ACE 2 CH:CHROMATOGRAPHY_SUMMARY Excel-C18 pfp, 100x2.1mm, 2um column with mobile phase A as 0.1% formic acid in CH:CHROMATOGRAPHY_SUMMARY water and mobile phase B as acetonitrile. This is a polar embedded stationary CH:CHROMATOGRAPHY_SUMMARY phase that provides comprehensive coverage, but does have some limitation is the CH:CHROMATOGRAPHY_SUMMARY coverage of very polar species. The flow rate was 350 µL/min with a column CH:CHROMATOGRAPHY_SUMMARY temperature of 25°C. 4 µL was injected for negative ions and 2 µL for CH:CHROMATOGRAPHY_SUMMARY positive ions. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Dionex CH:COLUMN_NAME ACE 5 C18-300 (100 x 2.1mm) CH:SOLVENT_A 100% water; 0.1% formic acid CH:SOLVENT_B 100% acetonitrile CH:SOLVENT_C - CH:FLOW_GRADIENT - CH:FLOW_RATE 350 µL/min CH:COLUMN_TEMPERATURE 25°C #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS All samples were analyzed in positive and negative heated electrospray MS:MS_COMMENTS ionization with a mass resolution of 35,000 at m/z 200 as separate injections. MS:MS_COMMENTS Separation was achieved on an ACE 18-pfp 100 x 2.1 mm, 2 µm column with mobile MS:MS_COMMENTS phase A as 0.1% formic acid in water and mobile phase B as acetonitrile. This is MS:MS_COMMENTS a polar embedded stationary phase that provides comprehensive coverage, but does MS:MS_COMMENTS have some limitation is the coverage of very polar species. The flow rate was MS:MS_COMMENTS 350 µL/min with a column temperature of 25°C. 4 µL was injected for negative MS:MS_COMMENTS ions and 2 µL for positive ions. MS:MS_RESULTS_FILE ST002706_AN004387_Results.txt UNITS:Peak height Has m/z:Yes Has RT:Yes RT units:Minutes #END