#METABOLOMICS WORKBENCH hormel101_20170623_145343 DATATRACK_ID:1099 STUDY_ID:ST000650 ANALYSIS_ID:AN000982 PROJECT_ID:PR000460 VERSION 1 CREATED_ON June 26, 2017, 8:16 am #PROJECT PR:PROJECT_TITLE The dystrophic muscle metabolome: effects of exercise and NO donor therapy PR:PROJECT_SUMMARY "In Duchenne and Becker muscular dystrophy (DMD, BMD), loss of the cytoskeletal PR:PROJECT_SUMMARY protein dystrophin weakens the sarcolemma and disrupts cellular signaling, PR:PROJECT_SUMMARY rendering the diseased muscles susceptible to contractioninduced damage. We and PR:PROJECT_SUMMARY others have shown that loss of neuronal nitric oxide synthase (nNOSμ) from the PR:PROJECT_SUMMARY sarcolemma of dystrophin-deficient muscle causes functional muscle ischemia PR:PROJECT_SUMMARY during exercise due to unopposed sympathetic vasoconstriction, thereby PR:PROJECT_SUMMARY exacerbating fatigue and injury of the diseased muscles. Genetic and PR:PROJECT_SUMMARY pharmacologic strategies targeting nNOSμ-NO signaling ameliorate functional PR:PROJECT_SUMMARY muscle ischemia, as well as many other features of the dystrophic phenotype in PR:PROJECT_SUMMARY the mdx mouse model of DMD/BMD. These findings suggest that the therapeutic PR:PROJECT_SUMMARY benefit of NO likely extends beyond its vascular effects. A growing body of PR:PROJECT_SUMMARY evidence indicates that NO directly influences muscle metabolism through effects PR:PROJECT_SUMMARY on glucose transport as well as mitochondrial biogenesis and function. Both PR:PROJECT_SUMMARY nNOS-/- mice and mdx mice exhibit muscle mitochondrial dysfunction, decreased PR:PROJECT_SUMMARY resistance to fatigue, and exercise-induced muscle injury, suggesting a causal PR:PROJECT_SUMMARY role of nNOSμ-NO deficiency. However, the specific metabolic changes resulting PR:PROJECT_SUMMARY from reduced NO signaling that might render dystrophic muscle susceptible to PR:PROJECT_SUMMARY fatigue and use-dependent injury remain poorly defined. Therefore, the goal of PR:PROJECT_SUMMARY this pilot metabolomics study is to identify the unique biochemical profiles of PR:PROJECT_SUMMARY skeletal and cardiac muscles of mdx mice to gain further mechanistic insight PR:PROJECT_SUMMARY into the pathophysiological role of NO deficiency in muscular dystrophy. In Aim PR:PROJECT_SUMMARY 1, we will characterize the skeletal and cardiac muscle metabolomes of mdx and PR:PROJECT_SUMMARY nNOS-/- mice at rest and following a single bout of treadmill exercise with the PR:PROJECT_SUMMARY goal of discovering common metabolic signatures caused by loss of NO signaling. PR:PROJECT_SUMMARY In Aim 2, we will evaluate the potential of a NO donor drug that is under PR:PROJECT_SUMMARY development as a therapeutic for DMD/BMD to improve the skeletal and cardiac PR:PROJECT_SUMMARY muscle metabolomes in mdx mice. As a result of this pilot study, we hope to gain PR:PROJECT_SUMMARY new understanding of the metabolic derangements in dystrophin-deficient muscle, PR:PROJECT_SUMMARY insight into the therapeutic effects of NO replacement, and to identify new PR:PROJECT_SUMMARY pathogenic mechanisms and putative therapeutic targets that will form the basis PR:PROJECT_SUMMARY of future grant applications." PR:INSTITUTE Mayo Clinic PR:LAST_NAME Thomas PR:FIRST_NAME Gail PR:ADDRESS Penn State Hershey Heart and Vascular Institute Penn State College of Medicine PR:ADDRESS 500 University Drive, MC H047 Hershey, PA 17033 PR:EMAIL gthomas4@hmc.psu.edu PR:PHONE 717-531-0003, ext. 287087 #STUDY ST:STUDY_TITLE Effects of NO Donor Therapy on the Dystrophic Mouse Muscle TCA Cycle (part VI) ST:STUDY_SUMMARY For this aim, we will only use male mdx mice. We will study three groups treated ST:STUDY_SUMMARY for 7 days with vehicle, naproxcinod (i.e., NO-naproxen), or naproxen (n = 10 ST:STUDY_SUMMARY each group). Two hours after the final treatment, half the mice in each group ST:STUDY_SUMMARY will be run to exhaustion on a treadmill. The heart and gastrocnemius, soleus, ST:STUDY_SUMMARY and quadriceps muscles of one hindlimb will be sent to the Mayo Clinic ST:STUDY_SUMMARY Metabolomics Resource Core. The heart and quadriceps muscle will be used for ST:STUDY_SUMMARY untargeted metabolomics profiling (LC/MS) while the gastrocnemius and soleus ST:STUDY_SUMMARY muscles will be used for targeted analyses of amino acids plus amino ST:STUDY_SUMMARY metabolites, non-esterified fatty acids, and citric acid cycle intermediates. ST:INSTITUTE Mayo Clinic ST:LAST_NAME Thomas ST:FIRST_NAME Gail ST:ADDRESS Penn State Hershey Heart and Vascular Institute Penn State College of Medicine ST:ADDRESS 500 University Drive, MC H047 Hershey, PA 17033 ST:EMAIL gthomas4@hmc.psu.edu ST:PHONE 717-531-0003, ext. 287087 #SUBJECT SU:SUBJECT_TYPE Mouse 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 - Sample # 1 Time point:Sedentary | Grouping:SED-Naproxcinod Date of study=9/23/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - Sample # 2 Time point:Sedentary | Grouping:SED-Naproxcinod Date of study=9/24/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - Sample # 3 Time point:Sedentary | Grouping:SED-Naproxcinod Date of study=10/1/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - Sample # 4 Time point:Sedentary | Grouping:SED-Naproxcinod Date of study=10/7/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - Sample # 5 Time point:Sedentary | Grouping:SED-Naproxcinod Date of study=10/9/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - Sample # 6 Time point:Post Run | Grouping:RUN-Naproxcinod Date of study=9/23/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - Sample # 7 Time point:Post Run | Grouping:RUN-Naproxcinod Date of study=9/24/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - Sample # 8 Time point:Post Run | Grouping:RUN-Naproxcinod Date of study=10/1/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - Sample # 9 Time point:Post Run | Grouping:RUN-Naproxcinod Date of study=10/7/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - Sample # 10 Time point:Post Run | Grouping:RUN-Naproxcinod Date of study=10/9/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - Sample # 11 Time point:Sedentary | Grouping:SED-Naproxen Date of study=9/23/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - Sample # 12 Time point:Sedentary | Grouping:SED-Naproxen Date of study=9/24/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - Sample # 13 Time point:Sedentary | Grouping:SED-Naproxen Date of study=10/1/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - Sample # 14 Time point:Sedentary | Grouping:SED-Naproxen Date of study=10/7/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - Sample # 15 Time point:Sedentary | Grouping:SED-Naproxen Date of study=10/9/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - Sample # 16 Time point:Post Run | Grouping:RUN-Naproxen Date of study=9/23/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - Sample # 17 Time point:Post Run | Grouping:RUN-Naproxen Date of study=9/24/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - Sample # 18 Time point:Post Run | Grouping:RUN-Naproxen Date of study=10/1/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - Sample # 19 Time point:Post Run | Grouping:RUN-Naproxen Date of study=10/7/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - Sample # 20 Time point:Post Run | Grouping:RUN-Naproxen Date of study=10/9/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - Sample # 21 Time point:Sedentary | Grouping:SED-Vehicle Date of study=9/23/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - Sample # 22 Time point:Sedentary | Grouping:SED-Vehicle Date of study=9/24/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - Sample # 23 Time point:Sedentary | Grouping:SED-Vehicle Date of study=10/1/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - Sample # 24 Time point:Sedentary | Grouping:SED-Vehicle Date of study=10/7/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - Sample # 25 Time point:Sedentary | Grouping:SED-Vehicle Date of study=10/9/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - Sample # 26 Time point:Post Run | Grouping:RUN-Vehicle Date of study=9/23/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - Sample # 27 Time point:Post Run | Grouping:RUN-Vehicle Date of study=9/24/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - Sample # 28 Time point:Post Run | Grouping:RUN-Vehicle Date of study=10/1/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - Sample # 29 Time point:Post Run | Grouping:RUN-Vehicle Date of study=10/7/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - Sample # 30 Time point:Post Run | Grouping:RUN-Vehicle Date of study=10/9/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse #COLLECTION CO:COLLECTION_SUMMARY Blood and Tissue Harvesting: Mice will be euthanized and blood will be taken by CO:COLLECTION_SUMMARY cardiac puncture, centrifuged, and the plasma will be stored at -80ºC. The CO:COLLECTION_SUMMARY heart, diaphragm, and muscles of both hindlimbs (gastrocnemius, soleus, CO:COLLECTION_SUMMARY quadriceps) will be dissected and snap frozen in liquid nitrogen. Tibialis CO:COLLECTION_SUMMARY anterior and plantaris muscles from both hindlimbs also will be dissected and CO:COLLECTION_SUMMARY one set will be snap frozen in liquid nitrogen while the other set will be CO:COLLECTION_SUMMARY mounted in OCT and frozen in isopentane cooled by liquid nitrogen. Cryosections CO:COLLECTION_SUMMARY will be used to evaluate disease activity and exercise-induced muscle injury by CO:COLLECTION_SUMMARY staining with: (a) hematoxylin and eosin to assess gross morphology, cellular CO:COLLECTION_SUMMARY infiltration, and necrosis, (b) anti-F4/80 to label macrophages, and (c) CO:COLLECTION_SUMMARY anti-IgG or IgM to label damaged muscle fibers. #TREATMENT TR:TREATMENT_SUMMARY "Studies will be performed in 12-16 week old male mdx mice, C57BL10 control TR:TREATMENT_SUMMARY mice, and nNOS-/- mice obtained from Jackson Laboratory. All protocols will be TR:TREATMENT_SUMMARY approved by the Penn State College of Medicine Institutional Animal Care and Use TR:TREATMENT_SUMMARY Committee. Drug Treatment: Mice will be treated with vehicle, naproxcinod (20 TR:TREATMENT_SUMMARY mg/kg/day) or equimolar naproxen (12.5mg/kg/day) once a day for 7 consecutive TR:TREATMENT_SUMMARY days. To avoid the stress of oral gavage or ip injections in mdx mice, drug or TR:TREATMENT_SUMMARY vehicle will be administered in a small volume of peanut butter. Most mice TR:TREATMENT_SUMMARY readily consume the dosed peanut butter within 30 min. Terminal experiments will TR:TREATMENT_SUMMARY be performed 2 hours after the final dose is consumed." #SAMPLEPREP SP:SAMPLEPREP_SUMMARY mouse gastrocnemius TCA cyle #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE GC CH:INSTRUMENT_NAME Agilent 7890B CH:COLUMN_NAME Agilent HP5-MS (30m × 0.25mm, 0.25 um) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME Agilent 5977A MS:INSTRUMENT_TYPE Single quadrupole MS:MS_TYPE EI MS:ION_MODE POSITIVE #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS picomoles/mg MS_METABOLITE_DATA_START Samples Sample # 1 Sample # 2 Sample # 3 Sample # 4 Sample # 5 Sample # 6 Sample # 7 Sample # 8 Sample # 9 Sample # 10 Sample # 11 Sample # 12 Sample # 13 Sample # 14 Sample # 15 Sample # 16 Sample # 17 Sample # 18 Sample # 19 Sample # 20 Sample # 21 Sample # 22 Sample # 23 Sample # 24 Sample # 25 Sample # 26 Sample # 27 Sample # 28 Sample # 29 Sample # 30 Factors Time point:Sedentary | Grouping:SED-Naproxcinod Time point:Sedentary | Grouping:SED-Naproxcinod Time point:Sedentary | Grouping:SED-Naproxcinod Time point:Sedentary | Grouping:SED-Naproxcinod Time point:Sedentary | Grouping:SED-Naproxcinod Time point:Post Run | Grouping:RUN-Naproxcinod Time point:Post Run | Grouping:RUN-Naproxcinod Time point:Post Run | Grouping:RUN-Naproxcinod Time point:Post Run | Grouping:RUN-Naproxcinod Time point:Post Run | Grouping:RUN-Naproxcinod Time point:Sedentary | Grouping:SED-Naproxen Time point:Sedentary | Grouping:SED-Naproxen Time point:Sedentary | Grouping:SED-Naproxen Time point:Sedentary | Grouping:SED-Naproxen Time point:Sedentary | Grouping:SED-Naproxen Time point:Post Run | Grouping:RUN-Naproxen Time point:Post Run | Grouping:RUN-Naproxen Time point:Post Run | Grouping:RUN-Naproxen Time point:Post Run | Grouping:RUN-Naproxen Time point:Post Run | Grouping:RUN-Naproxen Time point:Sedentary | Grouping:SED-Vehicle Time point:Sedentary | Grouping:SED-Vehicle Time point:Sedentary | Grouping:SED-Vehicle Time point:Sedentary | Grouping:SED-Vehicle Time point:Sedentary | Grouping:SED-Vehicle Time point:Post Run | Grouping:RUN-Vehicle Time point:Post Run | Grouping:RUN-Vehicle Time point:Post Run | Grouping:RUN-Vehicle Time point:Post Run | Grouping:RUN-Vehicle Time point:Post Run | Grouping:RUN-Vehicle Lactate 14596.64511 16420.75983 15126.27525 16619.35266 19997.61534 19556.71475 22035.84166 15516.69709 18657.12426 13890.31759 18515.1742 20781.34894 21125.14994 14804.14821 26545.65113 8987.286032 20955.02826 14359.45038 19600.56498 18546.67448 19987.22389 14795.49088 14004.70925 23941.41968 13768.44387 19809.28376 15756.93677 14659.87655 18097.78279 16134.22994 Succinate 44.01633422 44.63672492 24.4127468 34.4418587 39.18839369 45.84325129 52.52413725 50.95848631 58.41268935 27.29246514 27.42818412 29.39479483 29.83524329 16.81996589 56.58251072 16.02763062 50.5412443 22.79770221 33.68454134 30.17677559 35.47640262 19.35292991 37.61946861 57.17342453 20.06497305 29.9802799 16.7974661 32.30513499 35.64087901 62.26259665 Fumarate 127.2430232 154.1698097 152.1796124 114.4404551 144.8785357 203.3937752 239.7540176 211.8967311 173.2402948 103.9920769 172.6543471 140.846423 154.4585281 131.1940718 144.0891663 154.3924845 192.3715068 148.6130466 210.0063672 207.2661194 155.4493438 157.9170417 108.0915115 197.3208578 147.5172929 195.1305304 166.2379325 142.1466807 209.891747 150.4474527 Oxaloacetate 0.302156462 0.331549227 0.213636957 0.232613134 0.216575598 0.284003421 0.286940844 0.331851737 0.324075865 0.327793732 0.276883479 0.36578117 0.328351776 0.339625445 0.269680298 0.226843447 0.272980568 0.250027015 0.192654768 0.248192886 0.299789789 0.354374226 0.327781903 0.327422014 0.332158878 0.310166521 0.284072916 0.412354038 0.475129383 0.445001851 Ketoglutarate 4.058147269 0.888284681 0.337554008 0.244860583 0.183112618 0.125413665 0.150015532 0.20247586 0.165656685 0.182440629 0.118767281 0.114767885 0.140505008 0.143395016 0.118948331 0.097545059 0.187915903 0.108669324 0.097815555 0.168506967 0.168039891 0.125142349 0.072906121 0.359036879 0.101943355 0.154594965 0.158347176 0.099390884 0.189123609 0.132391065 Malate 114.6728535 139.3540793 134.6312574 102.1387162 127.2927452 178.5011073 210.1390342 186.4425887 152.465831 96.45748433 153.9469339 125.3246053 137.2693175 115.045303 125.7996698 140.0564758 170.4749068 133.6776947 185.9337925 185.6666698 137.7935408 140.4353031 95.55244955 175.036296 132.3052911 173.9270855 136.757557 125.7796362 186.6916021 132.601348 2-Hydroxyglutarate 5.772210803 4.896599217 6.833668806 8.611665194 5.67520246 7.037465781 6.140841289 5.548828021 5.187760446 4.018313441 4.471823337 3.777857881 3.92415097 4.287781888 4.738517965 3.849364655 5.694905974 4.406578802 5.534862731 8.495301408 5.794653003 4.974903909 3.53283756 7.943746858 4.909560917 5.871592884 5.333829761 5.376779685 6.479829786 8.82091876 cis-Aconitic Acid 1.863194344 1.332655822 1.534438887 1.739507279 1.722795158 1.315756369 1.971697938 1.549910097 1.911311903 1.169436045 1.395744277 1.360608215 1.180386354 1.243824431 1.141821224 1.058867509 2.266932166 1.500296393 1.27310081 1.75128231 1.665329161 1.803913611 0.666260104 2.55731557 1.077358542 2.31685421 1.278538044 0.775829275 2.610668773 1.621287821 Citrate 65.13873867 54.8124167 79.5190848 85.11545593 87.63940273 77.62639995 103.4953624 70.80183789 119.3361431 72.68088998 77.95616282 68.75581453 67.49675906 72.89367102 51.05610849 58.4899187 115.4305464 88.50698182 75.90352169 98.283215 90.4882571 84.38743472 37.78458597 132.1414579 62.94466182 100.4218888 82.736994 64.96383015 120.4373076 81.62003565 Isocitrate 10.83051153 9.174140486 10.9528954 11.36902688 12.38726008 8.682194076 10.36972558 7.751696903 12.39787639 8.501532609 10.44417435 9.493869484 6.93364463 7.660011787 7.182636151 4.052252012 12.72283046 6.582370659 8.044852403 6.385482631 10.33120857 8.87693766 4.814773003 11.3367726 7.708240047 8.402129937 8.377875439 5.635094858 10.28049366 5.670032493 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Lactate Succinate Fumarate Oxaloacetate Ketoglutarate Malate 2-Hydroxyglutarate cis-Aconitic Acid Citrate Isocitrate METABOLITES_END #END