#METABOLOMICS WORKBENCH hormel101_20170623_142013 DATATRACK_ID:1095 STUDY_ID:ST000646 ANALYSIS_ID:AN000978 PROJECT_ID:PR000460 VERSION 1 CREATED_ON June 25, 2017, 10:27 pm #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 Exercise on Dystrophic Mouse Muscle TCA Cycle (part II) ST:STUDY_SUMMARY We will use male C57BL10, mdx, and nNOS-/- mice (n = 10 each group) to ST:STUDY_SUMMARY characterize the skeletal and cardiac muscle metabolomes. Half of the mice in ST:STUDY_SUMMARY each group will remain sedentary while the other half will be subjected to a ST:STUDY_SUMMARY single bout of treadmill exercise to exhaustion. Mice will be euthanized ST:STUDY_SUMMARY immediately postexercise and blood, hearts, and hindlimb muscles will be ST:STUDY_SUMMARY harvested and frozen as detailed in the General Methods. The heart and ST:STUDY_SUMMARY gastrocnemius, soleus, and quadriceps muscles of one hindlimb will be sent to ST:STUDY_SUMMARY the Mayo Clinic Metabolomics Resource Core. The heart and quadriceps muscle will ST:STUDY_SUMMARY be used for untargeted metabolomics profiling (LC/MS) while the gastrocnemius ST:STUDY_SUMMARY and soleus 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-BL10 Control Date of study=10/15/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - Sample # 2 Time point:Sedentary | Grouping:SED-BL10 Control Date of study=10/21/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - Sample # 3 Time point:Sedentary | Grouping:SED-BL10 Control Date of study=10/28/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - Sample # 4 Time point:Sedentary | Grouping:SED-BL10 Control Date of study=11/23/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - Sample # 5 Time point:Sedentary | Grouping:SED-BL10 Control Date of study=11/25/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - Sample # 6 Time point:Post Run | Grouping:RUN-BL10 Control Date of study=10/20/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - Sample # 7 Time point:Post Run | Grouping:RUN-BL10 Control Date of study=10/21/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - Sample # 8 Time point:Post Run | Grouping:RUN-BL10 Control Date of study=10/28/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - Sample # 9 Time point:Post Run | Grouping:RUN-BL10 Control Date of study=10/30/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - Sample # 10 Time point:Post Run | Grouping:RUN-BL10 Control Date of study=11/25/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - Sample # 11 Time point:Sedentary | Grouping:SED-nNOS-/- Date of study=10/20/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - Sample # 12 Time point:Sedentary | Grouping:SED-nNOS-/- Date of study=10/21/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - Sample # 13 Time point:Sedentary | Grouping:SED-nNOS-/- Date of study=10/28/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - Sample # 14 Time point:Sedentary | Grouping:SED-nNOS-/- Date of study=11/23/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - Sample # 15 Time point:Sedentary | Grouping:SED-nNOS-/- Date of study=11/25/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - Sample # 16 Time point:Post Run | Grouping:RUN-nNOS-/- Date of study=10/20/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - Sample # 17 Time point:Post Run | Grouping:RUN-nNOS-/- Date of study=10/21/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - Sample # 18 Time point:Post Run | Grouping:RUN-nNOS-/- Date of study=10/28/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - Sample # 19 Time point:Post Run | Grouping:RUN-nNOS-/- Date of study=10/30/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - Sample # 20 Time point:Post Run | Grouping:RUN-nNOS-/- Date of study=11/25/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - Sample # 21 Time point:Sedentary | Grouping:SED-MDX Date of study=10/20/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - Sample # 22 Time point:Sedentary | Grouping:SED-MDX Date of study=10/21/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - Sample # 23 Time point:Sedentary | Grouping:SED-MDX Date of study=10/28/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - Sample # 24 Time point:Sedentary | Grouping:SED-MDX Date of study=11/23/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - Sample # 25 Time point:Sedentary | Grouping:SED-MDX Date of study=11/25/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - Sample # 26 Time point:Post Run | Grouping:RUN-MDX Date of study=10/20/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - Sample # 27 Time point:Post Run | Grouping:RUN-MDX Date of study=10/21/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - Sample # 28 Time point:Post Run | Grouping:RUN-MDX Date of study=10/28/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - Sample # 29 Time point:Post Run | Grouping:RUN-MDX Date of study=10/30/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - Sample # 30 Time point:Post Run | Grouping:RUN-MDX Date of study=11/25/2015; 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. Treadmill Exercise: Mice will run on a horizontal treadmill (Columbus TR:TREATMENT_SUMMARY Instruments) to assess fatigue by a single bout of exercise beginning at 5 m/min TR:TREATMENT_SUMMARY for 5 min followed by 1 m/min increases every minute until exhaustion. Electric TR:TREATMENT_SUMMARY shocks will not be used to stimulate running due to adverse effects in mdx TR:TREATMENT_SUMMARY mice." #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-BL10 Control Time point:Sedentary | Grouping:SED-BL10 Control Time point:Sedentary | Grouping:SED-BL10 Control Time point:Sedentary | Grouping:SED-BL10 Control Time point:Sedentary | Grouping:SED-BL10 Control Time point:Post Run | Grouping:RUN-BL10 Control Time point:Post Run | Grouping:RUN-BL10 Control Time point:Post Run | Grouping:RUN-BL10 Control Time point:Post Run | Grouping:RUN-BL10 Control Time point:Post Run | Grouping:RUN-BL10 Control Time point:Sedentary | Grouping:SED-nNOS-/- Time point:Sedentary | Grouping:SED-nNOS-/- Time point:Sedentary | Grouping:SED-nNOS-/- Time point:Sedentary | Grouping:SED-nNOS-/- Time point:Sedentary | Grouping:SED-nNOS-/- Time point:Post Run | Grouping:RUN-nNOS-/- Time point:Post Run | Grouping:RUN-nNOS-/- Time point:Post Run | Grouping:RUN-nNOS-/- Time point:Post Run | Grouping:RUN-nNOS-/- Time point:Post Run | Grouping:RUN-nNOS-/- Time point:Sedentary | Grouping:SED-MDX Time point:Sedentary | Grouping:SED-MDX Time point:Sedentary | Grouping:SED-MDX Time point:Sedentary | Grouping:SED-MDX Time point:Sedentary | Grouping:SED-MDX Time point:Post Run | Grouping:RUN-MDX Time point:Post Run | Grouping:RUN-MDX Time point:Post Run | Grouping:RUN-MDX Time point:Post Run | Grouping:RUN-MDX Time point:Post Run | Grouping:RUN-MDX Lactate 10358.40135 18099.87815 27534.90164 25097.2316 21444.55287 22867.38542 20707.5253 15287.96799 18106.04902 16298.49928 19262.33368 19759.24374 23163.57869 24792.16946 11672.60064 13732.9151 25868.56413 25786.05567 24115.79757 24905.34794 27977.62551 21533.31653 23370.69513 26612.89851 21684.63449 5068.268496 23416.99172 19079.6297 26265.93739 19480.29653 Succinate 25.65655785 27.76144813 45.53193355 37.40222794 24.07134759 57.44245296 32.40434018 27.95453682 25.83042963 20.7825877 103.3513209 137.3074882 81.49403704 85.15243631 39.02943967 16.18837065 24.31764166 20.30329197 25.76379144 31.85200278 53.95416904 29.44772966 31.54302108 39.71790179 57.57692517 51.95156258 75.94781452 93.26670489 73.30778809 70.89726455 Fumarate 105.3583297 91.66034543 185.7250698 176.7970245 169.3109371 233.4383808 200.7031131 197.3342477 188.8511351 185.4474509 168.2891977 293.1817614 228.6576877 218.398001 245.6529505 129.7408534 192.1177648 192.739371 221.5516336 240.7221106 249.8093997 228.0725274 190.1391049 237.1451437 184.231402 104.3900971 265.8708398 157.2695535 329.4987419 231.9172851 Oxaloacetate 1.625699006 1.4345169 0.054036556 0.060925262 0.069702018 0.057128173 0.125571176 0.063307194 0.079233814 0.06515436 0.093984829 0.09252981 0.071869677 0.080989662 0.07964049 0.117773156 0.12424322 0.105165022 0.058977534 0.120693073 0.140543402 0.058280443 0.130981791 0.067246117 0.071677919 0.126456506 0.111388537 0.188366477 0.089574592 0.128693444 Ketoglutarate 122.5231502 2072.284675 415.8293863 551.3231198 356.9830057 455.4783861 478.6782018 505.4518766 568.3484433 512.7956343 543.7595695 1154.309239 757.8808722 634.416959 200.6392273 298.6614436 456.2904937 482.7413732 513.7855057 580.287782 640.3003313 697.2600798 786.539125 834.4742826 864.1337046 286.5013598 751.548193 631.4872238 709.6430939 898.74346 Malate 94.48081994 88.31162032 167.4269068 157.7417513 155.87326 207.7619764 183.6245335 177.4793506 169.2142551 166.9504018 147.4593335 153.5267947 197.1364506 190.5147943 223.0377634 128.0755059 176.6974321 176.598628 196.2711536 215.1908533 228.2568732 200.6909512 174.0243689 207.9315692 160.4421671 91.55280531 235.5348453 139.4965937 292.3671499 208.0828436 2-Hydroxyglutarate 5.581983851 6.564139628 14.10591061 11.58285607 8.574840948 14.84660006 8.059359276 9.103980968 6.668697694 6.927779954 4.846493135 15.25368253 4.904842453 5.866556098 6.479489648 2.227083962 4.126855571 3.946451721 11.25774319 7.775153568 4.022450332 7.981593543 4.618962594 5.993676523 4.71780954 7.897273741 7.679007245 5.541736347 8.161475497 5.059612066 cis-Aconitic Acid 0.797394814 0.720571036 4.108569454 1.624636708 1.039004631 2.718277211 3.367795751 4.231761308 2.339764443 2.474371077 2.607402528 50.76705292 2.838084211 1.369182297 2.400262444 0.75560976 1.12177817 1.696697387 1.525059947 1.975774987 0.978335414 1.772342749 3.327171016 1.821641017 1.031070836 0.591588073 1.850898401 1.656685767 2.037458067 1.926283446 Citrate 47.33506614 59.73370078 110.0035562 87.74720623 58.72781848 131.1123145 194.2775075 167.695557 148.5862948 118.5790062 77.97652904 88.86772426 75.09734058 67.35903967 149.062591 68.42623242 101.3936247 93.50569222 108.3381063 188.3628626 98.03319962 85.13984712 89.21740196 88.52474911 72.46859469 51.69049403 100.2484327 85.85122068 119.5554602 100.3362553 Isocitrate 2.949971216 13.8840156 12.49711068 9.258464937 6.880420885 5.954756809 7.551977526 7.550259902 7.444409282 6.4359181 10.12371838 52.08149694 13.71004603 11.16026568 5.099604045 3.467931138 5.714618768 5.695346911 5.065143087 9.187612697 11.62789988 11.79633784 12.50858322 12.56630102 11.90177384 3.965662503 11.05984724 10.5781714 9.816362313 10.64320098 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