#METABOLOMICS WORKBENCH hormel101_20170623_143546 DATATRACK_ID:1096 STUDY_ID:ST000647 ANALYSIS_ID:AN000979 PROJECT_ID:PR000460 VERSION 1 CREATED_ON June 26, 2017, 7:59 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 Exercise on Dystrophic Mouse Muscle Non-Esterified Fatty Acids (part ST:STUDY_TITLE III) 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 - ms5944-1 Time point:Sedentary | Grouping:SED-BL10 Control Date of study=10/15/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - ms5944-2 Time point:Sedentary | Grouping:SED-BL10 Control Date of study=10/21/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - ms5944-3 Time point:Sedentary | Grouping:SED-BL10 Control Date of study=10/28/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - ms5944-4 Time point:Sedentary | Grouping:SED-BL10 Control Date of study=11/23/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - ms5944-5 Time point:Sedentary | Grouping:SED-BL10 Control Date of study=11/25/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - ms5944-6 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 - ms5944-7 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 - ms5944-8 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 - ms5944-9 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 - ms5944-10 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 - ms5944-11 Time point:Sedentary | Grouping:SED-nNOS-/- Date of study=10/20/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - ms5944-12 Time point:Sedentary | Grouping:SED-nNOS-/- Date of study=10/21/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - ms5944-13 Time point:Sedentary | Grouping:SED-nNOS-/- Date of study=10/28/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - ms5944-14 Time point:Sedentary | Grouping:SED-nNOS-/- Date of study=11/23/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - ms5944-15 Time point:Sedentary | Grouping:SED-nNOS-/- Date of study=11/25/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - ms5944-16 Time point:Post Run | Grouping:RUN-nNOS-/- Date of study=10/20/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - ms5944-17 Time point:Post Run | Grouping:RUN-nNOS-/- Date of study=10/21/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - ms5944-18 Time point:Post Run | Grouping:RUN-nNOS-/- Date of study=10/28/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - ms5944-19 Time point:Post Run | Grouping:RUN-nNOS-/- Date of study=10/30/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - ms5944-20 Time point:Post Run | Grouping:RUN-nNOS-/- Date of study=11/25/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - ms5944-21 Time point:Sedentary | Grouping:SED-MDX Date of study=10/20/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - ms5944-22 Time point:Sedentary | Grouping:SED-MDX Date of study=10/21/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - ms5944-23 Time point:Sedentary | Grouping:SED-MDX Date of study=10/28/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - ms5944-24 Time point:Sedentary | Grouping:SED-MDX Date of study=11/23/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - ms5944-25 Time point:Sedentary | Grouping:SED-MDX Date of study=11/25/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - ms5944-26 Time point:Post Run | Grouping:RUN-MDX Date of study=10/20/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - ms5944-27 Time point:Post Run | Grouping:RUN-MDX Date of study=10/21/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - ms5944-28 Time point:Post Run | Grouping:RUN-MDX Date of study=10/28/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - ms5944-29 Time point:Post Run | Grouping:RUN-MDX Date of study=10/30/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - ms5944-30 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 NEFA #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Waters Acquity CH:COLUMN_NAME Waters Acquity BEH C18 (150 x 2.1mm, 1.7um) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME Thermo Quantum Ultra MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE NEGATIVE #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS picomoles/mg MS_METABOLITE_DATA_START Samples ms5944-1 ms5944-2 ms5944-3 ms5944-4 ms5944-5 ms5944-6 ms5944-7 ms5944-8 ms5944-9 ms5944-10 ms5944-11 ms5944-12 ms5944-13 ms5944-14 ms5944-15 ms5944-16 ms5944-17 ms5944-18 ms5944-19 ms5944-20 ms5944-21 ms5944-22 ms5944-23 ms5944-24 ms5944-25 ms5944-26 ms5944-27 ms5944-28 ms5944-29 ms5944-30 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 EPA 2.085488794 3.960870674 2.7168319 6.288397133 10.05379854 6.082153922 5.04048841 5.823294658 7.333498149 6.042478884 6.801408218 5.635999919 13.25472458 7.000004302 3.414559817 2.48607193 4.91772162 5.149419354 5.162824095 6.528121618 7.45503801 4.639679296 4.80851927 7.184499972 9.778572855 5.005494002 6.423766863 4.576915048 6.290928892 11.40143535 Linolenic 4.349921304 19.68457337 13.46802617 29.68830549 47.93947937 23.11074859 23.17015161 26.18789094 51.20478404 24.93897287 29.07167599 29.46335897 27.94748701 25.48797126 13.32707712 9.90273763 16.63781824 21.33675646 45.58195778 43.14918918 25.64319784 18.82929635 13.77434426 19.41226508 35.6136951 23.9695567 19.78503455 13.89494175 18.94604962 48.0789016 DHA 9.495178307 9.088437195 11.2338996 15.81791398 26.37414157 23.56562059 19.81708056 23.93714709 21.31881616 21.93241995 23.39505391 24.91044959 20.56575981 22.61655439 31.93572707 14.44030855 22.70663206 21.44019909 28.45502691 27.73956906 17.52552425 15.94874553 14.7111246 18.39845206 19.91110353 12.31117817 19.09134129 13.7682488 22.58849143 17.86811934 Myristic 1.317999024 5.836531379 4.764905593 10.89554482 18.76720848 10.6989287 9.712218008 9.719676602 15.85385319 10.90945418 10.58854914 8.628248666 9.554270882 8.408650667 5.354848083 3.725203428 6.925490095 9.631397353 14.15671903 14.99345282 8.694923931 5.360024554 4.0227219 5.698723332 10.21189554 5.65745217 9.570170189 4.21489754 7.449805462 12.12314134 Palmitoleic 7.063099986 28.8051614 20.96815023 55.75337797 79.96912663 47.98438004 41.18880424 46.80774812 79.02368845 53.21654876 46.55540764 34.39885769 41.6179943 41.65856292 17.07221498 20.50027436 29.38966805 36.58843636 40.38508993 74.11008758 40.55342759 24.64889611 18.83172904 27.91650407 71.05751434 38.91252205 31.9484429 22.056481 30.02411465 80.45111619 Arachidonic 4.316581133 8.123392584 5.816898297 14.57635694 17.31138152 14.70953595 11.5463877 13.53944085 15.69086586 12.30224751 14.00702881 15.33670664 15.82862289 13.3363045 21.85700206 10.03091779 12.46973864 14.62560256 17.86238777 19.82085181 22.85863634 15.72690924 14.97209589 20.51703007 24.03749395 17.15466331 19.29351539 15.485211 21.85168975 28.55589363 Palmitelaidic 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Linoleic 67.85321016 129.7586541 103.7082429 179.8328633 348.5196472 135.5061878 125.586576 151.9395114 368.7113366 161.4905766 210.8494738 223.1522454 196.0166753 180.8519832 105.8630898 69.63832496 111.9041762 145.3360865 351.9890361 306.9988429 201.7945361 151.5116712 114.3399819 143.1058653 264.6697228 212.1153731 140.284756 108.2277654 159.5031477 352.124393 Palmitic 39.92693694 96.16045411 65.45875284 136.9145431 211.272371 129.1698578 112.0006381 121.0786746 194.4481444 131.0059711 142.0663926 134.7092407 157.8938159 124.3245934 107.7026793 67.46223296 99.29583111 127.7212343 211.207582 185.9047349 149.6761564 115.091941 96.21898592 138.4174434 178.9209422 106.0854642 134.8695111 104.2446849 131.7990791 208.8447515 Oleic 54.73492182 88.11562151 68.7167426 137.123903 226.3259295 98.77374389 89.33177664 103.2353396 215.6287192 119.3004574 130.3771334 129.5658191 124.6875619 121.9504967 74.30253424 55.61347244 84.53339799 98.76908092 198.1687476 186.4938086 132.3602105 97.80321671 75.99490029 92.08792832 193.0324121 133.4413201 92.69609398 79.3011023 102.3062285 256.7580031 Elaidic 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Stearic 5.381820856 10.50160876 10.24437093 16.2096841 27.09942378 16.50298278 13.64270991 20.39888425 24.63160753 19.35773475 15.23455385 17.27763721 27.2737116 11.79229808 28.5325238 10.71055304 13.98081712 28.25896355 34.37854515 28.79721532 23.30739883 18.27483537 19.51870816 24.61204794 24.440773 16.80493694 21.24280867 19.71056461 26.70383703 32.39748075 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name EPA Linolenic DHA Myristic Palmitoleic Arachidonic Palmitelaidic Linoleic Palmitic Oleic Elaidic Stearic METABOLITES_END #END