#METABOLOMICS WORKBENCH hormel101_20170623_144803 DATATRACK_ID:1098 STUDY_ID:ST000649 ANALYSIS_ID:AN000981 PROJECT_ID:PR000460 VERSION 1 CREATED_ON June 26, 2017, 8:10 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 Non-Esterified Fatty ST:STUDY_TITLE Acids (part V) 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 - 5863-1 Time point:Sedentary | Grouping:SED-Naproxcinod Date of study=9/23/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - 5863-2 Time point:Sedentary | Grouping:SED-Naproxcinod Date of study=9/24/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - 5863-3 Time point:Sedentary | Grouping:SED-Naproxcinod Date of study=10/1/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - 5863-4 Time point:Sedentary | Grouping:SED-Naproxcinod Date of study=10/7/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - 5863-5 Time point:Sedentary | Grouping:SED-Naproxcinod Date of study=10/9/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - 5863-6 Time point:Post Run | Grouping:RUN-Naproxcinod Date of study=9/23/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - 5863-7 Time point:Post Run | Grouping:RUN-Naproxcinod Date of study=9/24/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - 5863-8 Time point:Post Run | Grouping:RUN-Naproxcinod Date of study=10/1/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - 5863-9 Time point:Post Run | Grouping:RUN-Naproxcinod Date of study=10/7/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - 5863-10 Time point:Post Run | Grouping:RUN-Naproxcinod Date of study=10/9/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - 5863-11 Time point:Sedentary | Grouping:SED-Naproxen Date of study=9/23/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - 5863-12 Time point:Sedentary | Grouping:SED-Naproxen Date of study=9/24/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - 5863-13 Time point:Sedentary | Grouping:SED-Naproxen Date of study=10/1/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - 5863-14 Time point:Sedentary | Grouping:SED-Naproxen Date of study=10/7/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - 5863-15 Time point:Sedentary | Grouping:SED-Naproxen Date of study=10/9/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - 5863-16 Time point:Post Run | Grouping:RUN-Naproxen Date of study=9/23/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - 5863-17 Time point:Post Run | Grouping:RUN-Naproxen Date of study=9/24/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - 5863-18 Time point:Post Run | Grouping:RUN-Naproxen Date of study=10/1/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - 5863-19 Time point:Post Run | Grouping:RUN-Naproxen Date of study=10/7/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - 5863-20 Time point:Post Run | Grouping:RUN-Naproxen Date of study=10/9/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - 5863-21 Time point:Sedentary | Grouping:SED-Vehicle Date of study=9/23/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - 5863-22 Time point:Sedentary | Grouping:SED-Vehicle Date of study=9/24/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - 5863-23 Time point:Sedentary | Grouping:SED-Vehicle Date of study=10/1/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - 5863-24 Time point:Sedentary | Grouping:SED-Vehicle Date of study=10/7/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - 5863-25 Time point:Sedentary | Grouping:SED-Vehicle Date of study=10/9/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - 5863-26 Time point:Post Run | Grouping:RUN-Vehicle Date of study=9/23/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - 5863-27 Time point:Post Run | Grouping:RUN-Vehicle Date of study=9/24/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - 5863-28 Time point:Post Run | Grouping:RUN-Vehicle Date of study=10/1/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - 5863-29 Time point:Post Run | Grouping:RUN-Vehicle Date of study=10/7/2015; Gender=Male; Sample type=Tissue-Gastrocnemius; Species=Mouse SUBJECT_SAMPLE_FACTORS - 5863-30 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 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 5863-1 5863-2 5863-3 5863-4 5863-5 5863-6 5863-7 5863-8 5863-9 5863-10 5863-11 5863-12 5863-13 5863-14 5863-15 5863-16 5863-17 5863-18 5863-19 5863-20 5863-21 5863-22 5863-23 5863-24 5863-25 5863-26 5863-27 5863-28 5863-29 5863-30 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 EPA 2.20 0.89 2.18 1.41 2.53 2.58 4.35 1.90 2.88 1.14 2.32 1.17 0.85 3.14 2.42 0.86 1.39 2.57 3.37 3.00 1.09 1.91 1.25 2.64 2.41 1.49 1.79 0.83 2.24 1.35 Linolenic 8.24 6.24 11.73 7.23 9.95 8.95 14.77 11.53 12.48 10.17 9.85 8.71 6.23 12.58 11.90 3.63 8.02 9.36 14.49 16.18 7.04 9.77 6.81 12.92 17.14 11.76 10.23 9.99 12.10 9.30 DHA 5.45 2.56 5.47 2.10 6.89 6.04 7.33 5.24 8.54 3.75 6.20 3.77 3.58 8.76 6.47 3.96 4.34 6.70 6.46 6.07 3.79 7.60 3.23 5.24 5.40 6.14 6.06 3.44 6.88 4.44 Myristic 3.28 1.08 2.20 0.78 2.10 2.94 6.49 3.12 2.90 3.51 2.99 1.81 1.28 2.85 2.50 0.18 1.81 2.69 4.08 6.02 1.76 3.21 0.95 3.59 2.92 4.01 1.52 1.18 3.06 1.64 Palmitoleic 16.86 9.80 15.40 13.46 16.48 16.06 33.49 27.68 18.31 20.04 17.14 12.05 12.42 17.22 24.60 7.46 12.94 23.26 27.60 37.23 11.12 15.15 10.98 25.79 22.49 19.55 13.16 16.80 21.08 13.14 Arachidonic 9.34 6.37 10.25 5.87 12.49 12.24 12.88 10.65 18.16 10.01 10.02 8.41 7.57 16.60 13.19 6.08 9.54 12.15 11.52 13.01 7.56 11.01 7.50 10.83 10.72 10.93 9.60 8.33 13.93 9.71 Palmitelaidic 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Linoleic 58.55 45.81 87.61 49.41 75.24 69.23 93.84 81.63 91.58 82.06 71.99 58.45 48.76 99.50 83.71 33.65 60.11 72.36 98.43 102.57 48.02 84.79 56.75 86.04 134.25 86.32 80.36 90.90 110.86 78.57 Palmitic 69.43 57.63 82.03 62.15 82.77 69.97 97.56 80.28 91.43 73.24 80.39 64.29 58.61 93.19 91.00 42.52 63.77 82.66 100.10 92.53 60.03 83.50 53.86 77.31 86.05 79.70 75.18 64.68 95.45 69.28 Oleic 43.01 34.38 61.21 35.91 54.35 49.63 70.91 64.07 65.57 57.23 51.74 38.39 41.74 70.73 67.87 25.61 44.41 59.16 72.01 77.19 32.41 59.39 41.90 67.15 78.44 59.12 53.07 62.24 73.05 53.62 Elaidic 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Stearic 13.98 14.97 15.37 19.70 17.72 16.16 19.52 20.79 19.21 13.35 13.90 13.24 14.31 22.29 19.91 15.89 13.40 23.73 24.07 20.28 17.61 16.90 11.69 15.58 15.42 17.22 19.80 17.88 23.98 20.51 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name EPA Linolenic DHA Myristic Palmitoleic Arachidonic Palmitelaidic Linoleic Palmitic Oleic Elaidic Stearic METABOLITES_END #END