Improved Muscle Function in Duchenne Muscular Dystrophy through L-Arginine and Metformin: An Investigator-Initiated, Open-Label, Single-Center, Proof-Of-Concept-Study

Patricia Hafner, Ulrike Bonati, Beat Erne, Maurice Schmid, Daniela Rubino, Urs Pohlman, Thomas Peters, Erich Rutz, Stephan Frank, Cornelia Neuhaus, Stefanie Deuster, Monika Gloor, Oliver Bieri, Arne Fischmann, Michael Sinnreich, Nuri Gueven, Dirk Fischer, Patricia Hafner, Ulrike Bonati, Beat Erne, Maurice Schmid, Daniela Rubino, Urs Pohlman, Thomas Peters, Erich Rutz, Stephan Frank, Cornelia Neuhaus, Stefanie Deuster, Monika Gloor, Oliver Bieri, Arne Fischmann, Michael Sinnreich, Nuri Gueven, Dirk Fischer

Abstract

Altered neuronal nitric oxide synthase function in Duchenne muscular dystrophy leads to impaired mitochondrial function which is thought to be one cause of muscle damage in this disease. The study tested if increased intramuscular nitric oxide concentration can improve mitochondrial energy metabolism in Duchenne muscular dystrophy using a novel therapeutic approach through the combination of L-arginine with metformin. Five ambulatory, genetically confirmed Duchenne muscular dystrophy patients aged between 7–10 years were treated with L-arginine (3 x 2.5 g/d) and metformin (2 x 250 mg/d) for 16 weeks. Treatment effects were assessed using mitochondrial protein expression analysis in muscular biopsies, indirect calorimetry, Dual-Energy X-Ray Absorptiometry, quantitative thigh muscle MRI, and clinical scores of muscle performance. There were no serious side effects and no patient dropped out. Muscle biopsy results showed pre-treatment a significantly reduced mitochondrial protein expression and increased oxidative stress in Duchenne muscular dystrophy patients compared to controls. Post-treatment a significant elevation of proteins of the mitochondrial electron transport chain was observed as well as a reduction in oxidative stress. Treatment also decreased resting energy expenditure rates and energy substrate use shifted from carbohydrates to fatty acids. These changes were associated with improved clinical scores. In conclusion pharmacological stimulation of the nitric oxide pathway leads to improved mitochondria function and clinically a slowing of disease progression in Duchenne muscular dystrophy. This study shall lead to further development of this novel therapeutic approach into a real alternative for Duchenne muscular dystrophy patients.

Trial registration: ClinicalTrials.gov NCT02516085.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1. Study profile according CONSORT flow…
Fig 1. Study profile according CONSORT flow chart.
Fig 2. Muscle biopsy.
Fig 2. Muscle biopsy.
Muscle biopsy findings, individual control (C) concentrations and individual DMD changes before (PRE) and after (POST) treatment as well as point estimates with 95% confidence intervals. A = nitrotyrosine ELISA, concentration is provided in nmol / 7.9 μg protein, B = cGMP ELISA, concentration is provided in pmol / 6.4 μg protein, C = western blot succinat oxidoreductase (complex II) / GAPDH ratio, D = western blot cytochrome c oxidoreductase (complex III) / GAPDH ratio, E = western blot cytochrome c oxidase (Complex IV) / GAPDH ratio, F = western blot ATP synthase (Complex V) / GAPDH ratio, G = carbonylated protein ELISA (nmol / 1 μg protein).
Fig 3. Indirect calorimetry.
Fig 3. Indirect calorimetry.
Indirect calorimetry results demonstrating consistently decreased rates of resting energy expenditure per kg muscle in 24 hours (A), reduced usage of carbohydrates as oxidative fuel (B), and increased usages of fatty acids (C) in oxidative energy metabolism.
Fig 4. Clinical changes.
Fig 4. Clinical changes.
Clinical changes over treatment period of 16 weeks are shown. Four of the five patients showed improvements in 2 min walking distance (A), the MFM total score (B) and the MFM D1 subscore (C).

References

    1. Yoshida M, Ozawa E. Glycoprotein complex anchoring dystrophin to sarcolemma. Journal of biochemistry. 1990;108(5):748–52. .
    1. Campbell KP, Kahl SD. Association of dystrophin and an integral membrane glycoprotein. Nature. 1989;338(6212):259–62. 10.1038/338259a0 .
    1. Smeitink J, van den Heuvel L, DiMauro S. The genetics and pathology of oxidative phosphorylation. Nature reviews Genetics. 2001;2(5):342–52. 10.1038/35072063 .
    1. Scholte HR, Luyt-Houwen IE, Busch HF, Jennekens FG. Muscle mitochondria from patients with Duchenne muscular dystrophy have a normal beta oxidation, but an impaired oxidative phosphorylation. Neurology. 1985;35(9):1396–7. .
    1. Kemp GJ, Taylor DJ, Dunn JF, Frostick SP, Radda GK. Cellular energetics of dystrophic muscle. Journal of the neurological sciences. 1993;116(2):201–6. .
    1. Sperl W, Skladal D, Gnaiger E, Wyss M, Mayr U, Hager J, et al. High resolution respirometry of permeabilized skeletal muscle fibers in the diagnosis of neuromuscular disorders. Molecular and cellular biochemistry. 1997;174(1–2):71–8. .
    1. Kuznetsov AV, Winkler K, Wiedemann FR, von Bossanyi P, Dietzmann K, Kunz WS. Impaired mitochondrial oxidative phosphorylation in skeletal muscle of the dystrophin-deficient mdx mouse. Molecular and cellular biochemistry. 1998;183(1–2):87–96. .
    1. Braun U, Paju K, Eimre M, Seppet E, Orlova E, Kadaja L, et al. Lack of dystrophin is associated with altered integration of the mitochondria and ATPases in slow-twitch muscle cells of MDX mice. Biochimica et biophysica acta. 2001;1505(2–3):258–70. .
    1. Millay DP, Sargent MA, Osinska H, Baines CP, Barton ER, Vuagniaux G, et al. Genetic and pharmacologic inhibition of mitochondrial-dependent necrosis attenuates muscular dystrophy. Nature medicine. 2008;14(4):442–7. 10.1038/nm1736
    1. Hankard R, Gottrand F, Turck D, Carpentier A, Romon M, Farriaux JP. Resting energy expenditure and energy substrate utilization in children with Duchenne muscular dystrophy. Pediatric research. 1996;40(1):29–33. Epub 1996/07/01. 10.1203/00006450-199607000-00006 .
    1. Gaeta M, Messina S, Mileto A, Vita GL, Ascenti G, Vinci S, et al. Muscle fat-fraction and mapping in Duchenne muscular dystrophy: evaluation of disease distribution and correlation with clinical assessments. Preliminary experience. Skeletal radiology. 2012;41(8):955–61. Epub 2011/11/10. 10.1007/s00256-011-1301-5 .
    1. Hollingsworth KG, Garrood P, Eagle M, Bushby K, Straub V. Magnetic resonance imaging in Duchenne muscular dystrophy: longitudinal assessment of natural history over 18 months. Muscle & nerve. 2013;48(4):586–8. 10.1002/mus.23879 .
    1. Brenman JE, Chao DS, Xia H, Aldape K, Bredt DS. Nitric oxide synthase complexed with dystrophin and absent from skeletal muscle sarcolemma in Duchenne muscular dystrophy. Cell. 1995;82(5):743–52. Epub 1995/09/08. .
    1. MacAllister RJ, Whitley GS, Vallance P. Effects of guanidino and uremic compounds on nitric oxide pathways. Kidney international. 1994;45(3):737–42. .
    1. Nisoli E, Carruba MO. Nitric oxide and mitochondrial biogenesis. Journal of cell science. 2006;119(Pt 14):2855–62. 10.1242/jcs.03062 .
    1. McGee SL, Hargreaves M. AMPK-mediated regulation of transcription in skeletal muscle. Clin Sci (Lond). 2010;118(8):507–18. 10.1042/CS20090533 .
    1. Horster I, Weigt-Usinger K, Carmann C, Chobanyan-Jurgens K, Kohler C, Schara U, et al. The L-arginine/NO pathway and homoarginine are altered in Duchenne muscular dystrophy and improved by glucocorticoids. Amino acids. 2015;47(9):1853–63. 10.1007/s00726-015-2018-x .
    1. Garbincius JF, Michele DE. Dystrophin-glycoprotein complex regulates muscle nitric oxide production through mechanoregulation of AMPK signaling. Proceedings of the National Academy of Sciences of the United States of America. 2015;112(44):13663–8. 10.1073/pnas.1512991112
    1. Jahnke VE, Van Der Meulen JH, Johnston HK, Ghimbovschi S, Partridge T, Hoffman EP, et al. Metabolic remodeling agents show beneficial effects in the dystrophin-deficient mdx mouse model. Skeletal muscle. 2012;2(1):16 10.1186/2044-5040-2-16
    1. Ljubicic V, Miura P, Burt M, Boudreault L, Khogali S, Lunde JA, et al. Chronic AMPK activation evokes the slow, oxidative myogenic program and triggers beneficial adaptations in mdx mouse skeletal muscle. Human molecular genetics. 2011;20(17):3478–93. 10.1093/hmg/ddr265 .
    1. Pauly M, Daussin F, Burelle Y, Li T, Godin R, Fauconnier J, et al. AMPK activation stimulates autophagy and ameliorates muscular dystrophy in the mdx mouse diaphragm. The American journal of pathology. 2012;181(2):583–92. 10.1016/j.ajpath.2012.04.004 .
    1. Musi N, Hirshman MF, Nygren J, Svanfeldt M, Bavenholm P, Rooyackers O, et al. Metformin increases AMP-activated protein kinase activity in skeletal muscle of subjects with type 2 diabetes. Diabetes. 2002;51(7):2074–81. .
    1. Ljubicic V, Jasmin BJ. Metformin increases peroxisome proliferator-activated receptor gamma Co-activator-1alpha and utrophin a expression in dystrophic skeletal muscle. Muscle & nerve. 2015;52(1):139–42. 10.1002/mus.24692 .
    1. Langone F, Cannata S, Fuoco C, Lettieri Barbato D, Testa S, Nardozza AP, et al. Metformin protects skeletal muscle from cardiotoxin induced degeneration. PloS one. 2014;9(12):e114018 10.1371/journal.pone.0114018
    1. Koga Y, Akita Y, Nishioka J, Yatsuga S, Povalko N, Tanabe Y, et al. L-arginine improves the symptoms of strokelike episodes in MELAS. Neurology. 2005;64(4):710–2. 10.1212/01.WNL.0000151976.60624.01 .
    1. Bennett-Richards KJ, Kattenhorn M, Donald AE, Oakley GR, Varghese Z, Bruckdorfer KR, et al. Oral L-arginine does not improve endothelial dysfunction in children with chronic renal failure. Kidney international. 2002;62(4):1372–8. Epub 2002/09/18. 10.1111/j.1523-1755.2002.kid555.x .
    1. Berard C, Payan C, Hodgkinson I, Fermanian J. A motor function measure for neuromuscular diseases. Construction and validation study. Neuromuscular disorders: NMD. 2005;15(7):463–70. Epub 2005/08/18. .
    1. .
    1. Mazzone E, Vasco G, Sormani MP, Torrente Y, Berardinelli A, Messina S, et al. Functional changes in Duchenne muscular dystrophy: a 12-month longitudinal cohort study. Neurology. 2011;77(3):250–6. Epub 2011/07/08. 10.1212/WNL.0b013e318225ab2e .
    1. Elliott SA, Davidson ZE, Davies PS, Truby H. Predicting resting energy expenditure in boys with Duchenne muscular dystrophy. European journal of paediatric neurology: EJPN: official journal of the European Paediatric Neurology Society. 2012;16(6):631–5. 10.1016/j.ejpn.2012.02.011 .
    1. Skalsky AJ, Han JJ, Abresch RT, Shin CS, McDonald CM. Assessment of regional body composition with dual-energy X-ray absorptiometry in Duchenne muscular dystrophy: correlation of regional lean mass and quantitative strength. Muscle & nerve. 2009;39(5):647–51. 10.1002/mus.21212 .
    1. Dixon WT. Simple proton spectroscopic imaging. Radiology. 1984;153(1):189–94. Epub 1984/10/01. .
    1. Fischmann A, Hafner P, Gloor M, Schmid M, Klein A, Pohlman U, et al. Quantitative MRI and loss of free ambulation in Duchenne muscular dystrophy. Journal of neurology. 2013;260(4):969–74. Epub 2012/11/10. 10.1007/s00415-012-6733-x .
    1. de Castro Barbosa T, Jiang LQ, Zierath JR, Nunes MT. L-Arginine enhances glucose and lipid metabolism in rat L6 myotubes via the NO/ c-GMP pathway. Metabolism: clinical and experimental. 2013;62(1):79–89. 10.1016/j.metabol.2012.06.011 .
    1. Ouyang J, Parakhia RA, Ochs RS. Metformin activates AMP kinase through inhibition of AMP deaminase. The Journal of biological chemistry. 2011;286(1):1–11. 10.1074/jbc.M110.121806
    1. Lee-Young RS, Griffee SR, Lynes SE, Bracy DP, Ayala JE, McGuinness OP, et al. Skeletal muscle AMP-activated protein kinase is essential for the metabolic response to exercise in vivo. The Journal of biological chemistry. 2009;284(36):23925–34. 10.1074/jbc.M109.021048
    1. Dudley RW, Danialou G, Govindaraju K, Lands L, Eidelman DE, Petrof BJ. Sarcolemmal damage in dystrophin deficiency is modulated by synergistic interactions between mechanical and oxidative/nitrosative stresses. The American journal of pathology. 2006;168(4):1276–87; quiz 404–5. 10.2353/ajpath.2006.050683
    1. Hall DT, Ma JF, Marco SD, Gallouzi IE. Inducible nitric oxide synthase (iNOS) in muscle wasting syndrome, sarcopenia, and cachexia. Aging. 2011;3(8):702–15.
    1. Kruszelnicka O, Chyrchel B, Golay A, Surdacki A. Differential associations of circulating asymmetric dimethylarginine and cell adhesion molecules with metformin use in patients with type 2 diabetes mellitus and stable coronary artery disease. Amino acids. 2015;47(9):1951–9. 10.1007/s00726-015-1976-3 .
    1. Bestermann WH Jr. The ADMA-Metformin Hypothesis: Linking the Cardiovascular Consequences of the Metabolic Syndrome and Type 2 Diabetes. Cardiorenal medicine. 2011;1(4):211–9. 10.1159/000332382
    1. Radley-Crabb HG, Marini JC, Sosa HA, Castillo LI, Grounds MD, Fiorotto ML. Dystropathology increases energy expenditure and protein turnover in the mdx mouse model of duchenne muscular dystrophy. PloS one. 2014;9(2):e89277 10.1371/journal.pone.0089277
    1. Suhr F, Gehlert S, Grau M, Bloch W. Skeletal muscle function during exercise-fine-tuning of diverse subsystems by nitric oxide. International journal of molecular sciences. 2013;14(4):7109–39. 10.3390/ijms14047109
    1. Vuillerot C, Girardot F, Payan C, Fermanian J, Iwaz J, De Lattre C, et al. Monitoring changes and predicting loss of ambulation in Duchenne muscular dystrophy with the Motor Function Measure. Developmental medicine and child neurology. 2010;52(1):60–5. Epub 2009/05/21. 10.1111/j.1469-8749.2009.03316.x .
    1. Silva EC, Machado DL, Resende MB, Silva RF, Zanoteli E, Reed UC. Motor function measure scale, steroid therapy and patients with Duchenne muscular dystrophy. Arquivos de neuro-psiquiatria. 2012;70(3):191–5. Epub 2012/03/07. .
    1. Bonati U, Hafner P, Schadelin S, Schmid M, Naduvilekoot Devasia A, Schroeder J, et al. Quantitative muscle MRI: A powerful surrogate outcome measure in Duchenne muscular dystrophy. Neuromuscular disorders: NMD. 2015;25(9):679–85. 10.1016/j.nmd.2015.05.006 .
    1. Percival JM, Whitehead NP, Adams ME, Adamo CM, Beavo JA, Froehner SC. Sildenafil reduces respiratory muscle weakness and fibrosis in the mdx mouse model of Duchenne muscular dystrophy. The Journal of pathology. 2012;228(1):77–87. 10.1002/path.4054
    1. Witting N, Kruuse C, Nyhuus B, Prahm KP, Citirak G, Lundgaard SJ, et al. Effect of sildenafil on skeletal and cardiac muscle in Becker muscular dystrophy. Annals of neurology. 2014;76(4):550–7. 10.1002/ana.24216 .
    1. Ito N, Ruegg UT, Kudo A, Miyagoe-Suzuki Y, Takeda S. Activation of calcium signaling through Trpv1 by nNOS and peroxynitrite as a key trigger of skeletal muscle hypertrophy. Nature medicine. 2013;19(1):101–6. 10.1038/nm.3019 .
    1. Sciorati C, Staszewsky L, Zambelli V, Russo I, Salio M, Novelli D, et al. Ibuprofen plus isosorbide dinitrate treatment in the mdx mice ameliorates dystrophic heart structure. Pharmacological research: the official journal of the Italian Pharmacological Society. 2013;73:35–43. 10.1016/j.phrs.2013.04.009 .
    1. D'Angelo MG, Gandossini S, Martinelli Boneschi F, Sciorati C, Bonato S, Brighina E, et al. Nitric oxide donor and non steroidal anti inflammatory drugs as a therapy for muscular dystrophies: evidence from a safety study with pilot efficacy measures in adult dystrophic patients. Pharmacological research: the official journal of the Italian Pharmacological Society. 2012;65(4):472–9. 10.1016/j.phrs.2012.01.006 .
    1. Kayacelebi AA, Langen J, Weigt-Usinger K, Chobanyan-Jurgens K, Mariotti F, Schneider JY, et al. Biosynthesis of homoarginine (hArg) and asymmetric dimethylarginine (ADMA) from acutely and chronically administered free L-arginine in humans. Amino acids. 2015;47(9):1893–908. 10.1007/s00726-015-2012-3 .
    1. NCT01995032.
    1. NCT02018731.

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