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Exercise-induced angiogenesis correlates with the up-regulated expression of neuronal nitric oxide synthase (nNOS) in human skeletal muscle

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Abstract

The contribution of neuronal nitric oxide synthase (nNOS) to angiogenesis in human skeletal muscle after endurance exercise is controversially discussed. We therefore ascertained whether the expression of nNOS is associated with the capillary density in biopsies of the vastus lateralis (VL) muscle that had been derived from 10 sedentary male subjects before and after moderate training (four 30-min weekly jogging sessions for 6 months, with a heart-rate corresponding to 75% VO2max). In these biopsies, nNOS was predominantly expressed as alpha-isoform with exon-mu and to a lesser extent without exon-mu, as determined by RT-PCR. The mRNA levels of nNOS were quantified by real-time PCR and related to the capillary-to-fibre ratio and the numerical density of capillaries specified by light microscopy. If the VL biopsies of all subjects were co-analysed, mRNA levels of nNOS were non-significantly elevated after training (+34%; P > 0.05). However, only five of the ten subjects exhibited significant (P ≤ 0.05) elevations in the capillary-to-fibre ratio (+25%) and the numerical density of capillaries (+21%) and were thus undergoing angiogenesis. If the VL biopsies of these five subjects alone were evaluated, the mRNA levels of nNOS were significantly up-regulated (+128%; P ≤ 0.05) and correlated positively (r = 0.8; P ≤ 0.01) to angiogenesis. Accordingly, nNOS protein expression in VL biopsies quantified by immunoblotting was significantly increased (+82%; P ≤ 0.05) only in those subjects that underwent angiogenesis. In conclusion, the expression of nNOS at mRNA and protein levels was statistically linked to capillarity after exercise suggesting that nNOS is involved in the angiogenic response to training in human skeletal muscle.

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References

  1. Arany Z, Foo SY, Ma Y, Ruas JL, Bommi-Reddy A, Girnun G, Cooper M, Laznik D, Chinsomboon J, Rangwala SM, Baek KH, Rosenzweig A, Spiegelman BM (2008) HIF-independent regulation of VEGF and angiogenesis by the transcriptional coactivator PGC-1alpha. Nature 451:1008–1012

    PubMed  Article  CAS  Google Scholar 

  2. Baum O, Da Silva-Azevedo L, Willerding G, Wöckel A, Planitzer G, Gossrau R, Pries AR, Zakrzewicz A (2004) Endothelial NOS is main mediator for shear stress-dependent angiogenesis in skeletal muscle after prazosin administration. Am J Physiol Heart Circ Physiol 287:H2300–H2308

    PubMed  Article  CAS  Google Scholar 

  3. Bouchard C, Rankinen T (2001) Individual differences in response to regular physical activity. Med Sci Sports Exerc 33:446–451

    Article  Google Scholar 

  4. Bradley SJ, Kingwell BA, Canny BJ, McConell GK (2007) Skeletal muscle neuronal nitric oxide synthase micro protein is reduced in people with impaired glucose homeostasis and is not normalized by exercise training. Metabolism 56:1405–1411

    PubMed  Article  CAS  Google Scholar 

  5. Bray MS, Hagberg JM, Perusse L, Rankinen T, Roth SM, Wolfarth B, Bouchard C (2009) The human gene map for performance and health-related fitness phenotypes: the 2006–2007 update. Med Sci Sports Exerc 41:35–73

    PubMed  Google Scholar 

  6. Chinsomboon J, Ruas J, Gupta RK, Thom R, Shoag J, Rowe GC, Sawada N, Raghuram S, Arany Z (2009) The transcriptional coactivator PGC-1alpha mediates exercise-induced angiogenesis in skeletal muscle. Proc Natl Acad Sci U S A 106:21401–21406

    PubMed  Article  CAS  Google Scholar 

  7. Coffey VG, Hawley JA (2007) The molecular bases of training adaptation. Sports Med 37:737–763

    PubMed  Article  Google Scholar 

  8. Copp SW, Hirai DM, Schwagerl PJ, Musch TI, Poole DC (2010) Effects of neuronal nitric oxide synthase inhibition on resting and exercising hindlimb muscle blood flow in the rat. J Physiol 588:1321–1331

    PubMed  Article  CAS  Google Scholar 

  9. Da Silva-Azevedo L, Baum O, Zakrzewicz A, Pries AR (2002) Vascular endothelial growth factor is expressed in endothelial cells isolated from skeletal muscles of nitric oxide synthase knockout mice during prazosin-induced angiogenesis. Biochem Biophys Res Commun 297:1270–1276

    PubMed  Article  CAS  Google Scholar 

  10. Da Silva-Azevedo L, Jahne S, Hoffmann C, Stalder D, Heller M, Pries AR, Zakrzewicz A, Baum O (2009) Up-regulation of the peroxiredoxin-6 related metabolism of reactive oxygen species in skeletal muscle of mice lacking neuronal nitric oxide synthase. J Physiol 587:655–668

    PubMed  Article  CAS  Google Scholar 

  11. Egginton S (2009) Invited review: activity-induced angiogenesis. Pflugers Arch 457:963–977

    PubMed  Article  CAS  Google Scholar 

  12. Frandsen U, Hoffner L, Betak A, Saltin B, Bangsbo J, Hellsten Y (2000) Endurance training does not alter the level of neuronal nitric oxide synthase in human skeletal muscle. J Appl Physiol 89:1033–1038

    PubMed  CAS  Google Scholar 

  13. Gavin TP (2009) Basal and exercise-induced regulation of skeletal muscle capillarization. Exerc Sport Sci Rev 37:86–92

    PubMed  Article  Google Scholar 

  14. Gustafsson T, Rundqvist H, Norrbom J, Rullman E, Jansson E, Sundberg CJ (2007) The influence of physical training on the angiopoietin and VEGF-A systems in human skeletal muscle. J Appl Physiol 103:1012–1020

    Google Scholar 

  15. Hoppeler H, Howald H, Conley K, Lindstedt SL, Claassen H, Vock P, Weibel ER (1985) Endurance training in humans: aerobic capacity and structure of skeletal muscle. J Appl Physiol 59:320–327

    PubMed  CAS  Google Scholar 

  16. Hoppeler H, Klossner S, Fluck M (2007) Gene expression in working skeletal muscle. Adv Exp Med Biol 618:245–254

    PubMed  Article  Google Scholar 

  17. Hudlicka O (1998) Is physiological angiogenesis in skeletal muscle regulated by changes in microcirculation? Microcirculation 5:7–23

    PubMed  CAS  Google Scholar 

  18. Hudlicka O, Brown MD (2009) Adaptation of skeletal muscle microvasculature to increased or decreased blood flow: role of shear stress, nitric oxide and vascular endothelial growth factor. J Vasc Res 46:504–512

    PubMed  Article  CAS  Google Scholar 

  19. Hudlicka O, Brown MD, Silgram H (2000) Inhibition of capillary growth in chronically stimulated rat muscles by N(G)-nitro-l-arginine, nitric oxide synthase inhibitor. Microvasc Res 59:45–51

    PubMed  Article  CAS  Google Scholar 

  20. Jackson MJ, Pye D, Palomero J (2007) The production of reactive oxygen and nitrogen species by skeletal muscle. J Appl Physiol 102:1664–1670

    PubMed  Article  CAS  Google Scholar 

  21. Kobayashi YM, Rader EP, Crawford RW, Iyengar NK, Thedens DR, Faulkner JA, Parikh SV, Weiss RM, Chamberlain JS, Moore SA, Campbell KP (2008) Sarcolemma-localized nNOS is required to maintain activity after mild exercise. Nature 456:511–515

    PubMed  Article  CAS  Google Scholar 

  22. Kobzik L, Reid MB, Bredt DS, Stamler JS (1994) Nitric oxide in skeletal muscle. Nature 372:546–548

    PubMed  Article  CAS  Google Scholar 

  23. Laine R, de Montellano PR (1998) Neuronal nitric oxide synthase isoforms alpha and mu are closely related calpain-sensitive proteins. Mol Pharmacol 54:305–312

    PubMed  CAS  Google Scholar 

  24. Lau KS, Grange RW, Isotani E, Sarelius IH, Kamm KE, Huang PL, Stull JT (2000) nNOS and eNOS modulate cGMP formation and vascular response in contracting fast-twitch skeletal muscle. Physiol Genomics 2:21–27

    PubMed  CAS  Google Scholar 

  25. Leick L, Hellsten Y, Fentz J, Lyngby SS, Wojtaszewski JF, Hidalgo J, Pilegaard H (2009) PGC-1alpha mediates exercise-induced skeletal muscle VEGF expression in mice. Am J Physiol Endocrinol Metab 297:E92–E103

    PubMed  Article  CAS  Google Scholar 

  26. McConell GK, Bradley SJ, Stephens TJ, Canny BJ, Kingwell BA, Lee-Young RS (2007) Skeletal muscle nNOS mu protein content is increased by exercise training in humans. Am J Physiol Regul Integr Comp Physiol 293:R821–R828

    PubMed  Article  CAS  Google Scholar 

  27. Melikian N, Seddon MD, Casadei B, Chowienczyk PJ, Shah AM (2009) Neuronal nitric oxide synthase and human vascular regulation. Trends Cardiovasc Med 19:256–262

    PubMed  Article  CAS  Google Scholar 

  28. Percival JM, Anderson KN, Gregorevic P, Chamberlain JS, Froehner SC (2008) Functional deficits in nNOSmu-deficient skeletal muscle: myopathy in nNOS knockout mice. PLoS One 3:e3387

    PubMed  Article  Google Scholar 

  29. Rudnick J, Puttmann B, Tesch PA, Alkner B, Schoser BG, Salanova M, Kirsch K, Gunga HC, Schiffl G, Luck G, Blottner D (2004) Differential expression of nitric oxide synthases (NOS 1–3) in human skeletal muscle following exercise countermeasure during 12 weeks of bed rest. FASEB J 18:1228–1230

    PubMed  CAS  Google Scholar 

  30. Salanova M, Schiffl G, Puttmann B, Schoser BG, Blottner D (2008) Molecular biomarkers monitoring human skeletal muscle fibres and microvasculature following long-term bed rest with and without countermeasures. J Anat 212:306–318

    PubMed  Article  CAS  Google Scholar 

  31. Sarelius I, Pohl U (2010) Control of muscle blood flow during exercise: local factors and integrative mechanisms. Acta Physiol (Oxf) 199:349–365

    Article  CAS  Google Scholar 

  32. Silvagno F, Xia H, Bredt DS (1996) Neuronal nitric-oxide synthase-mu, an alternatively spliced isoform expressed in differentiated skeletal muscle. J Biol Chem 271:11204–11208

    PubMed  Article  CAS  Google Scholar 

  33. Stamler JS, Meissner G (2001) Physiology of nitric oxide in skeletal muscle. Physiol Rev 81:209–237

    PubMed  CAS  Google Scholar 

  34. Suter E, Hoppeler H, Claassen H, Billeter R, Aebi U, Horber F, Jaeger P, Marti B (1995) Ultrastructural modification of human skeletal muscle tissue with 6-month moderate-intensity exercise training. Int J Sports Med 16:160–166

    PubMed  Article  CAS  Google Scholar 

  35. Timmons JA, Sundberg CJ (2006) Oligonucleotide microarray expression profiling: human skeletal muscle phenotype and aerobic exercise training. IUBMB Life 58:15–24

    PubMed  Article  CAS  Google Scholar 

  36. Vassilakopoulos T, Deckman G, Kebbewar M, Rallis G, Harfouche R, Hussain SN (2003) Regulation of nitric oxide production in limb and ventilatory muscles during chronic exercise training. Am J Physiol Lung Cell Mol Physiol 284:L452–L457

    PubMed  CAS  Google Scholar 

  37. Wang Y, Newton DC, Robb GB, Kau CL, Miller TL, Cheung AH, Hall AV, VanDamme S, Wilcox JN, Marsden PA (1999) RNA diversity has profound effects on the translation of neuronal nitric oxide synthase. Proc Natl Acad Sci U S A 96:12150–12155

    PubMed  Article  CAS  Google Scholar 

  38. Wehling-Henricks M, Oltmann M, Rinaldi C, Myung KH, Tidball JG (2009) Loss of positive allosteric interactions between neuronal nitric oxide synthase and phosphofructokinase contributes to defects in glycolysis and increased fatigability in muscular dystrophy. Hum Mol Genet 18:3439–3451

    PubMed  Article  CAS  Google Scholar 

  39. Williams JL, Cartland D, Hussain A, Egginton S (2006) A differential role for nitric oxide in two forms of physiological angiogenesis in mouse. J Physiol 570:445–454

    PubMed  Article  CAS  Google Scholar 

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Acknowledgments

This study was funded by grants from the Swiss National Science Foundation (SNF, 320030-120269) and the Swiss Foundation for Research on Muscle Diseases (SSEM). We would like to thank Franziska Graber and Adolfo Adriozola for their skilful technical support, and Matthias Müller and Fabio Breil for their helpful discussions.

Conflict of interest

There is no conflict of interest.

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Correspondence to Oliver Baum.

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Communicated by Martin Flueck.

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Huber-Abel, F.A.M., Gerber, M., Hoppeler, H. et al. Exercise-induced angiogenesis correlates with the up-regulated expression of neuronal nitric oxide synthase (nNOS) in human skeletal muscle. Eur J Appl Physiol 112, 155–162 (2012). https://doi.org/10.1007/s00421-011-1960-x

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Keywords

  • Angiogenesis
  • Human exercise physiology
  • Neuronal nitric oxide synthase
  • Skeletal muscle