Ally provide benefit to all these conditions. Restoration and improvement of muscle mass have been reported in 6R-Tetrahydro-L-biopterin dihydrochloride site muscles of mice in which IGF-1 was specifically overexpressed, making hypertrophic myofibres that were able to elude age-related muscle atrophy [15]. Myostatin, a protein that negatively-regulates muscle mass, also appears to be a crucial regulator of muscle mass, as mutations in its gene cause muscle hypertrophy [16?2]. Blocking the myostatin pathway has been suggested as a potential way of intervention, since systemic delivery of myostatin 
antagonists [23], or inhibitors, induces muscle growth [24?6]. The role of satellite cells in adult muscle maintenance, as opposed to regeneration, has been controversial [27?0], but recent data have highlighted a subpopulation of satellite cells responsible for muscle growth and routine maintenance [8]. How their contribution is triggered and regulated remains to be investigated. Interestingly, signals responsible for muscle growth may originate from the fibre itself [31,32]. Shedding light on this key process is of fundamental importance in order to prevent muscle atrophy. Here, starting from our experimental observation that engraftment of single fibres in myotoxin-injured muscles causes an increase in the size of the grafted muscles, we have further explored this phenomenon. We found that grafting of a single fibre is able to trigger a hypertrophic muscle effect 
even in uninjured mdx mouse muscles and the presence of the fibre itself is an essential requirement for this effect.Hypertrophic Effect of Grafted Donor MyofibreFigure 1. Single myofibres grafted into BaCl2-treated host muscles give rise to no donor-derived muscle formation but cause muscle hypertrophy. Single 15481974 fibres (SF) isolated from a 3F-nlacZ-2E donor mouse were grafted into TA muscles that had been either irradiated 3 days before (n = 6) (A-I), or that had been BaCl2-injected three days before (n = 10) (A-II); as a control, DMEM was injected into untreated TA muscles (n = 10) (A-III). Donor-derived myofibres (identified by dystrophin expression and incorporation of X-gal positive myonuclei 4 weeks after grafting)Hypertrophic Effect of Grafted Donor Myofibrewere clearly observed in pre-irradiated grafted muscles (I), but to a trivial extent in BaCl2-injured grafted muscles (II) (B), as shown by representative pictures of dystrophin positive fibres with donor-derived myonuclei X-gal stained in serial sections (C and D respectively for I and II). H E staining of whole purchase SIS 3 transverse-sections from the largest middle part of grafted TA muscles highlighted the difference in size between muscles in I (E) and II (F). This difference was quantified in (G) showing that the cross-sectional area (CSA) of BaCl2-injured and SF-grafted muscles (II) was significantly bigger than in I and III. The number of fibres was not increased in II compared to control, but a loss of fibres was detected in I (H). Size bar = 100 mm. *p,0.05; ***p,0.0001. doi:10.1371/journal.pone.0054599.gMaterials and Methods Host Mice and Muscle InjuryBreeding of mice and experimental procedures were carried out in the Biological Services Unit of University College London, Institute of Child Health, in accordance with the Animals (Scientific Procedures) Act 1986. Experiments were performed under Home Office licence. Three-week-old mdx nude mice [33] were anaesthetised with hypnorm and hypnovel to irradiate their hindlimbs with 18Gy (at dose rate of 0.72Gy/minute) or isofl.Ally provide benefit to all these conditions. Restoration and improvement of muscle mass have been reported in muscles of mice in which IGF-1 was specifically overexpressed, making hypertrophic myofibres that were able to elude age-related muscle atrophy [15]. Myostatin, a protein that negatively-regulates muscle mass, also appears to be a crucial regulator of muscle mass, as mutations in its gene cause muscle hypertrophy [16?2]. Blocking the myostatin pathway has been suggested as a potential way of intervention, since systemic delivery of myostatin antagonists [23], or inhibitors, induces muscle growth [24?6]. The role of satellite cells in adult muscle maintenance, as opposed to regeneration, has been controversial [27?0], but recent data have highlighted a subpopulation of satellite cells responsible for muscle growth and routine maintenance [8]. How their contribution is triggered and regulated remains to be investigated. Interestingly, signals responsible for muscle growth may originate from the fibre itself [31,32]. Shedding light on this key process is of fundamental importance in order to prevent muscle atrophy. Here, starting from our experimental observation that engraftment of single fibres in myotoxin-injured muscles causes an increase in the size of the grafted muscles, we have further explored this phenomenon. We found that grafting of a single fibre is able to trigger a hypertrophic muscle effect even in uninjured mdx mouse muscles and the presence of the fibre itself is an essential requirement for this effect.Hypertrophic Effect of Grafted Donor MyofibreFigure 1. Single myofibres grafted into BaCl2-treated host muscles give rise to no donor-derived muscle formation but cause muscle hypertrophy. Single 15481974 fibres (SF) isolated from a 3F-nlacZ-2E donor mouse were grafted into TA muscles that had been either irradiated 3 days before (n = 6) (A-I), or that had been BaCl2-injected three days before (n = 10) (A-II); as a control, DMEM was injected into untreated TA muscles (n = 10) (A-III). Donor-derived myofibres (identified by dystrophin expression and incorporation of X-gal positive myonuclei 4 weeks after grafting)Hypertrophic Effect of Grafted Donor Myofibrewere clearly observed in pre-irradiated grafted muscles (I), but to a trivial extent in BaCl2-injured grafted muscles (II) (B), as shown by representative pictures of dystrophin positive fibres with donor-derived myonuclei X-gal stained in serial sections (C and D respectively for I and II). H E staining of whole transverse-sections from the largest middle part of grafted TA muscles highlighted the difference in size between muscles in I (E) and II (F). This difference was quantified in (G) showing that the cross-sectional area (CSA) of BaCl2-injured and SF-grafted muscles (II) was significantly bigger than in I and III. The number of fibres was not increased in II compared to control, but a loss of fibres was detected in I (H). Size bar = 100 mm. *p,0.05; ***p,0.0001. doi:10.1371/journal.pone.0054599.gMaterials and Methods Host Mice and Muscle InjuryBreeding of mice and experimental procedures were carried out in the Biological Services Unit of University College London, Institute of Child Health, in accordance with the Animals (Scientific Procedures) Act 1986. Experiments were performed under Home Office licence. Three-week-old mdx nude mice [33] were anaesthetised with hypnorm and hypnovel to irradiate their hindlimbs with 18Gy (at dose rate of 0.72Gy/minute) or isofl.