LETTERS. Sarcolemma-localized nnos is required to maintain activity after mild exercise

Transcription

1 Vol November 28 oi:1.138/nature7414 Sarcolemma-localize nnos is require to maintain activity after mil exercise Yvonne M. Kobayashi 1,2,3,4, Erik P. Raer 1,2,3,4, Robert W. Crawfor 1,2,3,4, Nikhil K. Iyengar 4, Daniel R. Theens 5, John A. Faulkner 7, Swapnesh V. Parikh 4, Robert M. Weiss 4, Jeffrey S. Chamberlain 8, Steven A. Moore 6 & Kevin P. Campbell 1,2,3,4 Many neuromuscular conitions are characterize by an exaggerate exercise-inuce fatigue response that is isproportionate to activity level. This fatigue is not necessarily correlate with greater central or peripheral fatigue in patients 1, an some patients experience severe fatigue without any emonstrable somatic isease 2. Except in myopathies that are ue to specific metabolic efects, the mechanism unerlying this type of fatigue remains unknown 2. With no treatment available, this form of inactivity is a major eterminant of isability 3. Here we show, using mouse moels, that this exaggerate fatigue response is istinct from a loss in specific force prouction by muscle, an that sarcolemmalocalize signalling by neuronal nitric oxie synthase (nnos) in skeletal muscle is require to maintain activity after mil exercise. We show that nnos-null mice o not have muscle pathology an have no loss of muscle-specific force after exercise but o isplay this exaggerate fatigue response to mil exercise. In mouse moels of nnos mislocalization from the sarcolemma, prolonge inactivity was only relieve by pharmacologically enhancing the cgmp signal that results from muscle nnos activation uring the nitric oxie signalling response to mil exercise. Our finings suggest that the mechanism unerlying the exaggerate fatigue response to mil exercise is a lack of contraction-inuce signalling from sarcolemma-localize nnos, which ecreases cgmp-meiate vasomoulation in the vessels that supply active muscle after mil exercise. Sarcolemmal nnos staining was ecrease in patient biopsies from a large number of istinct myopathies, suggesting a common mechanism of fatigue. Our results suggest that patients with an exaggerate fatigue response to mil exercise woul show clinical improvement in response to treatment strategies aime at improving exercise-inuce signalling. To unerstan the molecular basis of the exercise-inuce fatigue response, we stuie genetically efine mouse moels. We esigne an integrative in vivo assay to test conscious mice, subjecting the mice to brief low-spee treamill exercise followe by testing in an openfiel activity chamber (see Methos). We first assesse two ystrophic mouse lines, (moel for Duchenne muscular ystrophy) 4 an (moel for limb-girle muscular ystrophy type 2D that is eficient for the gene encoing a-sarcoglycan (Sgca)) 5. In the absence of previous exercise, activity in these mice was inistinguishable from that of wil-type mice (Fig. 1a, b, an Supplementary Vieos 1a ). After a single trial of mil exercise, significant ifferences were observe (Fig. 1a, b, an Supplementary Vieos 2a ): the an mice showe a significant ecrease in vertical activity. The ecrease in vertical activity among an mice i not correlate with ifferences in extensor igitorum longus (EDL)- specific force measurements relative to those taken in C57BL/6 mice before exercise (Fig. 1c). Moreover, mice o not evelop brain, heart or vascular pathology 6, an they have muscle-force values similar to those of control mice 7. Therefore, neither cariac eficiency nor an inability to prouce force was the cause of the postexercise inactivity in the mice. Because inflammation is a feature of ystrophinopathy 4, chronic fatigue is associate with muscle pain, an chronic pain is associate with fatigue 8, we treate mice with either eflazacort or ibuprofen. However, neither treatment resulte in improve post-exercise activity (Fig. 1), suggesting that the inactivity occurring immeiately after mil exercise in mice was not ue to inflammation or pain. Overall, the results of our exercise activity assay implie that the exaggerate fatigue response in these mice was not attributable to cariac eficiency, inflammation, pain or lack of muscle force. To test whether the exercise-inuce inactivity in the an mice was ue to the genetically etermine structural efect in muscle, we assaye two mouse moels in which the muscle pathology relate to the specific ystrophin glycoprotein complex (DGC) efect is rescue microystrophin/ an MCKeSG/. In microystrophin/ mice (a moel for mil Becker muscular ystrophy 9 the DGC has a mutate but functional ystrophin), microystrophin is expresse in mouse muscle. In the MCKeSG/ mice, e-sarcoglycan is expresse in mouse muscle that is eficient for Sgca (Supplementary Fig. 1). Neither rescue strain showe pathological signs of muscular ystrophy, an the skeletal muscle DGC of both was recovere at the biochemical, structural an functional levels (refs 9, 1 an Supplementary Figs 1 an 2). Despite having a structurally intact skeletal muscle DGC, microystrophin/ mice experience a substantial ecrease in activity after mil exercise, like their littermates (Fig. 1e). Because patients with Becker muscular ystrophy show profoun fatigue after light exertion 11, an loss of sarcolemma-localize nnos serves as a iagnostic inicator of some forms of Becker muscular ystrophy 12,a possible reason for the post-exercise inactivity is a loss of sarcolemma-localize nnos. To test this possibility we probe for nnos localization in microystrophin/ skeletal muscle an foun that the DGC generate in this rescue strain faile to recruit nnos to the sarcolemma (Fig. 1e, inset). These ata are in agreement with recent reports on microystrophin expression in ystrophineficient mouse moels 13. Moreover, the ata suggest that exercise- 1 Howar Hughes Meical Institute, 2 Department of Molecular Physiology an Biophysics, 3 Department of Neurology, 4 Department of Internal Meicine, 5 Department of Raiology, 6 Department of Pathology, University of Iowa, Roy J. an Lucille A. Carver College of Meicine, 4283 Carver Biomeical Research Builing, 285 Newton Roa, Iowa City, Iowa , USA. 7 Department of Molecular an Integrative Physiology, University of Michigan, 231 Biomeical Sciences Research Builing, Ann Arbor, Michigan , USA. 8 Department of Neurology, University of Washington School of Meicine, HSB, Room K243b, Seattle, Washington , USA. 28 Macmillan Publishers Limite. All rights reserve 511

2 NATURE Vol November 28 a C57BL/1 c 25 2 EDL specific force (kn m 2 ) Vertical activity Pre-exercise Post-exercise C57BL/6 e 1,2 f 3 min 3 min b 3 min 3 min C57BL/1 C57BL/ Microystrophin/ C57BL/6 MCKεSG/ Untreate Acute Chronic Acute eflazacort eflazacort ibuprofen Wil type C57BL/6 MCKεSG/ hom cm Microystrophin/ Figure 1 Loss of sarcolemma-localize nnos leas to skeletal muscle vascular narrowings, ecrease capillary perfusion an an exaggerate fatigue response after mil exercise in ystrophic an non-ystrophic mouse moels. a, Representative vertical activity tracings of zone maps for C57BL/1 an mice before an after exercise. b, Quantifie vertical activity before (fille columns) an after (open columns) exercise for C57BL/1, C57BL/6, an mouse strains (n 5 6 for each strain). Asterisk, P c, EDL muscle-specific force measurements from C57BL/6 (n 5 6), (n 5 4) an (n 5 4) mice. Asterisk, P,.5., Pre-exercise (fille columns) an post-exercise (open columns) vertical activity in untreate (n 5 7) an anti-inflammatory treate mice, acutely (n 5 4) or chronically (n 5 4) with eflazacort or acutely with ibuprofen (n 5 5). Asterisk, P,.3. e, Left panel: quantifie pre-exercise (fille columns) an post-exercise (open columns) vertical activity for microystrophin/ mice (n 5 6) an their littermates (n 5 4). Asterisk, P 5.5; two asterisks, P,.1. The right panels show representative immunofluorescence images of nnos etection in the gastrocnemius muscles from C57BL/6 an microystrophin/ mice. f, Quantifie pre-exercise (fille columns) an post-exercise (open columns) vertical activity for MCKeSG/ mice (n 5 6) an their littermates (n 5 6). Asterisk, P,.1. Inset: immunoblot etection of total nnos from homogenates (hom), an crue skeletal muscle membranes (cm). g, Representative Microfil image of skeletal muscle vessels of MCKeSG/ mice after exercise large arrowheas mark extene areas of vascular narrowing; the small arrow marks a shorter stretch of raial vascular narrowing. Error bars inicate s.e.m. inuce inactivity in the microystrophin/ mice is not cause irectly by a structurally efective muscle DGC, an that loss of sarcolemmal nnos oes not negatively affect muscle contractility. Thus, sarcolemmal nnos seems to act at the level of post-exercise activity. In contrast to the microystrophin/ mice, MCKeSG/ mice have structurally intact DGC in the brain an the vasculature, but express e-sarcoglycan instea of a-sarcoglycan in the DGC of muscle. Our exercise activity assay showe that post-exercise activity 512 g 28 Macmillan Publishers Limite. All rights reserve in the MCKeSG/ mice was substantially ecrease relative to that in C57BL/6 mice but similar to that in an mice (Fig. 1b, f). Because the microystrophin-containing DGC faile to recruit nnos, we speculate that the MCKeSG/ mice woul also fail to localize nnos to the sarcolemma. Inee, although total nnos levels in muscle homogenates from MCKeSG/ mice were similar to those in the wil type, nnos from the rescue moel faile to purify together with the e-sarcoglycan-containing DGC in the membrane preparation (Fig. 1f, inset). Taken together, these results are compatible with the notion that the exaggerate fatigue response is not irectly relate to a structurally efective muscle DGC or to muscle weakness, but rather to a failure in the sarcolemmal localization of nnos. Because sarcolemma-localize nnos is crucial for maintaining vasomoulation to contracting muscles 14, we teste whether communication from skeletal muscle to the local bloo supply is eficient after mil exercise by perfusing MCKeSG/ mouse arteries before or after exercise with Microfil an examine the skeletal muscle vasculature (Fig. 1g). We ientifie vascular narrowings of various lengths along the arteries that fee the skeletal muscles in the post-exercise samples only, an also note the lack of perfusion of capillaries. The an microystrophin/ mice similarly showe skeletal muscle vascular narrowings only after exercise an also a lack of perfusion of capillaries (Supplementary Fig. 3c an ata not shown). This phenotype is consistent with inefficient contraction-inuce muscle nnos signalling to local bloo vessels. Overall, these ata imply that loss of sarcolemma-localize nnos causes eficient exercise-inuce vasomoulation in skeletal muscle, an that these lea to prolonge inactivity after mil exercise. To irectly examine the contribution of NO generate by enothelial NOS (enos) or nnos to the exaggerate fatigue response, we teste both nnos-null an enos-null mice in our exercise activity assay. Mice eficient for nnos express normal levels of the DGC components at the sarcolemma an have histologically normal muscle Reports suggest that both mouse strains have efective vasoregulation 18,19 ; however, an nnos-null mice have a normal a-arenergic vasoconstrictive response to exercise 2. Vertical preexercise activities were similar in enos-null, nnos-null an C57BL/6 mice, suggesting that the loss of either NOS oes not affect mouse activity (Fig. 2a). After exercise, however, nnos-null vertical activity ecrease significantly (Fig. 2a). Serum creatine kinase levels before an after exercise for each of the NOS-null mice were similar to those in C57BL/6 mice an low compare with mice (Fig. 2b), an there were no signs of muscle pathology in sections from nnosnull quariceps muscle (Supplementary Fig. 4b), suggesting that muscle amage an necrosis were not the causes of the post-exercise inactivity. We then teste whether post-exercise muscle contractility affecte the ability of C57BL/6 an nnos-null skeletal muscle to prouce force after mil exercise. We foun that the specific force of EDL muscles after exercise was not significantly affecte in nnosnull muscle in comparison with C57BL/6 muscle (Supplementary Fig. 4c). Because lack of muscle contractility was not causing the inactivity in the nnos-null mice after exercise, we checke whether NOS-null mice ha post-exercise skeletal muscle vascular narrowings an lack of capillary perfusion similar to those in the ystrophic an rescue mice. Microfil perfusion of arteries from NOS-null mice before an after exercise reveale the lack of capillary perfusion an also the presence of vascular narrowings only in post-exercise nnos-null skeletal muscle (Fig. 2c). We also foun that treating wil-type mice with either the nnos-specific inhibitor 3-bromo-7- nitroinazole or the vasoconstrictor sarafotoxin 6c cause post-exercise inactivity (Fig. 2). These finings suggest that a eficiency of sarcolemma-localize nnos causes exercise-inuce narrowing of the vasculature that fees active muscles after exercise, thereby promoting prolonge inactivity after mil exercise. To test whether the vascular effect on post-exercise activity was from NO or was ownstream of the NO signal, we bypasse

3 NATURE Vol November 28 sarcolemmal nnos signalling for ecreasing vasoconstriction by treating mice with a panel of pharmacological agents that promote vasoilation; we foun that the exaggerate fatigue response was alleviate only by treatment with a phosphoiesterase (PDE) 5A inhibitor (Supplementary Fig. 6), suggesting that the fatigue that we saw epene on cgmp, which acts ownstream of NO prouction. PDE activity in mice is 2 6-fol higher than in C57BL/1 mice 21, which is consistent with the elevate PDE activity in human muscular isorers 18,21,22. We treate nnos-null, MCKeSG/ an mice with PDE5A inhibitors an teste them in our exercise activity assay; we foun that the treate MCKeSG/ an mice showe an increase in post-exercise activity (Fig. 2e an Supplementary Fig. 7a ). Because inhibition of PDE5A ha no effect on activity before exercise, our results suggest that PDE5A inhibition is alleviating the exaggerate fatigue response by enhancing the cgmp signal prouce by contraction-inuce nnos stimulation. Although ownstream effectors of cgmp are numerous a 3 min c e 3 min C57BL/6 enos-null nnos-null b 1, log[serum CK (U l 1 )] 1, 1, 1 C57BL/6 8 3 min 1 1 Untreate Untreate Treate Untreate Treate Untreate Treate nnos-null MCKεSG/ enos-null nnos-null 3-Bi-7-Ni Sarafotoxin Figure 2 Enhancing the cgmp signal resulting from muscle nnos activation ecreases the exaggerate fatigue response to mil exercise. a, Comparison of vertical activity before (fille columns) an after (open columns) exercise between C57BL/6, enos-null an nnos-null mice (n 5 6 for each). Two asterisks, P,.1. b, Serum creatine kinase (CK) levels before (fille columns) an after (open columns) exercise in C57BL/6 (n 5 6), enos-null (n 5 4) an nnos-null (n 5 6) mice, compare with mice (n 5 6). c, Representative Microfil image of nnos-null quariceps skeletal muscle arteries after exercise the large arrowhea marks the extene area of vascular narrowing; small arrows mark shorter areas of raial vascular narrowing. Scale bar, 1 mm., Pre-exercise (fille columns) an post-exercise (open columns) vertical activities in untreate wil-type (C57BL/6 an C57BL/1) mice (n 5 4), compare with 3-B-7-Ni-treate wil-type mice (n 5 3) an sarafotoxin-treate wil-type mice (n 5 4). Asterisk, P,.1. e, Quantifie pre-exercise (fille columns) an postexercise (open columns) activity with an without treatment with PDE5A inhibitor, in nnos-null (n 5 4), MCKeSG/ (n 5 4) an mice (n 5 6). Asterisk, P,.1. Pre-exercise an post-exercise vertical activity error bars are s.e.m an ivergent 23, the half-life of cgmp can be affecte by the activity of PDE5A. Our ata inicate that the elevate PDE activity in extracts from mice coul be PDE5A activity, an that PDE activity coul also be elevate in the rescue mouse moels we teste. Our ata suggest that the local resistance of arterioles that perfuse sarcolemmal nnos-eficient muscles increases uring exercise an that the lack of activity after mil exercise will lea to muscle oeema. We examine bloo flow before an after exercise with laser Doppler imaging an foun that bloo flow in mice faile to increase as it i in C57BL/6 mice (Fig. 3a an Supplementary Fig. 7a), but treatment of mice with a PDE5A inhibitor alleviate this efect (Fig. 3b) an increase muscle capillary perfusion (Fig. 3c). Given that insufficient relief of local vasoconstriction in active muscles can lea to muscle oeema 24, an that boys with Duchenne muscular ystrophy show muscle oeema 25, we looke for changes in water compartmentalization an ynamics in the hinleg muscles of nnos-null, C57BL/1 an mice before an after exercise by using spin spin relaxation time (T 2 )-magnetic resonance imaging. The nnos-null mice i not have muscle amage or loss of contractility after exercise (Fig. 2b an Supplementary Fig. 4b, c), nor i they have muscle oeema (Supplementary Fig. 8a), suggesting that their lack of muscle amage prevents water accumulation in the tissue. Similarly, C57BL/1 mice showe little to no oeema in hinleg muscles after exercise (.7 6.5% (mean 6 s.e.m.)) (Fig. 3f an a b c f Muscle oeema area (%) Macmillan Publishers Limite. All rights reserve Wil type +PDE5Ai Figure 3 Treatment with PDE5A inhibitor improves exercise-inuce vasomoulation an ecreases exercise-inuce oeema in mice. a, Representative images of coronal laser Doppler analysis of bloo flow in mice before (top) an after (bottom) exercise (n 5 3). b, Coronal laser Doppler analysis of bloo flow in mice, before (top) an after (bottom) exercise, treate with PDE5A inhibitor before exercise (n 5 3). c, Representative Microfil image of quariceps skeletal muscle arteries after exercise from mice treate with PDE5A inhibitor before exercise (n 5 3; scale bar, 1 mm)., e, Representative axial views, by magnetic resonance imaging, of hinlimb muscles before (left) an after (right) exercise (n 5 5) () an hinlimb muscles after exercise of mice treate with PDE5A inhibitor before exercise (n 5 5) (e). White arrowheas mark areas of increase water compartmentalization. f, Percentage muscle oeema area before (fille columns) an after (open columns) exercise, an with or without treatment with PDE5A inhibitor, in mice compare with that of the wil type. (Wil type an, n 5 3; plus PDE5A inhibitor, n 5 5; error bars are s.e.m.) Asterisk, P,.1. e 513

4 NATURE Vol November 28 Spectrin nnos Spectrin nnos Control LGMD-2B DMD UCMD BMD MDC1A LGMD-2I LGMD-2D Figure 4 nnos levels in sarcolemma are ecrease in human muscle iseases. Representative immunofluorescent staining in various human muscle iseases: primary ystrophinopathies, Duchenne an Becker muscular ystrophy (DMD an BMD, respectively); in several forms of limb-girle muscular ystrophy (LGMD); in two congenital muscular ystrophies (CMD) cause by mutations in extracellular matrix proteins (Ullrich CMD (UCMD), collagen VI an merosin-eficient CMD (MDC1A). Asterisks mark the same muscle fibres in some of the ajacent panels. Scale bar, 1 mm. Supplementary Fig. 8b). However, hinleg muscles of mice consistently showe significant changes in tissue hyration after exercise ( % (mean 6 s.e.m.)) (Fig. 3, f) that were inicative of exercise-inuce muscle oeema. The water accumulation observe in the muscles is probably ue to a combination of the increase local resistance in the arterioles that fee the active leg muscles an of muscle fibre fragility an amage. We also consistently foun that treatment with PDE5A inhibitor significantly ecrease exercise-inuce muscle oeema in mice ( % (mean 6 s.e.m.); Fig. 3e, f). Overall, our ata imply that treatment with PDE5A inhibitor can relieve the post-exercise inactivity by normalizing PDE activity, thereby allowing the available NO erive from muscle nnos to signal for cgmp-epenent vasoilation in active muscle; treatment with PDE5A inhibitor ecreases muscle amage in mice by improving moulation of vascular activity in active muscle, thus preventing muscle oeema from exacerbating the muscle amage that occurs uring the contraction of ystrophic muscle. Because more than 6% of all patients with neuromuscular isease suffer from severe fatigue 2, we teste for nnos localization to the sarcolemma in biopsies of patients representing ifferent myopathic isorers (Fig. 4, Supplementary Fig. 9 an Supplementary Table 1). In most myopathic biopsies assesse, sarcolemma-localize nnos was either reuce or not etecte, implying that many myopathic isorers may share a mechanism that results in severe exerciseinuce fatigue. Although increase fatigability inevitably occurs in patients with muscle weakness 1, our mouse ata imply that the exercise-inuce inactivity is istinct from muscle weakness an that loss of sarcolemma-localize nnos leas to an exaggerate fatigue response to mil exercise. Our mouse ata show that ecrease or mislocalize skeletal muscle nnos exacerbates the fatigue experience after mil exercise because the normal contraction-inuce cgmp-epenent attenuation of local vasoconstriction fails to occur, an that this failure causes vascular narrowing in muscles after exercise. In aition, our ata from mice suggest that, as a result of nnos mislocalization 514 an increase PDE activity 1,18,21, signalling for increase vasoilation to active muscle is eficient, causing muscle oeema. This, in turn, contributes to increase muscle amage as well as profoun postexercise ebility. Although the exact mechanism that leas to the inactivity after mil exercise has not been reuce to a single beginning an en pathway, our ata suggest that contraction-inuce cgmp-epenent attenuation of local vasoconstriction is pivotal in this mechanism. These finings coul lea to a better unerstaning of muscle fatigue uner other physiological conitions in which muscle nnos expression, localization or activity is affecte. METHODS SUMMARY Mouse moels. Animal care an proceures were approve an performe in accorance with the stanars set forth by the National Institutes of Health an the University of Iowa Animal Care an Use Committee. Treamill exercise an activity monitoring. Animals were milly exercise with an ajustable variable-spee belt treamill from AccuPacer. Activity base on ambulatory behaviour was assesse in an open-fiel test. Receive 16 January; accepte 29 August 28. Publishe online 26 October Macmillan Publishers Limite. All rights reserve 1. Schillings, M. L. et al. Experience an physiological fatigue in neuromuscular isorers. Clin. Neurophysiol. 118, (27). 2. Zwarts, M. J., Bleijenberg, G. & van Engelen, B. G. Clinical neurophysiology of fatigue. Clin. Neurophysiol. oi:1.116/j.clinph (27). 3. Kalkman, J. S., Schillings, M. L., Zwarts, M. J., van Engelen, B. G. & Bleijenberg, G. The evelopment of a moel of fatigue in neuromuscular isorers: A longituinal stuy. J. Psychosom. Res. 62, (27). 4. Raley, H. G., De Luca, A., Lynch, G. S. & Grouns, M. D. Duchenne muscular ystrophy: focus on pharmaceutical an nutritional interventions. Int. J. Biochem. Cell Biol. 39, (27). 5. Duclos, F. et al. Progressive muscular ystrophy in a-sarcoglycan-eficient mice. J. Cell Biol. 142, (1998). 6. Ozawa, E., Mizuno, Y., Hagiwara, Y., Sasaoka, T. & Yoshia, M. Molecular an cell biology of the sarcoglycan complex. Muscle Nerve 32, (25). 7. Consolino, C. M. et al. Muscles of mice eficient in a-sarcoglycan maintain large masses an near control force values throughout the life span. Physiol. Genomics 22, (25). 8. Yokoyama, T., Lisi, T. L., Moore, S. A. & Sluka, K. A. Muscle fatigue increases the probability of eveloping hyperalgesia in mice. J. Pain 8, (27).

5 NATURE Vol November Harper, S. Q. et al. Moular flexibility of ystrophin: implications for gene therapy of Duchenne muscular ystrophy. Nature Me. 8, (22). 1. Imamura, M., Mochizuki, Y., Engvall, E. & Takea, S. I. e-sarcoglycan compensates for lack of a-sarcoglycan in a mouse moel of limb-girle muscular ystrophy. Hum. Mol. Genet. 14, (25). 11. Phillips, B. A. & Mastaglia, F. L. Exercise therapy in patients with myopathy. Curr. Opin. Neurol. 13, (2). 12. Torelli, S. et al. Absence of neuronal nitric oxie synthase (nnos) as a pathological marker for the iagnosis of Becker muscular ystrophy with ro omain eletions. Neuropathol. Appl. Neurobiol. 3, (24). 13. Juge, L. M., Haraguchiln, M. & Chamberlain, J. S. Dissecting the signaling an mechanical functions of the ystrophin glycoprotein complex. J. Cell Sci. 119, (26). 14. Thomas, G. D., Shaul, P. W., Yuhanna, I. S., Froehner, S. C. & Aams, M. E. Vasomoulation by skeletal muscle-erive nitric oxie requires a-syntrophinmeiate sarcolemmal localization of neuronal nitric oxie synthase. Circ. Res. 92, (23). 15. Chao, D. S., Silvagno, F. & Bret, D. S. Muscular ystrophy in mice espite lack of neuronal nitric oxie synthase. J. Neurochem. 71, (1998). 16. Crosbie, R. H. et al. muscle pathology is inepenent of nnos perturbation. Hum. Mol. Genet. 7, (1998). 17. Suzuki, N. et al. NO prouction results in suspension-inuce muscle atrophy through islocation of neuronal NOS. J. Clin. Invest. 117, (27). 18. Asai, A. et al. Primary role of functional ischemia, quantitative evience for the two-hit mechanism, an phosphoiesterase-5 inhibitor therapy in mouse muscular ystrophy. PLoS ONE 2, e86 (27). 19. Huang, P. L. et al. Hypertension in mice lacking the gene for enothelial nitric oxie synthase. Nature 377, (1995). 2. Thomas, G. D. et al. Impaire metabolic moulation of a-arenergic vasoconstriction in ystrophin-eficient skeletal muscle. Proc. Natl Aca. Sci. USA 95, (1998). 21. Bloom, T. J. Age-relate alterations in cyclic nucleotie phosphoiesterase activity in ystrophic mouse leg muscle. Can. J. Physiol. Pharmacol. 83, (25). 22. Bloom, T. J. Cyclic nucleotie phosphoiesterase isozymes expresse in mouse skeletal muscle. Can. J. Physiol. Pharmacol. 8, (22). 23. Kass, D. A., Champion, H. C. & Beavo, J. A. Phosphoiesterase type 5: expaning roles in cariovascular regulation. Circ. Res. 11, (27). 24. Persson, J., Ekelun, U. & Grane, P. O. Enogenous nitric oxie reuces microvascular permeability an tissue oeema uring exercise in cat skeletal muscle. J. Vasc. Res. 4, (23). 25. Maren, F. A., Connolly, A. M., Siegel, M. J. & Rubin, D. A. Compositional analysis of muscle in boys with Duchenne muscular ystrophy using MR imaging. Skeletal Raiol. 34, (25). Supplementary Information is linke to the online version of the paper at Acknowlegements We thank M. Anerson an M. Henry for comments, an M. M. Kilburg, K. Uppal, B. J. Steinmann an S. Watkins an members of the Campbell laboratory for scientific contributions. This work was supporte in part by a Paul D. Wellstone Muscular Dystrophy Cooperative Research Center Grant. Y.M.K. was supporte by grants from the University of Iowa Cariovascular Interisciplinary Research/ National Research Service Awar (NRSA) Fellowship, from an iniviual NRSA Fellowship from the National Institute of Arthritis an Musculoskeletal an Skin Diseases, from the National Institutes of Health (NIH), an from a Senator Paul D. Wellstone Fellowship. E.P.R. was supporte by a Muscular Dystrophy Association Development Grant. R.M.W. was supporte by the NIH. K.P.C. is an investigator of the Howar Hughes Meical Institute. Author Information Reprints an permissions information is available at Corresponence an requests for materials shoul be aresse to K.P.C. (kevin-campbell@uiowa.eu). 28 Macmillan Publishers Limite. All rights reserve 515