2008 Research Article Sarcolemmal nNOS anchoring reveals a qualitative difference between and utrophin

Dejia Li1,*, Akshay Bareja2,*, Luke Judge3,*, Yongping Yue1, Yi Lai1, Rebecca Fairclough2, Kay E. Davies2, Jeffrey S. Chamberlain3 and Dongsheng Duan1,‡ 1Department of Molecular Microbiology and Immunology, School of Medicine, The University of Missouri, M610G Medical Science Building, Columbia, MO 65212, USA 2Department of Physiology Anatomy and Genetics, University of Oxford, Le Gros Clark Building, South Parks Road, Oxford OX1 3QX, UK 3Department of Neurology, The University of Washington, Health Sciences Building K234, 1959 NE Pacific Street, Box 357720, Seattle, WA 98195, USA *These authors contributed equally to this work ‡Author for correspondence ([email protected])

Accepted 17 March 2010 Journal of Cell Science 123, 2008-2013 © 2010. Published by The Company of Biologists Ltd doi:10.1242/jcs.064808

Summary Duchenne muscular dystrophy (DMD) is a lethal muscle disease caused by dystrophin deficiency. In normal muscle, dystrophin helps maintain sarcolemmal stability. Dystrophin also recruits neuronal (nNOS) to the . Failure to anchor nNOS to the membrane leads to functional ischemia and aggravates muscle disease in DMD. Over the past two decades, a great variety of therapeutic modalities have been explored to treat DMD. A particularly attractive approach is to increase utrophin expression. Utrophin shares considerable sequence, structural and functional similarity with dystrophin. Here, we test the hypothesis that utrophin also brings nNOS to the sarcolemma. Full-length utrophin cDNA was expressed in dystrophin-deficient mdx mice by gutted adenovirus or via transgenic overexpression. Subcellular nNOS localization was determined by immunofluorescence staining, in situ nNOS activity staining and microsomal preparation western blot. Despite supra-physiological utrophin expression, we did not detect nNOS at the sarcolemma. Furthermore, transgenic utrophin overexpression failed to protect mdx muscle from exercise-associated injury. Our results suggest that full-length utrophin cannot anchor nNOS to the sarcolemma. This finding might have important implications for the development of utrophin-based DMD therapies.

Key words: Dystrophin, Utrophin, nNOS, Duchenne muscular dystrophy

Journal of Cell Science Introduction ready diffusion of nitric oxide to the nearby vasculature to Duchenne muscular dystrophy (DMD) is the most common counteract a-adrenergic vasoconstriction during . childhood lethal muscle disease. It is caused by mutations in the In the absence of dystrophin, sarcolemmal nNOS expression is dystrophin gene (Kunkel, 2005). The dystrophin gene (DMD) lost. Consequently, the protective vessel relaxation mechanism is encodes a 427 kDa multiple-domain cytosolic . The N- compromised (Brenman et al., 1995; Chang et al., 1996; Lai et al., terminal domain of dystrophin interacts with cytosolic F-. The 2009; Sander et al., 2000; Thomas et al., 1998). In this regard, central rod domain contains 24 spectrin-like repeats and four contraction-associated ischemic injury has been recognized as one hinges. The C-terminal domain carries the binding motifs for of the earliest pathological changes in DMD muscle (Mendell et several cytosolic such as syntrophin and . A al., 1971; Parker and Mendell, 1974). The physiological relevance cysteine-rich domain sits between the central rod and the C-terminal of membrane-associated nNOS was further emphasized by several domains and it connects dystrophin to the extracellular matrix via recent reports (Kobayashi et al., 2008; Lai et al., 2009; Percival et . The dystrophin-dystroglycan complex is further al., 2008). In these studies, investigators found that sarcolemmal strengthened by the and . Together, nNOS prevented exercise-related fatigue and improved exercise dystrophin and its associated proteins protect the sarcolemma from performance in dystrophic subjects. In summary, restoring contraction-induced injury (for a review, see Blake et al., 2002; sarcolemmal nNOS could represent an important therapeutic Ervasti, 2007). endpoint. In DMD patients, dystrophin expression is abolished owing to Soon after the discovery of the dystrophin gene on the X- gene mutation. As a result, dystrophin-associated proteins chromosome, the utrophin gene (UTRN) was identified as an disassemble from the muscle membrane and the sarcolemma autosomal paralog of the dystrophin gene (Khurana et al., 1990; integrity is reduced. Although the loss of the physical support has Love et al., 1989; Tinsley et al., 1992). Similarly to dystrophin, certainly contributed to the muscle disease, recent studies have utrophin also contains four major functional domains, including begun to appreciate other pathogenic factors (Heydemann et al., the N-terminal, central rod, cysteine-rich and C-terminal domains. 2007). Among these, neuronal nitric oxide synthase (nNOS) is The N-terminal, cysteine-rich and C-terminal domains are 80% particularly interesting. In normal , nNOS is identical to those of dystrophin (Tinsley et al., 1992). Because of recruited to the sarcolemma by dystrophin and syntrophin (Adams the extraordinary sequence homology and structural resemblance, et al., 2000; Hillier et al., 1999; Kameya et al., 1999; Lai et al., it is not surprising that utrophin stabilizes the sarcolemma by 2009; Tochio et al., 1999). Membrane location of nNOS allows orchestrating dystrophin-associated proteins into a similar complex Utrophin cannot bring nNOS to sarcolemma 2009

linking the extracellular matrix with the cytoskeleton (for a review, see Blake et al., 2002; Ervasti, 2007). Considering the importance of sarcolemmal nNOS in DMD pathogenesis and therapy, we sought to determine whether utrophin was able to recruit nNOS to the sarcolemma. It has been well established that sarcolemmal nNOS anchoring is mediated by the syntrophin PDZ domain (Adams et al., 2001; Hillier et al., 1999; Tochio et al., 1999). We have recently shown that this process also requires dystrophin spectrin-like repeats 16 and 17 (R16/17) (Lai et al., 2009). Whereas dystrophin carries 24 spectrin-like repeats, utrophin contains 22 repeats. Although the repeats corresponding to dystrophin R16/17 appear to be preserved in utrophin, individual repeat units show considerable sequence divergence and it remains unclear whether utrophin repeats can interact with nNOS (Winder et al., 1995). Based on the known structural similarity, we hypothesized that utrophin could anchor nNOS to the sarcolemma. To test this hypothesis, we first overexpressed full-length utrophin using gutted adenoviral vectors in dystrophin-deficient mdx mouse muscles. Surprisingly, we did not detect sarcolemmal nNOS expression by immunofluorescence staining. To thoroughly address this issue, we compared nNOS expression in C57Bl/10 (BL10), mdx, utrophin-null, utrophin-dystrophin double knockout (u-dko) and full-length utrophin transgenic mdx mice. Through nNOS immunofluorescence staining, in situ activity staining and microsomal western analysis, we found that the loss of utrophin had a nominal effect on sarcolemmal nNOS localization. Furthermore, supra-physiological utrophin expression did not restore sarcolemmal nNOS expression. Importantly, chronic treadmill running resulted in apparent muscle degeneration- regeneration and reduction of specific muscle force in utrophin transgenic mdx mice, but not in normal control mice.

Results Journal of Cell Science Expression of gutted-adenovirus-mediated full-length dystrophin, but not utrophin, restores sarcolemmal nNOS To determine whether utrophin recruits nNOS to the sarcolemma, we delivered full-length mouse utrophin to the anterior tibialis Fig. 1. Adenovirus-mediated full-length utrophin expression does not (TA) muscle in mdx mice using a gutted adenoviral vector. Gutted restore sarcolemmal nNOS. (A)Representative dystrophin and nNOS adenoviral vectors carrying full-length cDNA encoding human or immunofluorescence staining photomicrographs from normal and mdx mouse dystrophin were included in the study as the positive control. muscles. Some reverent fibers can restore nNOS (arrowhead) whereas others Two months after gene transfer, we examined nNOS expression by cannot (arrow). (B)Representative immunofluorescence staining immunofluorescence staining. Robust utrophin and dystrophin photomicrographs at 2 months after mdx muscles were infected with gutted expression was detected in mdx muscles injected with the respective adenoviruses. Left panels are dystrophin and nNOS staining following human adenoviral vectors (Fig. 1). Although sarcolemmal nNOS staining full-length dystrophin gutted adenovirus infection. Right panels are utrophin and nNOS staining following mouse full-length utrophin gutted adenovirus was restored following viral-mediated expression of full-length infection. gAd, gutted adenovirus; HDys, human dystrophin; Utr, utrophin. human (Fig. 1B) or mouse (data not shown) dystrophin, we did not (C)Two examples of serial sections from full-length utrophin gutted observe membrane-associated nNOS immunoreactivity in mdx adenovirus infected muscles stained with antibodies against utrophin (left muscles infected by the full-length utrophin vector (Fig. 1B). As panels) and a-syntrophin (right panels). Myofibers expressing high levels of demonstrated before, some (but not all) revertant fibers showed utrophin at the sarcolemma also expressed a-syntrophin. Asterisks indicate the membrane-associated nNOS expression (Lai et al., 2009; Wells et same myofiber in serial sections. al., 2003). The absence of a-syntrophin compromises sarcolemmal nNOS localization (Adams et al., 2000; Kameya et al., 1999). If utrophin Transgenic overexpression of full-length utrophin fails to fails to recruit a-syntrophin, it might explain our finding. To test recover sarcolemmal nNOS this hypothesis, we examined serial muscle sections from gutted To further extend the intriguing finding seen with the gutted adenoviral utrophin-injected muscles (Fig. 1C). Consistent with adenovirus, we examined nNOS localization in utrophin-deficient our previous studies (Odom et al., 2008), full-length utrophin mice (utrophin knockout and u-dko) and full-length utrophin delivered by gutted adenovirus restored sarcolemmal a-syntrophin transgenic mdx mice. Immunostaining was performed on serial TA expression (Fig. 1C). muscle sections for dystrophin, utrophin, syntrophin and nNOS. In 2010 Journal of Cell Science 123 (12)

the case of syntrophin, a pan-syntrophin antibody was used to detect all syntrophin isoforms. To corroborate immunostaining results, we also stained for nNOS activity in adjacent muscle sections. In BL10 mice, we observed uniform dystrophin, syntrophin and nNOS expression at the sarcolemma. Consistent with previous reports (Blake et al., 2002; Nguyen et al., 1991; Rivier et al., 1997), utrophin was detected in the microvasculature in normal muscle (Fig. 2A). In dystrophin-deficient mdx mice, utrophin was upregulated and elevated utrophin resulted in low- level syntrophin staining at the sarcolemma. However, neither nNOS immunostaining nor nNOS activity staining generated positive signals (Fig. 2B). Eliminating utrophin alone had minimal influence on sarcolemmal nNOS expression (Fig. 2C). As expected, u-dko mice displayed much severe muscle disease (Deconinck et al., 1997; Grady et al., 1997). We observed substantial sarcolemmal damage in u-dko mice, as illustrated by extensive immunoglobulin infiltration (Lai et al., 2005). Nonetheless, the nNOS expression pattern was not altered (Fig. 2D). In the Fiona line of full-length utrophin transgenic mdx mice (Fiona mice) (Tinsley et al., 1998), strong utrophin expression was detected in every myofiber (Fig. 2E). Transgenic full-length utrophin overexpression also reduced central nucleation and inflammation (Fig. 2E) (Tinsley et al., 1998). Consistent with the results of the adenoviral vector experiment (Fig. 1), supra-physiological utrophin expression in transgenic mice did not restore nNOS to the sarcolemma (Fig. 2E). To confirm immunostaining results, we performed western analysis with total muscle lysates and membrane-enriched microsomal preparations. Using whole-muscle lysates, dystrophin was detected only in BL10 and utrophin-null muscles. Normal muscle showed little utrophin expression. Utrophin was completely eliminated in utrophin-null and u-dko muscles. Although utrophin levels were moderately increased in mdx muscles, the highest utrophin expression was observed in Fiona mouse muscles (Fig. 3A). Total cellular nNOS levels were substantially reduced in mdx

Journal of Cell Science and u-dko muscles, but not in utrophin-null and Fiona mouse muscles (Fig. 3A). Membrane-associated syntrophin and nNOS were evaluated by western blot analysis of muscle microsomes. Similarly to the immunostaining results, syntrophin levels were normalized in utrophin transgenic muscles. However, supra-physiological utrophin expression did not restore nNOS to the sarcolemma (Fig. 3B). In summary, utrophin overexpression or elimination had a nominal effect on subcellular nNOS distribution (Figs 1-3).

Chronic treadmill exercise leads to focal degeneration- regeneration and force reduction in utrophin transgenic Fiona mice To evaluate the physiological consequences, we challenged 4- week-old Fiona and BL10 mice with treadmill exercise for 8 weeks

Fig. 2. In situ evaluation of muscle and dystrophin, utrophin, syntrophin and nNOS expression. Serial muscle sections from normal BL10 (A), mdx (B), utrophin knockout (C), u-dko (D) and Fiona strain of utrophin transgenic mdx mice (E) were stained for general histology with hematoxylin and eosin (HE), and for dystrophin, utrophin, syntrophin, nNOS and nNOS activity. Representative photomicrographs from each mouse model are presented. nNOS (pAb), immunofluorescence staining for nNOS; Asterisks indicate the same myofiber in serial sections; empty arrowheads in C and E indicate neuromuscular junctions; arrows in D show damaged myofibers with infiltrated mouse immunoglobulin; filled arrowheads in E indicate myofibers with centrally located nucleus. Utrophin cannot bring nNOS to sarcolemma 2011

Fig. 3. Western blot analysis of nNOS expression in whole-muscle lysate and microsomal preparation. (A)Representative western blot of whole-muscle lysate. (B)Representative microsomal western blot results. Rapid blue staining of a duplicated gel was included as the loading control for microsomal preparation western blot.

(Grounds et al., 2008). Non-exercised mice were included as Discussion controls. At the end of the study, we isolated the extensor digitorium The sequence homology and the structural similarity between longus muscle and compared histology and force between sedentary utrophin and dystrophin have stirred a tremendous interest in and treadmill-challenged mice. developing utrophin-based therapies for DMD (for reviews, see In non-exercised Fiona mice, centrally nucleated myofibers were Khurana and Davies, 2003; Miura and Jasmin, 2006; Perkins and occasionally observed (Fig. 2E). This might reflect accumulated Davies, 2002; Tinsley and Davies, 1993). Numerous studies have functional ischemic damage from daily activity. Consistent with further established functional redundancy between the two proteins our previous publication (Lai et al., 2009), focal muscle (for reviews, see Blake et al., 1996; Blake et al., 2002; Ervasti, degeneration-regeneration was substantially aggravated in Fiona 2007). These studies suggest that the utrophin and dystrophin mice after chronic treadmill running (Fig. 4A). Large patches of genes are derived from a common evolutionary ancestor. Except myofibers now showed centrally located nuclei (Fig. 4A). Force for some spatial and/or temporal differences in the expression measurement further suggested muscle damage in exercised Fiona pattern and a minor difference in the rod-domain length, utrophin mice (Fig. 4B). Although treadmill challenge did not influence seems sufficient to correct virtually all the cellular defects caused force generation in BL10 mice, specific tetanic force was by dystrophin deficiency (Tinsley et al., 1998). Encouraging results significantly reduced in Fiona mice after continuous treadmill from gene therapy, protein therapy and pharmacological running (Fig. 4B). interventions have further substantiated the therapeutic promise of

Journal of Cell Science utrophin (Cerletti et al., 2003; Deol et al., 2007; Khurana and Davies, 2003; Miura and Jasmin, 2006; Odom et al., 2008; Sonnemann et al., 2009). In an effort to better understand the basic biology of utrophin- mediated DMD therapy, here we examined whether utrophin could recover sarcolemmal nNOS. Based on the striking similarity between dystrophin and utrophin, we initially hypothesized that utrophin could recruit nNOS to the sarcolemma. Our data however, show the hypothesis not to be true. For example, utrophin is upregulated in mdx muscles (Fig. 2B and Fig. 3A), but nNOS is not observed at the mdx sarcolemma (Fig. 1A and Fig. 2B). One possible explanation is that utrophin has a lower affinity for nNOS binding. If this were the case, the amounts of sarcolemmal nNOS in mdx muscle would be too low and beyond the detection threshold. To thoroughly test our hypothesis, we examined whether sarcolemmal nNOS expression could be restored when utrophin was overexpressed. Two different approaches were used to increase utrophin expression in mdx muscles, injection of gutted adenoviral vectors and the use of transgenic mice. We did not detect nNOS at the sarcolemma by immunofluorescence staining, despite robust full-length utrophin Fig. 4. Continuous treadmill running leads to aggravated degeneration- expression from the gutted adenoviral vectors (Fig. 1). Three regeneration and specific force reduction in the extensor digitorum longus independent approaches (immunofluorescence staining, in situ muscle of Fiona mice. Experimental mice were divided into running and no- running groups. In the running group, mice were challenged with treadmill activity staining and microsomal preparation western blot) were exercise for 8 weeks. (A)Representative photomicrographs of HE-stained then applied to determine membrane-associated nNOS expression muscle cross-sections from exercised mice. Arrows indicate areas of ischemic in transgenic mice. Several lines of utrophin transgenic mdx mice damage. (B)Specific muscle force (mean ± s.e.m., n6 per group). Asterisk have been described (Tinsley et al., 1998). Among these, the Fiona indicates a significantly lower value compared with other groups. line showed the highest utrophin expression (~10-fold higher than 2012 Journal of Cell Science 123 (12)

the endogenous level) (Perkins and Davies, 2002). Importantly, adenoviral vectors expressing full-length mouse utrophin (Fig. 1C). morphological and physiological defects were almost completely Immunofluorescence staining was performed with an a-syntrophin- corrected in Fiona mice (Fig. 2E) (Perkins and Davies, 2002; specific antibody (Adams et al., 2000; Peters et al., 1997). As shown Tinsley et al., 1998). Despite the supra-physiological utrophin levels, in Fig. 1C, we observed robust sarcolemmal a-syntrophin expression we were not able to detect nNOS at the sarcolemma (Figs 2 and 3). in myofibers that were transduced by the adenoviral vector. To gain more insight into the role utrophin might have, additional The finding that utrophin cannot recover sarcolemmal nNOS studies were performed in utrophin-null and u-dko mice. Subcellular might also reflect a poor interaction between nNOS and utrophin. nNOS localization was not altered in these mice (Fig. 2C,D and Dystrophin R16/17 constitutes the nNOS binding domain. Utrophin Fig. 3B). spectrin-like repeats 15 and 16 (R15/16) appear to be the We have recently shown that dystrophin-mediated sarcolemmal corresponding repeats of dystrophin R16/17 (Winder et al., 1995). nNOS localization prevents functional ischemia and enhances Our result offers a platform to dissect the molecular interaction exercise performance in dystrophin-deficient mice (Lai et al., between nNOS and dystrophin R16/17 or utrophin R15/16. Since 2009). Specifically, focal ischemic damage (such as degeneration the majority of the amino acid residues are highly conserved in and regeneration) was found in transgenic mdx mice expressing these repeats, reciprocal mutagenesis could help to identify residues the ⌬H2-R19 mini-dystrophin gene (this minigene cannot restore crucial for dystrophin-nNOS interaction. nNOS) but not in ⌬H2-R15 mini-dystrophin transgenic mdx mice, Taken together, our results reveal a qualitative difference between which contain sarcolemmal nNOS (Lai et al., 2009). In sedentary utrophin and dystrophin. Dystrophin can anchor nNOS to the Fiona muscle, we also observed sporadic focal degeneration and sarcolemma and maintain blood perfusion in contracting muscle. regeneration (Fig. 2E, arrowhead). This might reflect accumulated However, utrophin cannot recover membrane-associated nNOS functional ischemic damage from daily activity. To further evaluate expression. The absence of sarcolemmal nNOS is known to result the physiological alterations that might occur with utrophin- in functional ischemia during muscle contraction. To enhance overexpression therapy, mice were challenged with treadmill efficacy of utrophin-based therapies, innovative means are needed running for 8 weeks. Consistent with our previous observation (Lai to maintain muscle perfusion in exercise. et al., 2009), treadmill exercise induced significant degeneration- regeneration and compromised muscle force generation in Fiona Materials and Methods Animal studies mice, but not in normal mice (Fig. 4). Animal experiments were performed in accordance with the NIH and institutional Previous studies suggest that some quantitative functional guidelines of the University of Missouri, the University of Washington and Oxford differences between dystrophin and utrophin might exist. For University. C57Bl/6 (this strain was used as the normal control in adenoviral example, dystrophin binds actin via two separate contact sites experiments), BL10 and mdx mice were purchased from the Jackson Laboratory (Bar Harbor, ME). The original breeding pair of u-dko mice was kindly provided by Mark whereas utrophin interacts with actin through a single continuous Grady (Washington University, St Louis, MO) (Grady et al., 1997). U-dko mice were unit (for a review, see Ervasti, 2007). Furthermore, utrophin shows subsequently backcrossed with mdx mice to the BL10 background (Yue et al., 2006). a twofold lower affinity for b-dystroglycan (Ishikawa-Sakurai et Experimental u-dko mice were all on the BL10 background. Utrophin-null mice were generated by two rounds crossing of BL10 mice with BL10 background u-dko mice. al., 2004). Nevertheless, both proteins are fully capable of Fiona strain full-length utrophin transgenic mdx mice was reported before (Tinsley et interacting with F-actin and b-dystroglycan, and each alone is al., 1998). Treadmill exercise was performed according to a published protocol

Journal of Cell Science sufficient to preserve the sarcolemmal integrity. It appears that (Grounds et al., 2008). Briefly, mice were run on a horizontal treadmill at a speed of dystrophin and utrophin are functionally exchangeable. Our results 12 m/minute, twice a week for a total of 8 weeks. At the beginning of each session, mice received 10 minute warm-up exercise at the speed of 8 m/minute. identify for the first time a significant difference in the functional capacity of utrophin and dystrophin. Dystrophin can recruit nNOS Gutted adenoviral vector to the sarcolemma, but utrophin cannot. The full-length dystrophin and utrophin gutted adenoviral vectors have been described before (DelloRusso et al., 2002; Scott et al., 2002). High-titer gutted adenoviral Despite intensive investigation, the molecular mechanism(s) vectors was grown and purified as described (Hartigan-O’Connor et al., 2002). Local underlying sarcolemmal nNOS localization remains incompletely muscle injection was performed as reported previously to the TA muscle in adult understood. The current model suggests the involvement of mdx mice using 4ϫ1010 particles of vector per muscle (DelloRusso et al., 2002). syntrophin and dystrophin R16/17. The syntrophin family consists Muscles were analyzed for expression 2 months after injection. of five members, including a-, b1-, b2-, g1- and g2-syntrophin Immunofluorescence staining and in situ nNOS activity staining (Albrecht and Froehner, 2002). Only a-syntrophin has been Dystrophin was detected with a mouse monoclonal antibody against the C-terminal domain (Dys-2, 1:30, Novocastra, Newcastle, UK). Utrophin was examined with a conclusively shown to interact with nNOS (Adams et al., 2000; mouse monoclonal antibody against the utrophin N-terminal domain (1:20; Vector Adams et al., 2001; Hillier et al., 1999; Kameya et al., 1999; Laboratories, Burlingame, CA). Syntrophin was revealed with two different antibodies Tochio et al., 1999). Based on this model, one possible explanation including a polyclonal a-syntrophin specific antibody (1:200, a gift from Stanley for our results might relate to the specific syntrophin isoform(s) Froehner, University of Washington, Seattle, WA) and a pan-syntrophin mouse monoclonal antibody that recognizes the PDZ domain (1:200; Abcam, Cambridge, recruited by utrophin. MA) (Adams et al., 2000; Peters et al., 1997). nNOS was detected with a polyclonal It has been shown in wild-type muscle that dystrophin primarily antibody (1:2000; Santa Cruz Biotechnology, Santa Cruz, CA). Immunofluorescence interacts with a-syntrophin and b1-syntrophin whereas utrophin staining was performed using protocols we described before (Lai et al., 2009; Lai et al., 2005; Yue et al., 2003). In situ nNOS activity staining was performed according primarily interacts with b1-syntrophin and b2-syntrophin (Peters et to a published protocol (Lai et al., 2009). al., 1997). However, utrophin has also been shown to recruit a- syntrophin to the sarcolemma at places where utrophin is highly Western blot Whole-muscle lysate was extracted from limb muscle and western blot was performed expressed (such as the and regenerating according to a previously published protocol (Lai et al., 2009; Li et al., 2009; Li et myofibers) (Peters et al., 1997). We have recently shown that a al., 2008). Membrane-enriched microsomal preparations were produced from limb synthetic micro-utrophin protein also interacts with a-syntrophin muscle according to a previously described protocol (Ervasti and Campbell, 1991; (Odom et al., 2008). To determine whether the failure to recruit a- Lai et al., 2009). Proteins were resolved on a 6% SDS-polyacrylamide gel and transferred to a PVDF membrane. Dystrophin was detected with the anti-Dys-2 syntrophin underlies our observation, we examined a-syntrophin antibody (1:100; Novocastra). Utrophin was detected with a mouse monoclonal expression in mdx muscles that have been transduced with gutted antibody (1:200; BD Biosciences, San Jose, CA). Syntrophin was revealed with a Utrophin cannot bring nNOS to sarcolemma 2013

mouse monoclonal antibody (1:2000; Abcam). nNOS was detected with a rabbit anti- Kobayashi, Y. M., Rader, E. P., Crawford, R. W., Iyengar, N. K., Thedens, D. R., nNOS polyclonal antibody (1:2000; Upstate, Lake Placid, NY). a-tubulin (1:3000; Faulkner, J. A., Parikh, S. V., Weiss, R. M., Chamberlain, J. S., Moore, S. A. et al. Sigma, St Louis, MO) was used as the loading control in whole-muscle-lysate western (2008). Sarcolemma-localized nNOS is required to maintain activity after mild exercise. blot. Rapid blue staining (Geno Technology, St Louis, MO) of a duplicated gel was Nature 456, 511-515. used as the loading control in microsomal preparation western blot. Kunkel, L. M. (2005). 2004 William Allan Award address. Cloning of the DMD gene. Am. J. Hum. Genet. 76, 205-214. Lai, Y., Yue, Y., Liu, M., Ghosh, A., Engelhardt, J. F., Chamberlain, J. S. and Duan, This work was supported by grants from the National Institutes of D. (2005). Efficient in vivo gene expression by trans-splicing adeno-associated viral Health (AR-49419, D.D; AR-44533, J.S.C.), the Muscular Dystrophy vectors. Nat. Biotechnol. 23, 1435-1439. Association (D.D. and J.S.C.), the Clarendon Fund and Kokil Pathak Lai, Y., Thomas, G. D., Yue, Y., Yang, H. T., Li, D., Long, C., Judge, L., Bostick, B., Chamberlain, J. S., Terjung, R. L. et al. (2009). carrying spectrin-like scholarship (A.B.). The authors (D.L., Y.Y., Y.L. and D.D.) thank repeats 16 and 17 anchor nNOS to the sarcolemma and enhance exercise performance Robert J. McDonald, Jr, for the generous support to Duchenne muscular in a mouse model of muscular dystrophy. J. Clin. Invest. 119, 624-635. dystrophy research in the Duan lab. We thank Stanley Froehner for Li, D., Yue, Y. and Duan, D. (2008). Preservation of muscle force in mdx3cv mice providing the polyclonal anti-a-syntrophin antibody. We also thank correlates with low-level expression of a near full-length dystrophin protein. Am. J. Pathol. 172, 1332-1341. Sarah Squire, Brian Bostick, Chun Long, Dave Powell and Yadong Li, D., Long, C., Yue, Y. and Duan, D. (2009). Sub-physiological expression Zhang for technical help. Deposited in PMC for release after 12 contributes to compensatory muscle protection in mdx mice. Hum. Mol. Genet. 18, months. 1209-1220. Love, D. R., Hill, D. F., Dickson, G., Spurr, N. K., Byth, B. C., Marsden, R. F., Walsh, References F. S., Edwards, Y. H. and Davies, K. E. (1989). An autosomal transcript in skeletal muscle with homology to dystrophin. Nature 339, 55-58. Adams, M. E., Kramarcy, N., Krall, S. P., Rossi, S. G., Rotundo, R. L., Sealock, R. Mendell, J. R., Engel, W. K. and Derrer, E. C. (1971). Duchenne muscular dystrophy: and Froehner, S. C. (2000). Absence of alpha-syntrophin leads to structurally aberrant functional ischemia reproduces its characteristic lesions. Science 172, 1143-1145. neuromuscular synapses deficient in utrophin. J. Cell Biol. 150, 1385-1398. Miura, P. and Jasmin, B. J. (2006). Utrophin upregulation for treating Duchenne or Adams, M. E., Mueller, H. A. and Froehner, S. C. (2001). In vivo requirement of the Becker muscular dystrophy: how close are we? Trends Mol. Med. 12, 122-129. alpha-syntrophin PDZ domain for the sarcolemmal localization of nNOS and aquaporin- Nguyen, T. M., Ellis, J. M., Love, D. R., Davies, K. E., Gatter, K. C., Dickson, G. and 4. J. Cell Biol. 155, 113-122. Morris, G. E. (1991). Localization of the DMDL gene-encoded dystrophin-related Albrecht, D. E. and Froehner, S. C. (2002). Syntrophins and dystrobrevins: defining the protein using a panel of nineteen monoclonal antibodies: presence at neuromuscular dystrophin scaffold at synapses. Neurosignals 11, 123-129. junctions, in the sarcolemma of dystrophic skeletal muscle, in vascular and other Blake, D. J., Tinsley, J. M. and Davies, K. E. (1996). Utrophin: a structural and functional smooth muscles, and in proliferating brain cell lines. J. Cell Biol. 115, 1695-1700. comparison to dystrophin. Brain Pathol. 6, 37-47. Odom, G. L., Gregorevic, P., Allen, J. M., Finn, E. and Chamberlain, J. S. (2008). Blake, D. J., Weir, A., Newey, S. E. and Davies, K. E. (2002). Function and genetics of Microutrophin delivery through rAAV6 increases lifespan and improves muscle function dystrophin and dystrophin-related proteins in muscle. Physiol. Rev. 82, 291-329. in dystrophic dystrophin/utrophin-deficient mice. Mol. Ther. 16, 1539-1545. Brenman, J. E., Chao, D. S., Xia, H., Aldape, K. and Bredt, D. S. (1995). Nitric oxide Parker, J. M. and Mendell, J. R. (1974). Proximal myopathy induced by 5-HT-imipramine synthase complexed with dystrophin and absent from skeletal muscle sarcolemma in simulates Duchenne dystrophy. Nature 247, 103-104. Duchenne muscular dystrophy. Cell 82, 743-752. Percival, J. M., Anderson, K. N., Gregorevic, P., Chamberlain, J. S. and Froehner, S. Cerletti, M., Negri, T., Cozzi, F., Colpo, R., Andreetta, F., Croci, D., Davies, K. E., C. (2008). Functional deficits in nNOSmu-deficient skeletal muscle: myopathy in Cornelio, F., Pozza, O., Karpati, G. et al. (2003). Dystrophic phenotype of canine X- nNOS knockout mice. PLoS ONE 3, e3387. linked muscular dystrophy is mitigated by adenovirus-mediated utrophin gene transfer. Perkins, K. J. and Davies, K. E. (2002). The role of utrophin in the potential therapy of Gene Ther. 10, 750-757. Duchenne muscular dystrophy. Neuromuscul. Disord. 12 Suppl. 1, S78-S89. Chang, W. J., Iannaccone, S. T., Lau, K. S., Masters, B. S., McCabe, T. J., McMillan, Peters, M. F., Adams, M. E. and Froehner, S. C. (1997). Differential association of K., Padre, R. C., Spencer, M. J., Tidball, J. G. and Stull, J. T. (1996). Neuronal nitric syntrophin pairs with the dystrophin complex. J. Cell Biol. 138, 81-93. oxide synthase and dystrophin-deficient muscular dystrophy. Proc. Natl. Acad. Sci. USA Rivier, F., Robert, A., Hugon, G. and Mornet, D. (1997). Different utrophin and 93, 9142-9147. dystrophin properties related to their vascular distributions. FEBS Lett. Deconinck, A. E., Rafael, J. A., Skinner, J. A., Brown, S. C., Potter, A. C., Metzinger, 408, 94-98. L., Watt, D. J., Dickson, J. G., Tinsley, J. M. and Davies, K. E. (1997). Utrophin- Sander, M., Chavoshan, B., Harris, S. A., Iannaccone, S. T., Stull, J. T., Thomas, G. dystrophin-deficient mice as a model for Duchenne muscular dystrophy. Cell 90, 717-727. Journal of Cell Science D. and Victor, R. G. (2000). Functional muscle ischemia in neuronal nitric oxide DelloRusso, C., Scott, J. M., Hartigan-O’Connor, D., Salvatori, G., Barjot, C., synthase-deficient skeletal muscle of children with Duchenne muscular dystrophy. Robinson, A. S., Crawford, R. W., Brooks, S. V. and Chamberlain, J. S. (2002). Proc. Natl. Acad. Sci. USA 97, 13818-13823. Functional correction of adult mdx mouse muscle using gutted adenoviral vectors Scott, J., Li, S., Harper, S., Welikson, R., Bourque, D., DelloRusso, C., Hauschka, S. expressing full-length dystrophin. Proc. Natl. Acad. Sci. USA 99, 12979-12984. and Chamberlain, J. (2002). Viral vectors for gene transfer of micro-, mini-, or full- Deol, J. R., Danialou, G., Larochelle, N., Bourget, M., Moon, J. S., Liu, A. B., Gilbert, length dystrophin. Neuromuscul. Disord. 12 Suppl, S23. R., Petrof, B. J., Nalbantoglu, J. and Karpati, G. (2007). Successful compensation Sonnemann, K. J., Heun-Johnson, H., Turner, A. J., Baltgalvis, K. A., Lowe, D. A. and for dystrophin deficiency by a helper-dependent adenovirus expressing full-length Ervasti, J. M. (2009). Functional substitution by TAT-utrophin in dystrophin-deficient utrophin. Mol. Ther. 15, 1767-1774. mice. PLoS Med. 6, e1000083. Ervasti, J. M. (2007). Dystrophin, its interactions with other proteins, and implications Thomas, G. D., Sander, M., Lau, K. S., Huang, P. L., Stull, J. T. and Victor, R. G. for muscular dystrophy. Biochim. Biophys. Acta 1772, 108-117. (1998). Impaired metabolic modulation of alpha-adrenergic vasoconstriction in Ervasti, J. M. and Campbell, K. P. (1991). Membrane organization of the dystrophin- dystrophin-deficient skeletal muscle. Proc. Natl. Acad. Sci. USA 95, 15090-15095. glycoprotein complex. Cell 66, 1121-1131. Tinsley, J. M. and Davies, K. E. (1993). Utrophin: a potential replacement for dystrophin? Grady, R. M., Teng, H., Nichol, M. C., Cunningham, J. C., Wilkinson, R. S. and Neuromuscul. Disord. 3, 537-539. Sanes, J. R. (1997). Skeletal and cardiac myopathies in mice lacking utrophin and Tinsley, J. M., Blake, D. J., Roche, A., Fairbrother, U., Riss, J., Byth, B. C., Knight, dystrophin: a model for Duchenne muscular dystrophy. Cell 90, 729-738. A. E., Kendrick-Jones, J., Suthers, G. K., Love, D. R. et al. (1992). Primary structure Grounds, M. D., Radley, H. G., Lynch, G. S., Nagaraju, K. and De Luca, A. (2008). of dystrophin-related protein. Nature 360, 591-593. Towards developing standard operating procedures for pre-clinical testing in the mdx Tinsley, J., Deconinck, N., Fisher, R., Kahn, D., Phelps, S., Gillis, J. M. and Davies, mouse model of Duchenne muscular dystrophy. Neurobiol. Dis. 31, 1-19. K. (1998). Expression of full-length utrophin prevents muscular dystrophy in mdx Hartigan-O’Connor, D., Barjot, C., Salvatori, G. and Chamberlain, J. S. (2002). mice. Nat. Med. 4, 1441-1444. Generation and growth of gutted adenoviral vectors. Methods Enzymol. 346, 224-246. Tochio, H., Zhang, Q., Mandal, P., Li, M. and Zhang, M. (1999). Solution structure of Heydemann, A., Doherty, K. R. and McNally, E. M. (2007). Genetic modifiers of the extended neuronal nitric oxide synthase PDZ domain complexed with an associated muscular dystrophy: Implications for therapy. Biochim. Biophys. Acta 172, 216-228. peptide. Nat. Struct. Biol. 6, 417-421. Hillier, B. J., Christopherson, K. S., Prehoda, K. E., Bredt, D. S. and Lim, W. A. Wells, K. E., Torelli, S., Lu, Q., Brown, S. C., Partridge, T., Muntoni, F. and Wells, (1999). Unexpected modes of PDZ domain scaffolding revealed by structure of nNOS- D. J. (2003). Relocalization of neuronal nitric oxide synthase (nNOS) as a marker for syntrophin complex. Science 284, 812-815. complete restoration of the dystrophin associated protein complex in skeletal muscle. Ishikawa-Sakurai, M., Yoshida, M., Imamura, M., Davies, K. E. and Ozawa, E. Neuromuscul. Disord. 13, 21-31. (2004). ZZ domain is essentially required for the physiological binding of dystrophin Winder, S. J., Gibson, T. J. and Kendrick-Jones, J. (1995). Dystrophin and utrophin: and utrophin to beta-dystroglycan. Hum. Mol. Genet. 13, 693-702. the missing links! FEBS Lett. 369, 27-33. Kameya, S., Miyagoe, Y., Nonaka, I., Ikemoto, T., Endo, M., Hanaoka, K., Nabeshima, Yue, Y., Li, Z., Harper, S. Q., Davisson, R. L., Chamberlain, J. S. and Duan, D. (2003). Y. and Takeda, S. (1999). alpha1-syntrophin gene disruption results in the absence of Microdystrophin gene therapy of cardiomyopathy restores dystrophin-glycoprotein neuronal-type nitric-oxide synthase at the sarcolemma but does not induce muscle complex and improves sarcolemma integrity in the Mdx mouse heart. Circulation 108, degeneration. J. Biol. Chem. 274, 2193-2200. 1626-1632. Khurana, T. S. and Davies, K. E. (2003). Pharmacological strategies for muscular Yue, Y., Liu, M. and Duan, D. (2006). C-terminal truncated microdystrophin recruits dystrophy. Nat. Rev. Drug Discov. 2, 379-390. dystrobrevin and syntrophin to the dystrophin-associated glycoprotein complex and Khurana, T. S., Hoffman, E. P. and Kunkel, L. M. (1990). Identification of a chromosome reduces muscular dystrophy in symptomatic utrophin/dystrophin double knock-out 6-encoded dystrophin-related protein. J. Biol. Chem. 265, 16717-16720. mice. Mol. Ther. 14, 79-87.