The Polyproline Site in Hinge 2 Influences the Functional Capacity of Truncated Dystrophins

Mutations in dystrophin can lead to Duchenne muscular dystrophy or the more mild form of the disease, Becker muscular dystrophy. The hinge 3 region in the rod domain of dystrophin is particularly prone to deletion mutations. In-frame deletions of hinge 3 are predicted to lead to BMD, however the severity of disease can vary considerably. Here we performed extensive structure-function analyses of truncated dystrophins with modified hinges and spectrin-like repeats in mdx mice. We found that the polyproline site in hinge 2 profoundly influences the functional capacity of a microdystrophinΔR4-R23/ΔCT with a large deletion in the hinge 3 region. Inclusion of polyproline in microdystrophinΔR4-R23/ΔCT led to small myofibers (12% smaller than wild-type), Achilles myotendinous disruption, ringed fibers, and aberrant neuromuscular junctions in the mdx gastrocnemius muscles. Replacing hinge 2 of microdystrophinΔR4-R23/ΔCT with hinge 3 significantly improved the functional capacity to prevent muscle degeneration, increase muscle fiber area, and maintain the junctions. We conclude that the rigid α-helical structure of the polyproline site significantly impairs the functional capacity of truncated dystrophins to maintain appropriate connections between the cytoskeleton and extracellular matrix.


Introduction
Duchenne muscular dystrophy (DMD) is a lethal X-linked recessive disease caused by mutations in the 2.2 MB dystrophin gene [1][2][3]. In skeletal muscle, dystrophin provides a flexible connection between the cytoskeleton and the dystrophinglycoprotein complex at the sarcolemma, myotendinous junction (MTJ) and neuromuscular junction (NMJ) [4][5][6]. Mutations that affect the mechanical integrity of this molecular scaffold render muscles more susceptible to contraction-induced injury leading to cycles of necrosis and regeneration [3].
While the microdystrophin DR4-R23 transgene provides a clear benefit to dystrophic muscles [24], more detailed analyses have revealed a potentially serious abnormality in some muscle groups. The microdystrophin DR4-R23 /mdx transgenic mice have chronic Achilles myotendinous strain injury, which leads to the formation of ringed fibers and fragmentation of the neuromuscular junctions [33,36]. In the present study we examined whether the domain composition or the small size of microdystrophin DR4-R23/DCT led to this myopathy in mdx mice. We found that the hinge regions of microdystrophin, rather than its small size can profoundly influence skeletal muscle maintenance, maturation and structure.

Dystrophin hinge domains influence the maintenance and maturation of skeletal muscles
We initially screened several truncated dystrophins and found that inclusion of hinge 2, but not hinge 3 could lead to the structural abnormalities we observed in some muscles of the microdystrophin DR4-R23 transgenic mice (Text S1; Figures S1, S2, S3). We subsequently compared the efficacy of two microdystrophins that differ only in their inclusion of hinge 2 (microdystrophin DR4-R23/DCT ) or hinge 3 (microdystrophin DH2-R23+H3/DCT ) ( Figure 1A) to examine whether the hinge composition of microdystrophin could influence various aspects of muscle disease.

Dystrophin hinge domains influence myotendinous junction injury and formation of ringed fibers
The tendon extends deep folds into wild-type skeletal muscles to minimize membrane stress under shear ( Figure 2) [37]. Most of the folds in the mdx junctions did not extend as far into the gastrocnemius muscles ( Figure 2). rAAV6-microdystrophin DR4-R23/DCT severely disrupted the Achilles myotendinous junctions in mdx mice. Many of the junctional folds were missing and myofibril degeneration was evident ( Figure 2). Approximately 17% of the adjoining mdx gastrocnemius muscles had ringed fibers. In contrast, rAAV6microdystrophin DH2-R23+H3/DCT with hinge 3 retained the normal architecture of the Achilles myotendinous junction and we found no ringed fibers in the adjoining gastrocnemius muscles ( Figure 2). Thus, the hinge domains influenced whether microdystrophin was capable of maintaining the myotendinous junction and myofibril structure in mdx gastrocnemius muscles.

Dystrophin hinge domains influence neuromuscular synapse structure
We also examined neuromuscular synapses in mdx mice treated with rAAV6-microdystrophins. Most neuromuscular synapses in wild-type mice (,97%) form a continuous tertiary structure as shown by staining whole muscle fibers with a-bungarotoxin ( Figure 3A). Neuromuscular synapses in mdx mice begin to fragment temporally coincident with muscle degeneration [38]. Approximately 89% of neuromuscular synapses were fragmented in the gastrocnemius muscles of mdx mice ( Figure 3B). We had previously shown that the neuromuscular synapses in transgenic microdystrophin DR4-R23 /mdx gastrocnemius muscles fragmented temporally coincident with the formation of ringed fibers [36]. In the present study we found that rAAV6-microdystrophin DR4-R23/DCT containing hinge 2 maintained continuous synapses in only 46% of the mdx gastrocnemius muscles ( Figure 3A and 3B). In contrast, approximately 84% of synapses were continuous in mdx gastrocnemius muscles treated with rAAV6-microdystrophin DH2-R23+H3/DCT containing hinge 3 ( Figure 3A and 3B).
Neuromuscular synapses also contain folds in the postsynaptic membrane that align directly adjacent to vesicle release sites (active zones) in the pre-synaptic nerve terminal (arrows; Figure 3C). The number of synaptic folds in mdx mice was significantly reduced compared to wild-type (P,0.01; Figure 3C and 3D) as previously described [4,39]. The number of folds was restored in microdystrophin DR4-R23/DCT and microdystrophin DH2-R23+H3/DCT treated muscles ( Figure 3C and 3D). The synaptic folds extended significantly further into microdystrophin DR4-R23/DCT treated mdx muscles compared to wild-type muscles (P,0.001; Figure 3C and 3E), as previously described in transgenic microdystrophin DR4-R23 / mdx mice [36]. In contrast, the number and length of synaptic folds in microdystrophin DH2-R23+H3/DCT treated mdx muscles was similar to wild-type ( Figure 3C-3E). Thus, microdystrophin DH2-R23+H3/DCT containing hinge 3 can maintain the structure of neuromuscular junctions in mdx muscles.
Mechanical properties of muscles expressing microdystrophins with either hinge 2 or hinge 3 Contraction-induced injury can initiate muscle degeneration in mdx mice [40]. Skeletal muscles from mdx mice have a lower force

Author Summary
Dystrophin functions like a large molecular spring between the muscle cytoskeleton and the extracellular matrix in order to protect the membrane from contraction-induced injury. Mutations in dystrophin can lead to a severe muscle wasting disease called Duchenne muscular dystrophy (DMD) in young boys. DMD patients are typically wheelchair bound by 9-13 years of age and die at approximately 30 years. There are also mutations within the dystrophin gene that lead to internal truncations of non-essential regions, such as the internal rod domain that leads to a mild form of the disease called Becker Muscular Dystrophy. However, these internal truncations frequently occur at a ''hot spot'' within the rod domain where the resulting disease severity is difficult to predict. Here we found that consecutive proline residues, that function much like a molecular ruler, can dramatically influence the function of these internally truncated dystrophins within skeletal muscles. Using this information, we designed a dystrophin mini-gene that can accommodate the limited packaging size of recombinant adeno-associated virus. This virus can deliver the dystrophin mini-gene to most muscles throughout a dystrophic mouse to prevent muscle degeneration and partially restore muscle function.
producing capacity than wild-type muscles and are more susceptible to contraction-induced injury ( Figure 4). We found that sub-optimal doses of both rAAV6-microdystrophin DR4-R23/DCT and rAAV6-microdystrophin DH2-R23+H3/DCT maintained the peak force producing capacity of mdx gastrocnemius and tibialis anterior muscles ( Figure 4A). Both microdystrophins also significantly improved the specific force (force per cross sectional area of muscle) production in mdx muscles (P,0.05; Figure 4B). The specific force was not restored to wild-type partly because the sub-optimal dose of rAAV6-microdystrophin did not prevent the pseudo hypertrophy normally found in mdx muscles (P = 0.454 when comparing the muscle mass between mdx and treated mdx muscles; one-way  [15]). ABD1 at the N-terminus is composed of two calponin homology domains denoted by the two circles. The central rod domain contains 24 spectrin-like repeats (R1-24), 4 hinge domains, a 20 amino acid insertion between spectrin-like repeats 15 and 16, and a central actin-binding domain (ABD2). A cluster of basic repeats forms ABD2 that bind to actin through an electrostatic interaction [20]. The hinge domains vary in that hinge 2 contains a polyproline site and hinge 4 contains a WW motif that is required for binding to b-dystroglycan [19,68]. The cysteinerich region contains two EF hands and a ZZ domain that is also required for binding to b-dystroglycan. The microdystrophins used in this study are shown below the full-length dystrophin. Microdystrophin DR4-R23/DCT lacks a large portion of the central rod domain between spectrin-like repeats 4 and 23 and also lacks the C-terminal domain (DR4-R23/DCT) [24]. Note that microdystrophin DR4-R23/DCT and microdystrophin DH2-R23+H3/DCT differ by a single hinge domain. (B) Transverse sections of gastrocnemius muscles from wild-type, mdx and mdx mice expressing either microdystrophin DR4-R23/DCT or microdystrophin DH2-R23+H3/DCT . Scale bar = 100 mm. (C) Expression of truncated dystrophins in treated mdx gastrocnemius muscles was similar. The western blots were performed using frozen muscles extracted from OCT. All lanes were loaded equally as shown by a-sarcomeric actin. (D) Shown is the mean +/2 S.D. percentage of dystrophin-positive muscle fibers. TA is tibialis anterior. (E) Shown is the mean +/2 S.D. of the percentage of muscle fibers containing centrally-located nuclei. Microdystrophin DH2-R23+H3/DCT with hinge 3 was more effective at preventing muscle degeneration in mdx mice compared to microdystrophin DR4-R23/DCT with hinge 2. The treated muscles show the mean +/2 SD for dystrophin-positive fibers only. ***P,0.001 compared to untreated mdx mice. # P,0.05 compared to microdystrophin DR4-R23/DCT treated mdx muscles. (F) Hinge regions of dystrophin influence muscle fiber cross-sectional area. Shown is the mean +/2 distribution (25th and 75th percentile (box) in addition to the farthest (whiskers) area of muscle fibers. *P,0.05 and ***P,0.001 compared to wild-type. doi:10.1371/journal.pgen.1000958.g001 ANOVA). Each microdystrophin significantly protected the treated limb muscles from contraction-induced injury (P,0.001; Figure 4C and 4D). However, we found no significant difference between the peak force, specific force or protection from contraction-induced injury when comparing between the two microdystrophins with either hinge 2 or hinge 3.

Polyproline in hinge 2 influences the pathology of skeletal muscle fibers
Together, our results suggested that the structural abnormalities observed in some treated mdx muscles could be traced to the presence of hinge 2 within the microdystrophin. We next examined the molecular composition of the hinges to define  what was unique about hinge 2. The hinges in dystrophin are defined as such because of the higher concentration of proline residues, which function to limit the continuation of the a-helical coiled-coils of the spectrin-like repeats through the entire length of the dystrophin rod domain [19]. Both hinge 2 and hinge 3 have six proline residues and the lengths of these hinges are similar [19]. We hypothesized that the placement of the prolines most likely results in their different functions [5,19]. Hinge 2 has 5 consecutive proline residues (polyproline; Figure 5A) whereas the proline residues in hinge 3 are more evenly distributed throughout the hinge [19]. Polyproline residues are thought to have their own defined rigid helical structure [41,42], and this could affect the functional capacity of microdystrophin DR4-R23/DCT .
To test this hypothesis we compared muscles expressing the original microdystrophin DR4-R23/DCT with a newly developed microdystrophin DpolyP/DR4-R23/DCT that lacks the polyproline site in hinge 2 ( Figure 5A). We delivered 6610 10 vg of each microdystrophin into mdx gastrocnemius muscles at 2 days of age and examined the mice 7 weeks after treatment. Both microdystrophins were expressed in a similar percentage of muscle fibers ( Figure 5B; 59-68%), and were expressed at similar levels ( Figure 5C). Each microdystrophin significantly reduced muscle fiber degeneration ( Figure 5D). As expected, the original microdystrophin DR4-R23/DCT limited muscle fiber cross-sectional area ( Figure 5E), was associated with disrupted myotendinous junctions ( Figure 5F), led to the formation of ringed fibers ( Figure 5F), and perturbed neuromuscular junctions ( Figure 5G-5I). In contrast, the mdx muscles treated with microdystro-phin DpolyP/DR4-R23/DCT did not show any abnormalities in muscle fiber maturation or structure ( Figure 5). Thus, the presence of this polyproline site in hinge 2 of microdystrophin DR4-R23/DCT prevented the appropriate integration of muscles into the nervetendon environment.

Discussion
Most gene therapy strategies for DMD require the generation of highly functional truncated dystrophins. rAAV is an efficient and safe vector for systemically delivering truncated dystrophins to striated muscles to prevent muscle degeneration in animal models of DMD ( [28]; reviewed in [43]). We had previously generated a microdystrophin DR4-R23 that was highly capable of mitigating muscle degeneration and improving the mechanical function of mdx skeletal muscles [24,28]. However, the microdystrophin DR4-R23 transgene leads to chronic strain injury at the Achilles myotendinous junction [33]. This led to the formation of ringed fibers that function to protect skeletal muscles from contraction-induced injury, even better than wild-type mice [33]. The formation of the rings led to fragmentation of the neuromuscular junctions [36]. Other effects of the transgene included smaller muscle fibers [24], and increased length of synaptic folds [36]. Here we found that each of these phenotypic changes was recapitulated in mdx gastrocnemius muscles treated with rAAV6-microdystrophin DR4-R23/DCT . A screen of several newly developed dystrophin mini-genes revealed that the hinge 2 region influenced the functional capacity of microdystrophin DR4-R23/DCT . Replacing hinge 2 with hinge 3 led to several specific force. ***P,0.001 compared to wild-type. # P,0.05 and ## P,0.01 compared to mdx. The contractile performance of (C) gastrocnemius muscles and (D) tibialis anterior muscles immediately prior to increasing length changes during maximal force production. Bars represent the mean +/2 S.D. percentage of the initial optimal muscle contraction. rAAV6-microdystrophin DR4-R23/DCT treated muscles were significantly (*P,0.05; **P,0.01; ***P,0.001) protected from contraction-induced injury, as were rAAV6-microdystrophin DH2-R23/DCT+H3 ( # P,0.05; ## P,0.01; ### P,0.001) treated muscles when compared to mdx mice. doi:10.1371/journal.pgen.1000958.g004 advantages such as better protection of skeletal muscles (only 1-2% central nuclei 6 months post treatment), larger muscle fibers and normal junctions. Deleting the polyproline site from hinge 2 of microdystrophin DR4-R23/DCT also prevented these structural abnormalities.

Mechanical properties of skeletal muscles expressing microdystrophins
Microdystrophin DH2-R23+H3/DCT with hinge 3 significantly increased peak force, specific force and protected muscles from contraction-induced injury. However, the morphological improve- ments of microdystrophin DH2-R23+H3/DCT treated muscles did not translate into a functional improvement compared to microdystrophin DR4-R23/DCT treated muscles. This could result from the molecular and cellular responses to myotendinous strain injury that help protect the rAAV6-microdystrophin DR4-R23/DCT treated muscles from contraction-induced injury [33]. Another possibility is that the presence of some dystrophin negative fibers masked any functional difference between the two proteins. The inclusion of hinge 2 in microdystrophin limited muscle fiber area whereas the inclusion of hinge 3 increased muscle fiber area (Figure 1). Larger muscle fibers in microdystrophin DH2-R23+H3/DCT treated mice could have two distinct advantages: They could replace some of the muscle mass lost in advanced stages of disease and they could be better protected from contraction-induced injury [44]. However, the sub-optimal dose of either rAAV6-microdystrophin did not prevent the pseudo hypertrophy in mdx mice and no mechanical advantages could be discerned when comparing treatments. Saturating levels of rAAV6-microdystrophins or transgenic mice will most likely be required to detect minor differences in the mechanical properties of muscles expressing various truncated dystrophins.

How does polyproline influence the functional capacity of truncated dystrophins?
Our most effective truncated dystrophins developed for gene therapy have been designed to maximize functional interactions between specific spectrin-like repeats and hinge domains. This design has been influenced by genetic studies in mice and man as well as biophysical studies in vitro on the structure, folding and physical properties of both individual and tandemly expressed spectrin-like repeats and hinge domains [24,[45][46][47][48][49][50][51][52]. Individual spectrin-like repeats are not all interchangeable, and ones adjacent to hinges have distinct properties from those flanked by other spectrin-like repeats [21,24,47,51,52]. Also, spectrin-like repeats rarely function as isolated units [15,24,[50][51][52][53]. Instead, they appear to fold into nested domains interrupted by various insertions (hinges) that disrupt the uniformity and rigidity of the spectrin-like repeat rod domain [24,[45][46][47][48][53][54][55]. These interruptions appear important for the elastic and flexible structure that dystrophin requires in its role as a force transducer and shock absorber in muscle [56][57][58][59]. Our studies suggest that the most functional truncations of dystrophin retain a central hinge domain that is flanked by spectrin-like repeats found adjacent to a hinge in the wild-type dystrophin [24]. Disruption of this linkage could influence protein folding, stability and function leading to the variable phenotypes in patients associated with deletions at or near hinge 3, which is encoded on exons 50-51 [21].
Individual spectrin-like repeats are composed of 3 helical domains connected by non-helical linkers, which fold into a triple helical coiled coil structure ( Figure 5A; [45,47]). The linker regions between discreet repeats are also typically short and relatively unstructured to allow a smooth connection between the third helix of a preceding repeat and the first helix of the next repeat ( Figure 6A). However, hinge domains interrupt the nested nature of adjacent spectrin-like repeats and allow more flexibility in the rod domain ( Figure 6B). This degree of flexibility appears to be significantly different when hinge 2 or hinge 3 is present. While both hinges contain 6 prolines, which act to disrupt alpha helical structures, in hinge 3 they are dispersed whereas 5 of the 6 prolines in hinge 2 are clustered together ( Figure 5A, Figure 6C and 6D; [10]). Polyproline residues form a rigid a-helix [41,42], much like a molecular ruler [60]. We suggest that the location of this polyproline sequence within a highly truncated rod domain induces a severe structural disruption that can affect the ability of dystrophin to form a mechanically flexible connection between F-actin and bdystroglycan. Spectrin-like repeats 1-3 have been shown to associate with the sarcolemmal membrane, while the WW domain in hinge 4 forms a critical portion of the b-dystroglycan binding domain [45,61]. A rigid rod domain induced by polyproline in hinge 2 may directly impair the ability of microdystrophin to form a flexible interaction with either or both of these structures ( Figure 6C). In contrast, when hinge 2 is present in full-length dystrophin, a significantly greater number of spectrin-like repeats are present between the hinge and the b-dystroglycan binding domain, allowing greater flexibility in the overall structure.
It is difficult to predict the function of the polyproline site from patients with in frame deletions of exon 17 (hinge 2) of dystrophin. The described deletions (Leiden Muscular Dystrophy Pages) usually encompass larger regions of dystrophin than the polyproline site and it is not clear how these deletions affect protein stability. Our finding that hinge 3 microdystrophin can prevent muscle degeneration suggests that the polyproline site is not a necessary component of dystrophin, similar to previous reports on longer forms of truncated dystrophins [24,62].
Flanigan et. al., 2009 has proposed that approximately 62% of all DMD patients could be treated with oligonucleotides that skip exons 45-55 (from spectrin-like repeat [18][19][20][21][22] [14]. This would create a truncated dystrophin that contains hinge 2 but not hinge 3, similar to, but much larger than our microdystrophin DR4-R23 transgene. It will therefore be of interest to determine whether the polyproline site in hinge 2 can influence the functional capacity of larger, truncated dystrophins. It will also be of interest to examine whether the polyproline site affects the functional capacity of truncated utrophin constructs that are designed for gene therapy of DMD [63,64].

Mice and ethics statement
We utilized C57Bl/10 wild-type mice and mdx 4cv mice. All experiments are in accordance with the institution of animal care and use committee (IACUC) of the University Of Washington.

Generation of constructs
The expression vector CMV-DR4-R23/DCT which uses the cytomegalovirus immediate early promoter and enhancer to drive expression of a microdystrophin cDNA was generated as previously described [32]. We generated the DH2-R24/DCT, DR2-R23+R18-H3/DCT, DH2-R23+H3/DCT and DPolyP/ DR4-R23/DCT constructs using recombination PCR with CMV-DR4-R23/DCT as the template [65]. The primers used to generate DH2-R24/DCT, DR2-R23+R18-H3/DCT, DH2-R23+H3/DCT and DPolyP/DR4-R23/DCT are found in Table  S1. The resulting expression vectors were sequenced and cotransfected with the pDGM6 packaging plasmid into HEK293 cells to generate recombinant AAV vectors comprising serotype 6 capsids that were harvested, purified, and quantitated as described previously [29]. The resulting titer was determined by comparison to previously known concentrations of rAAV6-CMV-lacZ and DR4-R23/DCT by Southern analyses with a probe to the CMV promoter. The rAAV6-microdystrophins were delivered intravenously by tail vein injection at two weeks of age or directly into the mdx gastrocnemius muscles at 2 days of age while the mice were anaesthetized.

Electron microscopy
Electron microscopy was performed as previously described [33]. The junctional fold number and lengths were measured from n = 4 mice at 6 months of age using Image J (NIH) and compared using Students t-test (Prism). The counts represent the fold numbers and lengths from all fibers (dystrophin positive and negative).

Quantitation of ringed fibers
We quantitated the number of ringed myofibers in EM images and thick (1 mm) toluidine blue sections from at least 4 animals per group. At least 300 muscle fibers from n = 4 gastrocnemius muscles were examined from wild-type, mdx 4cv and mdx 4cv mice expressing the various microdystrophins.

Quantification of neuromuscular synapses
Neuromuscular synapses were analyzed in whole mount immunofluorescence stained muscles and quantitated as previously described [36]. The acetylcholine receptor clusters were stained with TRITC conjugated a-bungarotoxin (aBTX; 1:800; Molecular Probes). Synapses were classified as continuous if they presented with 3 or less continuous regions of AChR clustering and discontinuous if they presented with more than 3 regions of AChR clustering. More than 50 synapses were analyzed from treated and untreated gastrocnemius skeletal muscle fibers from n = 4 mice. The counts in treated muscles include both dystrophin positive and negative fibers. We compared the proportion of continuous synapses using a Students t-test.

Muscle physiology
Muscle physiology was performed as previously described for tibialis anterior [29] and gastrocnemius [33] muscles. We examined six-month-old wild-type, mdx, and mdx mice treated with rAAV6-microdystrophin DR4-R23/DCT or rAAV6-microdystrophin DH2-R23+H3/DCT (n = 5). Figure S1 The molecular structure of truncated dystrophins. ABD1 at the N-terminus is composed of two calponin homology domains denoted by the two circles. The central rod domain contains 24 spectrin-like repeats (R1-24), 4 hinge domains, a 20 amino acid insertion between spectrin repeats 15 and 16, and a central actin-binding domain (ABD2). A cluster of basic repeats forms ABD2 that bind to actin through an electrostatic interaction. The hinge domains vary in that hinge 2 contains a polyproline site and hinge 4 contains a WW motif that is required for binding to dystroglycan. The cysteine rich region contains two EF hands and a ZZ domain that is also required for binding to dystroglycan. The microdystrophins used in this study are shown below the fulllength dystrophin. Microdystrophin DR4-R23/DCT has a large portion of the central rod domain missing between spectrin repeats 4 and 23 and also lacks the C-terminal domain (DR4-R23/ DCT). Note that microdystrophin DR4-R23/DCT and microdystrophin DH2-R23+H3/DCT differ by a single hinge domain.  Figure S3 Microdystrophin DR4-R23/DCT leads to ringed fibers when hinge 2 is present. Shown are transverse sections from wildtype, mdx and mdx gastrocnemius muscles expressing various microdystrophins. Arrows point to ringed fibers in mdx muscles treated with microdystrophin DR4-R23/DCT . Scale bars = 2 mm for electron microscopy images and 50 mm for the other images. Found at: doi:10.1371/journal.pgen.1000958.s003 (9.93 MB TIF) Figure S4 The hinge domains of dystrophin do not influence restoration of the dystrophin glycoprotein complex. Shown is the localization of dystrophin-associated proteins in transverse sections from wild-type, mdx and mdx mice treated with rAAV6-microdystrophin DR4-R23/DCT or rAAV6-microdystrophin DH2-R23/DCT+H3 . Scale bar = 200 mm.