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Human Molecular Genetics Advance Access originally published online on June 22, 2007
Human Molecular Genetics 2007 16(17):2105-2113; doi:10.1093/hmg/ddm158
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Functional capacity of dystrophins carrying deletions in the N-terminal actin-binding domain

Glen B. Banks, Paul Gregorevic, James M. Allen, Eric E. Finn and Jeffrey S. Chamberlain*

1 Department of Neurology, Senator Paul D. Wellstone Muscular Dystrophy Cooperative Research Center, University of Washington, Seattle, WA 98195, USA

* To whom correspondence should be addressed. Tel: +1 2062215363; Fax: +1 2066168272; Email: jsc5{at}u.washington.edu

Received March 12, 2007; Accepted June 19, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Duchenne muscular dystrophy and Becker muscular dystrophy (BMD) are caused by mutations in the dystrophin gene. Although many in-frame deletions in the dystrophin gene lead to mild cases of BMD, truncations within the N-terminal actin-binding domain (ABD1) typically decrease dystrophin expression and lead to more severe cases of BMD. Because of the large reduction in protein expression, the functional capacity of dystrophin proteins deleted for subportions of ABD1 has been difficult to ascertain. ABD1 contains three actin-binding sequences designated ABS1–3. In the present study, we examined the pathophysiological effects of in-frame actin-binding sequence deletions in the context of a highly functional microdystrophin ({Delta}R4–R23/{Delta}CT). We delivered microdystrophins into the tibialis anterior muscles of 2-day-old dystrophin-deficient mdx mice using recombinant adeno-associated viral vectors. Muscles expressing microdystrophin with an intact ABD1 displayed normal morphology and specific force generation and were partially protected from contraction-induced injury when evaluated at 4 months of age. In contrast, muscles expressing microdystrophins lacking ABS2 and 3 or ABS3 alone developed significantly lower levels of specific force and were highly susceptible to contraction-induced injury. Microdystrophins with deletions within ABD1 were also less able to protect myofibers from degeneration than was a microdystrophin with the complete ABD1. We conclude that an intact ABD1 is required to support normal contractile properties of skeletal muscle and to protect against myofiber necrosis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) result from mutations in the dystrophin gene and together are the most common form of dystrophy (1,2). DMD typically results from mutations that severely disrupt the expression of dystrophin, such as null alleles or deletions of critical functional domains, whereas the milder BMD results from mutations that lead to expression of internally truncated dystrophins (‘in-frame deletions’) or from expression of lower than normal levels of dystrophin (3,4). Dystrophin provides a mechanically strong connection between {gamma}-actin filaments and the dystrophin–glycoprotein complex (DGC) at the sarcolemma in muscle (5–7). Mutations that affect the mechanical integrity of this highly structured protein assembly render skeletal muscles susceptible to contraction-induced injury, leading to cycles of muscle necrosis and regeneration (8,9).

Dystrophin consists of four domains including an N-terminal actin-binding domain (ABD1), a central rod domain (which includes a second actin-binding domain, ABD2), a cysteine-rich region and the C-terminal domain (Fig. 1; reviewed in 10). The 5' end of the dystrophin gene is one of two regions in the gene associated with a high frequency of deletion mutations (the other in the central portion of the locus) (11–17). Mutations in the region encoding ABD1 typically result in low levels (10–20%) of dystrophin expression and are associated with more severe forms of BMD (18–26). The functional capacity of dystrophins with small deletions that remove portions of ABD1 is not clear because the expression levels are typically below that which would normally protect muscles from contraction-induced injury (19,27).


Figure 1
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  		Figure 1. Dystrophin has an N-terminal actin-binding domain (ABD1) that contains 2 calponin homology domains (CH1 and CH2) (56). The calponin homology domains contain three actin-binding sequences (ABS1–3) (68). ABD1 is followed by a large central rod domain that contains 24 spectrin-like repeats (R1–24), four hinges (H1–4) and a second actin-binding domain (ABD2) (69,70). The rod domain is followed by a cysteine-rich region and a C-terminal domain, which bind to the dystrophin-associated glycoprotein complex (reviewed in 10). The microdystrophin used in this study has a large portion of the central rod domain missing between spectrin repeats 4 and 23 and also lacks the C-terminal domain ({Delta}R4-23/{Delta}CT) (9). ABS1µDys is the same as microdystrophin except that it lacks both ABS2 and ABS3. ABS1,2µDys lacks ABS3.

 
Studies in transgenic mdx mice have shown that the presence of at least one actin-binding domain is required for dystrophin to protect muscles from degeneration (28–30). Deletion of ABD2 while maintaining ABD1 has a minimal effect on the function of dystrophin in DMD patients and in mdx mice (9,28,29,31,32). Deletion of ABD1 while maintaining ABD2 leads to a mild BMD-like phenotype in muscles of mdx mice (33). ABD1 is composed of two tandem calponin-homology (CH) domains, in which three actin-binding sequences (ABS1–3) have been identified (Fig. 1) (34–37). Actin-binding studies performed in vitro have shown that even a single ABS is able to bind actin (35–43). However, in vivo studies involving expression of dystrophins deleted for portions of ABD1 have not clearly defined the functional requirements for an intact ABD1. For example, although a transgenic/mdx mouse expressing a dystrophin deleted for ABS2 and 3 displayed a mild BMD-like phenotype, that particular dystrophin construct was also deleted for a portion of hinge 1 (27). Similarly, Fabb et al. (44) generated a truncated dystrophin missing ABS2 and 3, hinge 1, spectrin-like repeats 3–18 and the C-terminal domain and showed that this protein was able to ameliorate dystrophic pathology in mdx myofibers expressing the construct. Although these two previous studies suggested that a single actin-binding sequence might be sufficient to protect muscles from contraction-induced injury when expressed at levels above those that would normally provide physiological benefit, neither of these constructs contained an intact hinge 1 (27,44). Importantly, in-frame deletions that remove hinge 1 from dystrophin often lead to more severe cases of BMD than those removing adjacent sequences (26), thus complicating interpretation of the importance of the ABS1 sequences. Thus, the functional capacity of a single actin-binding sequence within dystrophin in terms of supporting specific force generation and protecting muscles from contraction-induced injury remains unclear.

Rational design of truncated dystrophins provides a source of mini-genes that may prove easier to deliver systemically to muscles of DMD patients for gene therapy (9,45–49). Recombinant adeno-associated viral vectors pseudotyped with serotype 6 capsids (rAAV6) infect most muscles following intravenous injection into mice and are therefore a promising method to deliver truncated dystrophins to striated muscles (48,49). Because of the enormous size of dystrophin, only dystrophin mini-genes are compatible with the limited cloning capacity of AAV vectors (9,46,50). Large in-frame deletions within the central rod domain and the C-terminal domain minimally affect the function of dystrophin (9,28,29,31). A particularly promising microdystrophin is the one that lacks spectrin-like repeats 4–23 as well as the C-terminal domain (Fig. 1) (9). This microdystrophin prevents muscle degeneration in mdx and dystrophin:utrophin double knockout mice (mdx:utrn–/–) (48,49,51–53). Microdystrophin also restores specific force in mdx mice when only a small portion of the tibialis anterior (TA) muscle fibers is infected prior to the onset of dystrophy (53) and protects TA muscles from contraction-induced injury in mdx mice (48). Therefore, comparing the functional capacity of microdystrophin with the same protein that lacks subportions of the actin-binding sequences within ABD1 provides a controlled method for analyzing the functional capacity of the N-terminal actin-binding domain in vivo.

In the present study, we show that in-frame deletions of actin-binding sequences in the N-terminal domain of microdystrophin significantly diminished its functional capacity in mdx mice. Our results suggest the more severe clinical phenotypes associated with N-terminal mutations in dystrophin are caused by both reduced protein expression and impaired functional capacity of the mutated dystrophin.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Impaired functional capacity of microdystrophins with deletions in ABD1
We introduced two deletions into the {Delta}R4–R23/{Delta}CT microdystrophin cDNA to produce dystrophins lacking amino acids 36–252 (ABS1µDys) or amino acids 131–252 (ABS1,2µDys) in the N-terminal domain (Fig. 1). The retained amino acid sequences (1–35 and 1–130) of ABD1 have previously been shown to bind filamentous actin in vitro (41). We administered 6 x 1010 vector genomes of an rAAV6-pseudotyped vector expressing either microdystrophin, ABS1µDys or ABS1,2µDys into the TA muscles of 2-day-old mdx mice, as previously described (51) (Fig. 1). Four months after injection, we measured the specific force generating capacity of the various injected muscles (Table 1). Specific force, which is the force normalized for the cross-sectional area of muscle, was reduced by 29% in mdx mice when compared with wild-type mice (n = 4; P < 0.05; Table 1). Injection of mdx muscles with the microdystrophin vector restored specific force generation to ~96% of wild-type levels (n = 5; P < 0.01 compared with mdx muscles; Table 1). Neither ABS1µDys nor ABS1,2µDys restored specific force (n = 5; P > 0.05; Table 1). Notably, force production in muscles expressing ABS1,2µDys was significantly reduced when compared with muscles of wild-type and mdx mice (n = 5; P < 0.05; Table 1). Therefore, deleting actin-binding sequences from microdystrophin adversely affected muscle strength.


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  		Table 1. Contractile properties of TA muscles expressing microdystrophins with actin-binding sequence deletions

 
Next, we tested whether the entire N-terminal domain of dystrophin is required to protect skeletal muscles from contraction-induced injury (Fig. 2). Here, we used a protocol that strains the muscle in a series of ramped stretches from 5–45% above its optimal length during muscle contraction (49). This protocol is a more robust test of contraction-induced injury when compared with the two lengthening contractions performed previously (9,48). Consequently, we revealed a difference in contraction-induced injury between wild-type mice and microdystrophin-treated mdx mice that was not previously apparent. However, microdystrophin expression partially protected muscles from injury when compared with mdx muscles that were stretched between 20 and 45% beyond their optimal length while maximally stimulated (n = 5; Fig. 2). At 20% strain, the muscle contractile force was reduced by 18% in microdystrophin expressing muscles when compared with 30% reduction in untreated muscles. Neither ABS1µDys nor ABS1,2µDys protected skeletal muscles from contraction-induced injury (n = 3–5; Fig. 2). These results show that ABS deletions prevented the ability of microdystrophin to protect muscles from contraction-induced injury.


Figure 2
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  		Figure 2. Deletion of actin-binding sequences from microdystrophin diminished its ability to protect muscles from contraction-induced injury. The contractile performance of TA muscles immediately prior to increasing length changes during maximal force production was also shown. Bars represent the mean ± SD percentage of the initial optimal muscle contraction.

 
N-terminally truncated microdystrophins prevent degeneration in fewer muscle fibers
To confirm that the level of dystrophin expression was similar before the onset of dystrophy, we analyzed transverse sections of muscles 2 weeks after vector injection (Fig. 3). Expression of microdystrophin typically peaks approximately 2 weeks after injection into muscles (54), and this time point is prior to the onset of dystrophy in mdx muscles (54,55). More than 95% of the muscle fibers in each TA were positive for dystrophin expression in all muscles injected with the three types of microdystrophin (Fig. 3). Therefore, the percentage of muscle fibers expressing microdystrophin, ABS1µDys and ABS1,2µDys was similar at 2 weeks.


Figure 3
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  		Figure 3. Transduction of muscles with rAAV6-pseudotyped vectors before the onset of muscular dystrophy in mdx mice. (A) mdx TA muscles were injected with vectors at 2 days of age and analyzed before the onset of muscular dystrophy at day 16. Transverse sections show the expression of microdystrophin on the sarcolemma that was similar to ABS1µDys and ABS1,2µDys 2 weeks after injection. (B) Transverse sections from each TA muscle show that the expression of microdystrophin, ABS1µDys and ABS1,2µdys was uniform throughout each muscle. Scale bars = 100 µm.

 
The percentage of mdx muscle fibers expressing truncated dystrophins decreased over time (Fig. 4A and B). Four months after injection, microdystrophin was found in ~74% of muscle fibers. ABS1,2µDys was found in ~59%, and ABS1µDys was found in ~32% of muscle fibers (Fig. 4B). The proportion of dystrophin positive fibers with central nuclei, a sign of muscle degeneration and regeneration, was ~7% for each truncated dystrophin (Fig. 4A). Approximately 80% of muscle fibers that were dystrophin negative had central nuclei consistent with mdx controls.


Figure 4
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  		Figure 4. Deletion of actin-binding sequences from microdystrophin reduces the number of myofibers protected from degeneration. (A) mdx TA muscles were injected at 2 days of age with vectors expressing truncated dystrophins and analyzed at 4 months for the presence of central nuclei. Transverse sections of TA muscles stained for dystrophin in green and nuclei in blue are shown. Transverse sections were also stained with hematoxylin and eosin. Scale bar = 100µm (B) Microdystrophin prevented central nuclei accumulation in positive fibers. Deletion of actin-binding sequences from microdystrophin did not change the percentage of dystrophin-positive fibers with central nuclei. ***P < 0.001 when compared with mdx (C) However, deletion of actin-binding sequences significantly reduced the overall percentage of dystrophin-positive fibers at 4 months of age. Bars represent the mean ± SD percentage of myofibers with centrally located nuclei in dystrophin-positive fibers. *P < 0.05 when compared with microdystrophin and #P < 0.05 when compared with ABS1,2µDys.

 
Abnormal accumulations of ABS1µDys and ABS1,2µDys were found, surrounding a few myonuclei in transverse sections of muscles 4 months after injection (Fig. 4A). Therefore, the amount of protein on the sarcolemma could be reduced and may not be localized in sufficient quantities to restore specific force or to protect muscles from contraction-induced injury. To test this possibility, we compared the relative intensity of fluorescence at the sarcolemma in transverse sections of muscle processed in parallel for immunofluorescence staining using confocal microscopy (Fig. 5). Quantification of the plot profiles revealed the maximal fluorescence at the sarcolemma in each mouse, which we normalized to microdystrophin-treated mice (see Materials and Methods; Fig. 5). The fluorescence intensity of wild-type dystrophin was between that of mdx mice (which denotes background fluorescence) and microdystrophin transgenic mice, which over-expressed the truncated dystrophin. This ensured that we could measure both increases and decreases in fluorescence intensity relative to wild-type sarcolemma. The maximal fluorescence intensity of the sarcolemma was the same between wild-type and ABS1µDys (78 and 77% of rAAV6-microdystrophin, respectively). The maximal fluorescence intensity of ABS1,2µDys was significantly greater than wild-type (22% increase; P < 0.01). The maximal fluorescence intensity was significantly less in ABS1µDys when compared with microdystrophin-treated mdx mice (23% less; P < 0.001). These results suggest that although ABS deletions may reduce the amount of protein at the sarcolemma, their lack of function was not caused by insufficient protein at the sarcolemma.


Figure 5
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  		Figure 5. Relative maximum fluorescence intensity of dystrophin at the sarcolemma. Fluorescence intensity of the sarcolemma was normalized to microdystrophin-treated mdx muscles as 100%. The mdx is background fluorescence. The µDys tg is microdystrophin transgenic mice, which over expresses the truncated dystrophin (9). **P < 0.01 and ***P < 0.001 when compared with wild-type.

 
Because of the abnormal accumulations of protein at the sarcolemma, we tested whether these truncated dystrophins were properly localized in skeletal muscle. Four months after injection, all truncated dystrophins concentrated to costameres, myotendinous junctions and neuromuscular synapses similar to the location of full-length dystrophin in wild-type mice (Fig. 6). The longitudinal bands (L-bands) in costameres were not as consistently present in mdx muscle expressing the truncated dystrophins when compared with wild-type mice, but in other aspects, the costameric localization was the same as wild-type mice in the TA muscle. mdx mice have previously been shown to have fewer M and L bands stained with ß-spectrin when compared with wild-type mice (56). How L-bands form, their significance and function are yet to be established.


Figure 6
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  		Figure 6. Distribution of wild-type dystrophin, microdystrophin, ABS1µDys and ABS1,2µDys in skeletal muscle. Note the normal distribution of truncated dystrophins at costameres, neuromuscular junctions (NMJ) and myotendinous junctions (MTJ) when compared with wild-type mice. Dystrophin is shown in green. Acetylcholine receptors are stained with rhodamine-bungarotoxin in red to define the location of neuromuscular synapses. Scale bars = 30µm.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Conclusions drawn by considering structure–function relationships of N-terminally truncated dystrophins in humans are complicated by the reduction in protein expression below that which would normally provide physiological benefit to the muscle (19,27). In the present study, we showed that microdystrophins with deletions in ABD1 are impaired in their ability to protect myofibers from degeneration even when expressed at physiologically relevant levels.

ABS1µDys and ABS1,2µDys did not prevent the loss of specific force generating capacity or protect against contraction-induced injury in skeletal muscles of mdx mice. Four months after injecting the various AAV vectors, ABS1µDys was retained in 32% and ABS1,2µDys was retained in 59% of TA muscle fibers, whereas the larger microdystrophin was retained in 77% of myofibers. Microdystrophin has been previously shown to restore specific force generation when expressed in only 20% of the muscle fibers of mice injected with vector prior to the onset of dystrophy (53). Furthermore, expression of microdystrophin in ~50–60% of mdx myofibers restored specific force generation and protected muscles from contraction-induced injury when injected into older mdx mice (52). Similarly, expression of microdystrophin in 60% of mdx:utrn–/– double knockout TA muscles provided substantial physiological benefit to striated muscles (49). On the basis of these studies, our results suggest that deleting actin-binding sequences from the N-terminal domain of microdystrophin impairs the physiological capacity of microdystrophin.

Microdystrophins with N-terminal truncations were able to properly localize within skeletal myofibers and slowed, but did not halt, muscle degeneration. We have previously generated transgenic mice expressing Dp116, which is a truncated dystrophin that contains R24, but which is deleted for all N-terminal actin-binding sequences as well as the central actin-binding domain (30). In addition, DP116 does not contain spectrin repeats 1–3 and hinge 2 of microdystrophin and these regions do not contribute to actin binding (57,58). Dp116 assembles the DGC at the sarcolemma, but does not protect mdx skeletal muscles from degenerating (30). Therefore, in the present study, we conclude that the reduced muscle degeneration in mdx mice treated with ABS1µDys or ABS1,2µDys is dependent on residual actin binding through the truncated N-terminal domains rather than the restoration of the DGC. The percentage of muscle fibers expressing the N-terminally truncated microdystrophins significantly diminished over time. The most likely explanation that more fibers were dystrophin positive in microdystrophin-injected muscles is because microdystrophin is more protective than the ABD1-deleted constructs. Myofibers expressing functionally impaired microdystrophins are more susceptible to necrosis, resulting in the loss of the episomal rAAV vector and subsequent regeneration without dystrophin expression.

The abnormal accumulations of ABS1µDys and ABS1,2µDys in the vicinity of a few myonuclei (Fig. 3A) suggest that the lower percentage of positive fibers may also stem from problems with folding of the N-terminal domain (59). Aberrant folding may adversely affect trafficking and/ or the ability of dystrophin to maintain its position at the sarcolemma. Similar ‘inclusion bodies’ have been shown in muscles of mdx mice expressing mostly non-functional-truncated dystrophin transgenes (9). Most N-terminally truncated dystrophins accumulate to very low levels on the sarcolemma of BMD patient muscles (19). Similarly, previous attempts to generate transgenic/mdx mice expressing an N-terminally truncated dystrophin required screening 12 separate lines of mice to obtain a single line that expressed dystrophin near wild-type levels (27). The central actin-binding domain (ABD2) may partially compensate for the N-terminal deletions when the protein is expressed near wild-type levels (27,33). One exceptional example is a person presenting a very mild form of dystrophy with an exon 3–9 deletion (60). No problems in obtaining consistent protein expression were encountered when generating DP116 transgenic mice that have no actin-binding domains (30). Therefore, partial deletions of the N-terminal domain may have a detrimental effect on protein stability or trafficking.

We conclude that the N-terminal ABD1 deletions considerably diminish the functional capacity of dystrophin to protect muscles from contraction-induced injury. ABD1 deletions moderately reduce the affinity for actin, which could lead to a propensity for muscle degeneration in vivo, as we have seen here (41,42,58,61). ABD1 deletions may also affect the binding of other cytoskeletal proteins such as talin (62,63), aciculin (64), keratin (65) or plectin (66) that may stabilize dystrophin at the sarcolemma and help protect muscles from contraction-induced injury. ABD1 may have multiple roles important for dystrophin expression, trafficking, stability and function and defining these roles will lead to therapeutic strategies required for DMD caused by the hot spot in deletions at the N-terminal domain.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Mice
Wild-type C57Bl/6 mice and mdx4Cv mice were utilized in this study. Two-day-old mice were anaesthetized on a chilled temperature pack for intramuscular injections, as described previously (51). A 31-gage needle was used to inject approximately 6 x 1010 vector genomes into the TA muscles of 2-day and 4-week-old mdx mice. All experiments were approved by the University of Washington Institutional Animal Care and Use Committee.

Generation of constructs
CMV microdystrophin was generated as previously described (48,49). We generated the ABS1µDys and ABS1,2µDys constructs using recombination PCR with CMV microdystrophin as the template (67). ABS1,2µDys was designed to contain the entire CH1 domain (59). The primers used to generate ABS1µDys were Forward1-GATCCAGCCTCCGCGGTTTTT, Reverse1-TAGGTGGCCTTGGCAACATCTGTAGGTCACTGAAGAGGT, Forward2-ACCTCTTCAGTGACCTACAGATGTTGCCAAGGCCACCTA and Reverse2-ATGCTAGCTACCCTGAGGCATT. The primers used to generate ABS1,2µDys were Forward1-GATCCAGCCTCCGCGGTTTTT, Reverse1-TAGGTGGCCTTGGCAACATTCCAGCCATGATATTTTTCA, Forward2-ACCTCTTCAGTGACCTACAGATGTTGCCAAGGCCACCTA and Reverse2-ATGCTAGCTACCCTGAGGCATT. The resulting two transcripts for each ABS1µDys or ABS1,2µDys were combined and amplified with Forward1 and Reverse2 primers. The final transcripts were digested with SacII–NheI restriction enzymes and ligated into the same sites found in the microdystrophin plasmid and sequenced. The resulting constructs were co-transfected into HEK293 cells with the pDGM6 packaging plasmid to generate recombinant AAV vectors comprising serotype 6 capsids that were harvested, purified and quantitated as described previously (54). The resulting titer was determined in comparison with previously known concentrations of rAAV6-CMV-lacZ and microdystrophin using Southern analysis with a probe to the CMV promoter.

Histology
For analyses of central nucleation and dystrophy, TA muscles were placed in OCT-embedding medium and frozen in 2-methylbutane in liquid nitrogen. Ten micrometer transverse sections were incubated in blocking buffer [1 x bovine serum albumin, 0.05% Triton X-100 in 1 x phosphate-buffered saline (PBS)] for 30 min and immunostained with Dys3 primary antibodies for 1 h (1:20 dilution; Novocastra). Because Dys3 is human specific, we used a rabbit polyclonal dystrophin antibody that recognizes the N-terminal domain of dystrophin, which was previously generated in our laboratory, to immunostain wild-type mouse sections (1:800 dilution; Rafael, 1996). The sections were then washed five times with 1 x PBS and incubated with goat antimouse Alexa 488 secondary antibodies for 30 min (1:1200; Molecular Probes). We added rhodamine bungarotoxin (1:800; Molecular Probes) to the Alexa 488 secondary antibody incubation to analyze whether dystrophin was concentrated at neuromuscular synapses. Sections were washed again five times in 1 x PBS and mounted in medium containing DAPI (Vector Labs), which stains nuclei. Dystrophin staining was viewed using a Nikon Eclipse E1000 fluorescent microscope and imaged with ImageQ camera and software (Media Cybernetics Inc.).

For staining of costameres, muscles were fixed in 2% paraformaldehyde for more than 2 h and teased using fine forceps. Individual muscles were then incubated in 0.1 M glycine for 1 h and blocking buffer overnight. Muscles were then stained with Dys3 primary antibody and Alexa 488 secondary antibody as described earlier except the primary antibody was incubated for 2 h and the secondary antibody for 1 h. Neuromuscular synapses, myotendinous junctions and costameres were all viewed and photographed using a Leica SL confocal microscope.

Quantitation of maximal sarcolemmal dystrophin fluorescence intensity
Transverse frozen sections of skeletal muscle were processed for immunostaining in parallel, as described earlier. Images were taken using confocal microscopy (Leica SL, Exton PA). Images were taken with the same settings and the images were processed in an identical way. We placed limits on the image ‘gain’ using the over/under glo function on the confocal microscope to ensure we had not saturated the immunofluorescence intensity of the images and this setting was maintained through all image processing. The intensity of fluorescence was measured using Image J computer software (NIH). A line was drawn across the sarcolemma of two adjacent muscle fibers and a plot profile was obtained. The maximum fluorescence intensity was recorded and compared between the mice. We chose to quantify fluorescent sections rather than western blots, as western blots would include the protein aggregates shown in Figure 4, which are unlikely to be mechanically functional.

An available antibody that recognizes the remaining epitopes of the ABS-deleted constructs is the human specific Dys3 antibody (Novocastra), which recognizes amino acids 321–494 (Hinge1-Repeat2). Using the Dys3 antibody, we recorded the plot profiles from the sarcolemma of 161 rAAV6-microdystrophin, 63 ABS1µDys and 85 ABS1,2µDys fibers from n = 4 mice each. We also recorded the plot profiles of 72 mdx muscles from four mice to control for background fluorescence. Because Dys3 does not recognize mouse dystrophin, we also performed a separate experiment to quantify the fluorescence in wild-type dystrophin using a rabbit polyclonal antibody to the N-terminus of dystrophin. Using the N-terminal dystrophin antibody, we recorded the plot profiles from the sarcolemma of 82 wild-type, 33 mdx, 54 microdystrophin transgenic and 106 rAAV6/microdystrophin-treated mdx sarcolemma positive for microdystrophin. Four mice were used for each treatment. The maximum fluorescence intensity was compared using a one-way analysis of variance (ANOVA), with a Tukey post-test using the Prism statistics program. To compare the fluorescence between the two experiments, we normalized the maximal fluorescence intensity of the sarcolemma to rAAV6-microdystrophin-treated mdx mice.

Muscle physiology
Muscle physiology was performed as previously described (49). Briefly, mice were anaesthetized with 2,2,2-tribromoethanol (Sigma) such that they were insensitive to tactile stimuli. Peak isometric force of the TA muscle was analyzed in situ via nerve stimulation. First, we found the maximum force-producing capacity of each muscle at its optimum length according to maximal stimulation over 300 ms to elicit tetanic contraction. The peak force was then divided by the unit area of muscle to obtain specific force (kN/m2). The equation is shown in Table 1. Next, we measured the protection from contraction-induced injury. The force-producing capacity of the muscle was measured immediately prior to increased length changes during maximal stimulation at 20 s intervals. Length changes were increased in 5% increments from 5 to 45% of muscle fiber length to produce injury. The rate of length change was 2 lengths/s.

Statistics
All data were compared using a one-way ANOVA with a Tukey post-test that compares all data sets with a Student's t-test. All data analyses were performed using the PRISM software.


   ACKNOWLEDGEMENTS
 
We are grateful to Leonard Meuse for animal husbandry, Greg Martin at the Keck Imaging Center University of Washington for help with confocal microscopy and Chamberlain lab members for critically reviewing the manuscript. This work was supported by grants from the National Institutes of Health (AR44533) and the Muscular Dystrophy Association (to J.S.C.). G.B.B. was supported by a CJ Martin Post-Doctoral Fellowship from the National Health and Medical Research Council of Australia (372212). P.G. was supported by a Development Grant from the Muscular Dystrophy Association.

Conflict of Interest statement. The authors declare no conflicts of interest.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Hoffman E.P., Brown R.H. Jr, Kunkel L.M. Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell (1987) 51:919–928.[CrossRef][Web of Science][Medline]

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G. B. Banks, A. C. Combs, J. R. Chamberlain, and J. S. Chamberlain
Molecular and cellular adaptations to chronic myotendinous strain injury in mdx mice expressing a truncated dystrophin
Hum. Mol. Genet., December 15, 2008; 17(24): 3975 - 3986.
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