DOI: 10.1113/jphysiol.2007.141481
2007 The Authors. Journal compilation
2007 The Physiological Society
Downloaded from J Physiol (jp.physoc.org) by guest on June 8, 2011

G. DAntona and others

J Physiol 584.3
human muscles (Gabellini et al. 2002). Very recently we demonstrated that transgenic overexpression of FRG1 in skeletal muscle was sufcient to cause MD in mice, whereas mice overexpressing the two other candidate genes, FRG2And ANT-1, did not show clear signs of MD (Gabellini et al. 2006). Moreover, the link between MD manifestations and FRG1 overexpression could not be ascribed to a plasma membrane defect, suggesting a novel pathogenetic mechanism for FSHD. However, similarly to other mouse models of MD, mice overexpressing FRG1 (FSHD mice) showed impaired mobility in vivo and dystrophic features, which appeared differentially distributed among skeletal muscles. In this respect, the analysis of the structure and function of muscle bres from the FSHD mice is relevant for at least two reasons. It will further the understanding of a novel mouse model of one of the main human MDs which will be used in the future to further our knowledge on the pathogenesis of the disease and to develop therapeutic strategies. Furthermore, the FSHD mouse model may help understanding of whether and to what extent fundamental features of the dystrophic process at muscle bre level, such as the intrinsic loss of contractile strength, the preferential involvement of fast bres, and the fast to slow shift in muscle phenotype are necessarily linked to alterations of the DCG complex or can be a common path of alternative pathogenetic mechanisms. The FSHD mouse represents in fact one model of MD displaying histological and functional dystrophic features similar to the most studied models of MDs (Gabellini et al. 2006) which cannot be linked to a plasma membrane defect and ultimately to altered calcium homeostasis across the membrane (Allen et al. 2005). The absence of a clear plasmamembrane defect, as demonstrated by no change in serum creatine kinase (Richard et al. 2000) and lack in Evans blue uptake, is found in other murine models of MDs, as calpain-3 deciency (Richard et al. 2000; Fougerousse et al. 2003). Nevertheless, murine models in which mutations do not involve proteins of the DCG complex either ultimately show membrane alterations such as the collagen VI decient mouse (Bonaldo et al. 1998; Irwin et al. 2003) or do not show any clear sign of skeletal muscle involvement, such as the Six5 decient mouse, a model of myotonic MD (Klesert et al. 2000; Sarkar et al. 2000). Notwithstanding the large body of information accumulating on murine models of MD, key features of the disease are still far from being fully understood. In particular, muscle weakness is generally attributed to replacement of degenerating and necrotic muscle bres with non contractile material (connective tissue and fat), which ultimately causes a loss of contractile muscle mass (quantitative mechanism) even in the presence of increased muscle weight (pseudo-hypertrophy) (Watchko et al. 2002). Whether a qualitative mechanism, namely a loss of the intrinsic capacity to develop force by a given

amount of muscle mass, is also involved in generating muscle weakness is unclear. In fact, whereas some earlier studies suggested no loss in specic force of single muscle bres of mdx mice (Takagi et al. 1992; Lynch et al. 2000), more recently we consistently showed a clear Po/CSA decit in several mouse models of MD: mdx (Denti et al. 2006), scid-mdx (Torrente et al. 2004), -sarcoglycan null mice (Sampaolesi et al. 2003). Interestingly, in humans affected by Duchenne MD, whereas Fink et al. (1990) found an impairment of specic force of muscle bres, Horowits et al. (1990) failed to show such impairment. The origin of force loss in muscular dystrophies appears to be far from understood. In particular an imbalance between protein synthesis and breakdown towards increased proteolysis (McKeran et al. 1977; Elia et al. 1981; Warnes et al. 1981) and the replacement of necrotic cells by weaker regenerating bres which show different functional characteristics from uninjured bres may contribute to reduced capacity to generate force (Gregorevic et al. 2004). Furthermore, in intact bres, decreased evoked calcium release due to ongoing triadic disruption might also contribute to force loss at least in mdx mice (Allen et al. 2005). In any case, in all muscular dystrophies the loss in force and specic force appear as common endpoints of diverse primary defects. In addition, it is generally believed that the dystrophic processes preferentially strike fast bres, and among fast bres the fastest, type 2B bres (Webster et al. 1988). However, such a preferential involvement has not been denitely demonstrated by the comparative analysis of strength of slow and fast muscle bres. As regards the origin of bre deterioration in MDs, an increased calcium inux across membrane tears and calcium permeable leak channels followed by activation of proteolysis is a candidate mechanism (Allen et al. 2005; Yeung et al. 2005). Nevertheless it is still unclear what is the link between the genetic defect and muscle bre deterioration, which are the cellular pathways involved and whether alternative mechanisms can be identied in the process (Gillis, 1999). In this work, structure and function of identied types of single muscle bres from the mouse model of FSHD were studied in comparison to corresponding types of WT bres. The existence of several lines of FSHD, expressing FRG1 at different levels and showing a variable progression of MD, enabled us to relate structural and functional properties of muscle bres to the progression of the disease. Results of this study (i) provide a detailed characterization of skeletal muscle bres of a novel mouse model of one of the main human MDs and (ii) point toward the existence of alternative pathogenetic mechanisms, besides altered calcium homeostasis through the membrane and increased number of weaker regenerating bres, which can give rise to fundamental features of the dystrophic process, common to other MDs, such as the intrinsic loss

Muscle bre function in facioscapulohumeral muscular dystrophy
of contractile strength of muscle bres, the preferential involvement of fast bres and the shift towards a slow muscle phenotype. Methods

Animals

Morphological analysis
Mice overexpressing FRG1 to variable extents under the control of the human skeletal -actin (HSA) gene promoter were previously generated (Gabellini et al. 2006). Briey, FRG1 open reading frames were PCR-amplied using the following primer: FRG1, HSA-FRG1-F 50-GATCTAGCGGCCGCCATGGCCGAGTACTCCTATGTGAAGTCT-300. PCR products were sequenced and cloned into pBSX-HSAvpA, provided by J. Chamberlain. Transgene was excised from agarose gels and puried for microinjection into fertilized eggs recovered from C57BL/6 females crossed with C57BL/6 males. All procedures were performed at the University of Massachusetts Medical School Transgenic Animal Modelling Core Facility. In this work, we used the same three lines of mice we previously characterized (Gabellini et al. 2006) in which the transgene was overexpressed at levels within the range previously reported for FRG1 overexpression in FSHD patients (Gabellini et al. 2002): FRG1-low, FRG1-med, FRG1-high. In these lines RT-PCR and Southern blot analysis were used to quantify the FRG1 expression levels and the transgene copy number (3 for FRG1-low, 5 for FRG1-med; 9 for FRG1-high) which corresponded to 10- (FRG1-low), 30- (FRG1-med), and 50-fold (FRG1-high) over the endogenous level of the murine FRG1 gene (Gabellini et al. 2006). This study was carried out on female FRG1 transgenic mice at 12 weeks of age; C57bL/6 mice of matched age served as controls. Animals were initially housed in the animal house facility of the Program in Gene Function and expression of the University of Massachusetts Medical School, under the care of the Department of Animal Medicine. At the age of 8 weeks mice were shipped (World Courier) to Italy avoiding extreme temperature during transportation. Once in Italy animals were individually housed in the animal house facility of the Department of Experimental Medicine (University of Pavia, Italy), maintained on a 12 : 12 h lightdark cycle and given unlimited access to food (Dottori Piccioni Italy) and water for the duration of the study. On the day of the experiment, the animals were weighed and killed by cervical dislocation. Muscles (facials: masseter; respiratory: intercostals and diaphragm; anterior limbs: deltoids, biceps and triceps; posterior limbs: soleus, tibialis, vastus and gastrocnemius) were carefully dissected under a stereomicroscope, blotted on lter paper, weighed and stored for analysis. The experimental protocols were approved by the Animal Ethics Committee of the University of Pavia.

Muscle samples were included in OCT (Tissue-tek) embedding medium, frozen in liquid nitrogen and serial transverse sections (8 m thick) were cut using a Leica CM 1850 cryostat. Sections were used for haematoxylin-eosin (H&E) staining, according to standard procedures (Gabellini et al. 2006). The percentage of centrally nucleated bres was calculated in non-consecutive sections (at least ve for each muscle) as (number of centrally nucleated bres/total number of bres considered) 100 (no less than 600 for each group of mice, n = 5).
Analysis of force and velocity of single muscle bres
Absolute (Po) and specic force (Po over the bre cross-sectional area, Po/CSA) of single muscle bres were measured in skinned preparations using an approach routinely used for years in our laboratory. All the procedures used have been previously described in detail (Bottinelli et al. 1991, 1996; DAntona et al. 2006) except for the solutions which were modied. The skinning solution previously described (DAntona et al. 2006) (150 mm potassium propionate, 5 mm KH2 PO4 , 5 mm magnesium acetate, 3 mm Na2 ATP, 5 mm EGTA, pCa 9.0) was enriched with protease inhibitors (leupeptin 20 m and E-m). Pre-activating (PA) and activating (A) solutions had the following composition: PA: imidazole-propionate 10 mm, magnesium propionate 2.5 mm, potassium propionate 170 mm, K2 -EGTA 5 mm; A: Imid-p 10 mm, magnesium propionate 2.2 mm, potassium phosphate 170 mm, K2 -EGTA 0.11 mm, Ca-EGTA 4.8 mm. In both solutions, Na2 ATP 5 mm and protease inhibitors leupeptin and E-64 were added. Ionic strength was 200 mm in all solutions. The pH of all solutions was set at 7.0. Po was determined at pCa 4.5, 12 C and optimal sarcomeric length for force developing (2.5 m) (Pellegrino et al. 2003). Po/CSA was expressed as kN m2. The set-up for single muscle bre analysis was located on the stage of an inverted microscope which enabled us to view the specimen at 320 magnication. CSA was determined, assuming a circular shape, from the mean of three diameters without correction for the swelling which is know to occur in demembranated specimens (Godt & Maughan, 1977). As discussed before (DAntona et al. 2006), skeletal muscle bres do not always have a circular cross-section and, due to the procedure used to mount the bre in the set-up, only the largest of the two main diameters could be routinely measured. Therefore, an overestimation of CSA could occur and the degree of overestimation was bound to depend on the shape of the cross-section. To make sure that variation in the shape of the cross-section between WT and dystrophic bres did not introduce a systematic error in the determination
of CSA and affect the reliability of Po/CSA comparisons we used a procedure previously described (DAntona et al. 2006). Briey, immediately after dissection, muscles were frozen in liquid nitrogen for subsequent histological analysis. On such sections the larger and smaller diameter of a large population of muscle bres from WT and FRG1-high mice were determined using freely available image analysis software (Scion Image, Scion Corp., USA). CSA was determined either using the largest diameter and assuming a circular shape or using both the largest and the smallest diameters and considering an elliptical shape for both WT and FRG1-med bres. The ratio between the measurement based on a circular shape and the measurement based on an elliptical shape was not signicantly different among WT (1.23 0.05 n = 40 from 4 mice), FRG1-low (1.21 0.07 n = 40) and FRG1-med (1.32 0.06 n = 40 from 4 mice) bres. As expected, the CSA determination assuming a circular shape somewhat underestimated Po/CSA, but could not introduce any systematic error in the comparison of Po/CSA (DAntona et al. 2006). To determine maximum shortening velocity (V o ), the slack test technique was employed (Bottinelli et al. 1996); V o was expressed as bre lengths per second (L s1 ). In skinned bres, disorder of the striation pattern can occur during maximal activation and this might affect determination of V o. To ensure that specimens did not go through any signicant sarcomere disorganization: (i) muscle bres were viewed before, during and after each activation at 320 and were discarded if non-uniformities were seen; (ii) bres which went through a force loss of more than 10% between the rst and the last (fthsixth) activation used to determine V o were discarded even in the absence of clear non-uniformities. Indeed, as the bre segments used for functional analysis were kept short (1.0 mm), were chosen on the basis of uniform CSA and were quickly and uniformly activated using a previously described procedure (Bottinelli et al. 1994), few bres did not pass the quality controls and had to be discarded. The slack test manoeuvres enable us to determine the series compliance. As expected, series compliance ranged between 3.0% and 5.5% with no signicant differences between corresponding bre types of WT and FSHD mice. At the end of the mechanical experiment, bres were put in 20 l of Leammli buffer (Laemmli, 1970) and stored at 20 C for MHC isoform content analysis.

Myosin heavy chain isoform distribution of FRG1 mice muscles
The distribution of MHC isoforms, a widely used index of bre type distribution, was determined in 10 muscles (facials: masseter; respiratory: intercostals and diaphragm; upper limbs: deltoids, biceps and triceps; lower limbs: soleus, tibialis, vastus and gastrocnemius) of WT and FRG1-med mice by densitometric analysis of MHC bands separated by SDS-PAGE (Table 2). Consistent with the observation that type 1, slow bres were relatively spared by the dystrophic process whereas type 2B bres were the most affected bre type, FRG1-med mice showed a signicant shift towards the expression of slower MHC isoforms in all muscles studied. In masseter the shift was from MHC-2B and 2X towards 2A, in intercostals and deltoid from MHC-2B towards MHC-2X and 2A; in diaphragm the shift was from MHC-2B, 2X and 2A towards MHC-1. As regards anterior and posterior limb muscles, in biceps, triceps, vastus and gastrocnemius the shift was from MHC-2B towards
Table 2. Myosin heavy chain (MHC) composition (%) of muscles from controls (WT) and FRG1-med transgenic mice at 12 week of age MHC-1 (%) District Facial Respiratory Anterior limb Muscle Masseter WT 4.7 5.5 FRG1-m 9.3 10.4 15.7 11.9 1.8 13.0 2.28.7 3.0 64.1 3.5 9.7 10.4 2.3 0.3 24.2 2.2 0.8 MHC-2A (%) WT 00 39.5 0.5 17.3 3.2 8.0 1.2 16.1 2.9 18.2 1.9 51.5 5.5 5.4 7.4 6.8 1.9 7.3 7.9 FRG1-m 2.0 MHC-2X (%) WT FRG1-m 2.3 MHC-2B (%) WT 17.9 12.6 11.1 1.9 56.7 2.3 66.4 0.6 58.4 4.8 58.2 3.72.7 4.2 82.1 1.3 76.6 6.8 FRG1-m 5.7 6.1 1.8 0.7 30.3 3.7 50.2 4.7 45.8 1.2 45.7 4.55.1 22. 52.0 1.3 52.1 3.5
16.1 17.7 77.3 8.0 68.9 5.7 54.7 24.7 3.9 15.7 1.4 27.2 0.27.6 3.6 7.4.9 8.0 17.0 0.8 11.4 12.3 37.7 3.9 27.7 19.9 4.1.5
Diaphragm 11.6 2.5 Intercostal 6.1 2.3 Deltoid Biceps Triceps Soleus Tibialis Vastus Gastrocn. 11.6 1.00 37.1 1.00 00
14.0 1.2 21.0 1.5 24.7 1.3 27.2 1.0 23.5 2.9 27.6 3.1 11.4 4.7 8.2 2.1 21.9 2.0 27.8 4.8 11.1 2.0 28.7 0.5 16.1 2.4 23.7 2.7

Posterior limb

Signicantly
different from correspondent isoforms. Means S.D. P < 0.05, n = 10.
MHC-1; nally in soleus the shift was from MHC-2X and especially MHC-2A towards MHC-1 (Table 2). To clarify whether a shift of muscle phenotype could be considered an early sign of FSHD, the MHC isoform
distribution of soleus, as an example of a slow, mildly affected muscle, and of biceps and vastus (Fig. 2) as examples of fast signicantly affected muscles was studied in all lines of FRG1 mice. Interestingly the overall shift

Po/CSA (kN/m 2)

WT FRG1-low FRG1-med

CSA (m2)

fiber types

% force deficit

FRG1-low

FRG1-med

Vo (L/s)

2A 2X 2B

Figure 1. Cross-sectional area (CSA, A), specic force (Po/CSA, B), maximum shortening velocity (V o , C) and tension decit ((FRG1/force of WT) 100, D) of single muscle bres from WT, FRG1-low and FRG1-med mice All bres (n = 329) were identied on the basis of MHC isoforms content determined by SDS-PAGE in pure type 1, 2A and 2B. CSA is expressed in m, Po/CSA in kN m1 , V o in length per second (L s1 ) and force decit as percentage force loss of corresponding bres from WT mice. The height of each bar represents the mean ( S.E.M.). Signicantly different from corresponding bres type of WT; signicantly different from corresponding bres of WT and FRG1-low; signicantly different from type 1 and type 2A; #signicantly different from type 1, 2A and 2X; P < 0.05 n = 10.

soleus

100 75

vastus

biceps
WT FRG1-low FRG1-med FRG1-high

MHC-2X MHC-2B

* * * * *

MHC-1 MHC-2A

MHC-2A

MHC-2X

MHC-2B
Figure 2. Myosin heavy chain (MHC) distribution of soleus, vastus and biceps in WT, FRG1-low, FRG1-med and FRG1-high mice MHC distribution determined from muscle samples (n = 5) of the four groups of mice by SDS-PAGE separation followed by densitometric analysis of MHC bands. The height of each bar represents the mean ( S.E.M.). Signicantly different from the other groups; signicantly different from WT and FRG1-low; P < 0.05, n = 5.
towards a slower phenotype appeared evident in all muscles analysed and in all lines, even in soleus muscles (Fig. 2) of FRG1-low mice which did not show clear histological signs of MD (Fig. 3). Thus, change in MHC composition of the muscle can be considered an early sign of the disease.
Muscle morphology of slow and fast muscles
The observation that the dystrophic process preferentially affected size and function of the fastest bre type (2B)
and relatively spared the slowest bre type (1) should be reected not only in a fast to slow transition in bre type composition of the muscles, but also in a differential dystrophic deterioration of fast and slow muscle. To clarify whether this was actually the case, the morphology of the muscles, the ratio between muscle weight and body weight and the percentage of centrally nucleated bres were compared through muscles and FRG-1 lines. As expected (Gabellini et al. 2006), dystrophic histological features, namely bre necrosis, number of centrally nucleated bres, connective tissue, and
Figure 3. Heamatoxilyneosin stained sections from soleus, vastus and biceps muscle of WT, FRG1-low, FRG1-med and FRG1-high mice Distribution of muscle damage appeared to depend on the muscle considered and on the level of FRG1 expression. In all muscles no clear histological signs of muscular dystrophy were detectable in FRG1-low mice, whereas signs of bre degeneration and necrosis (open arrows) as well as centrally nucleated bres (close arrows) were detectable in biceps and vastus from FRG1-med and FRG1-high mice and appeared to be more evident in FRG1-high than FRG1-med mice. Bar represents 30 m.
2007 The Authors. Journal compilation 2007 The Physiological Society

MW/BW percent of WT

FRG1-low FRG1-med

* * *

FRG1-high
Figure 4. Ratio between muscle weight (MW) and body weight (BW) expressed relative to the MW/BW ratio of the same muscle of age-matched WT, FRG1-low, FRG1-med and FRG1-high mice S, soleus; Ta, tibialis anterior; Ga, gastrocnemius; Tr, triceps; Bc, biceps; Dia, diaphragm. The height of each bar represents the mean ( S.E.M.). Signicantly different from the other groups; signicantly different FRG1- high; P < 0.05, n = 10.

Muscle weakness in MDs is not only due to a loss of contractile muscle mass
% of total number of fibers

0 S Bc V

Figure 5. Percentage of centrally nucleated bres in soleus (S), biceps (Bc) and vastus (V) of WT, FRG1-low, FRG1-med and FRG1-high mice At least 600 bres were analysed from each group of mice (n = 5). The height of each bar represents the mean ( S.E.M.). Signicantly different from WT and FRG1- low, P < 0.05, n = 10.
In the mouse model of FSHD, impaired mobility was ascribed to muscle atrophy (quantitative mechanism) which was observed at early stages of the disease in the absence of any, even transient, signicant compensatory hypertrophy (Gabellini et al. 2006). The present ndings, showing a loss of specic force (Po/CSA) of single muscle bres, indicate the existence of a qualitative mechanism of muscle weakness based on a loss of intrinsic contractile strength. As type 2B bres, which are the most represented bre type in mice muscles (Pellegrino et al. 2003), lose approximately half of their intrinsic strength even in FRG1-low mice, the latter mechanism can play a major
role, together with a loss of muscle mass, in determining muscle weakness in the FSHD mice in vivo. As muscle weakness in vivo, the loss of specic force of muscle bres appeared related to the progression of the disease being more evident in FRG1-med than in FRG1-low mice. The results obtained in this work conrm and extend previous ndings. Earlier studies showed a loss of specic force in some population of skinned muscle bres in mdx mice (Williams et al. 1993) and in an earlier murine model of MD, 129ReJ mice (Fink et al. 1986). Very recently, we consistently showed a loss of specic force in the fastest type 2B bre type in several mouse models of MDs: mdx mice (Denti et al. 2006), -sarcoglycan KO mice (Sampaolesi et al. 2003), scid-mdx mice (Torrente et al. 2004). It appears that the intrinsic loss of contractile strength is a major mechanism underlying muscle weakness in MD. Moreover, the latter phenomenon could be a fundamental feature of the dystrophic process as it is common to MDs having different pathogenetic mechanism and it is an early manifestation of the disease, at least in the only model, the FSHD mice, in which specic force decit could be related to the severity of the disease. The loss in specic force of muscle bres observed in recent studies on mdx mice (Denti et al. 2006) is not in agreement with some earlier ndings on the same mouse model (Takagi et al. 1992; Lynch et al. 2000). The dependence of muscle bre alterations on bre type and on the progression of the disease suggests possible causes for such discrepancy. In fact, in earlier works, muscle bres were not precisely typed and results might have been affected by pooling and comparing muscle bres of different types and therefore differentially affected by the disease. Mechanisms underlying the loss of specic force are still unclear. Interestingly, the loss of Po/CSA in atrophic muscle bres of elderly sedentary and immobilized humans has been shown to be linearly related to a decrease in myosin concentration within the bres (Hook et al. 2001; DAntona et al. 2003; DAntona et al. 2006). As in MDs the balance between myobrillar protein synthesis and degradation is shifted towards degradation (McKeran et al. 1977; Elia et al. 1981; Warnes et al. 1981), the latter phenomenon might be involved in MDs as well. Moreover a possible contribution to reduced specic force generation may also arise from increased number of weaker regenerating bres which, in the presence of degenerationregeneration cycles, initially replace necrotic bres (Gregorevic et al. 2004). Nevertheless in FRG1 mice the observation of a limited amount of necrosis and regeneration, even in FRG1-high mice, suggests that this mechanism might play a minor role. The observation that dystrophic bres have not only lower specic force (Po/CSA), but also lower unloaded shortening velocity (V o ) than WT bres is relevant as it

Interestingly abnormal mRNA processing plays a pathogenetic role in other neuromuscular disorders such as oculopharyngeal muscular dystrophy (Calado et al. 2000) and myotonic MD (Ranum & Day, 2004) and a recent study on muscle bres of patients affected by myotonic MD (Krivickas et al. 2000) showed a loss of specic force, although limited to slow bres. The latter observation strengthens the idea that abnormal mRNA processing can determine the structural and functional alterations at the bre level. However the causal link between aberrant pre-mRNA processing and loss in force of dystrophic bres is still unknown. In conclusion, the analysis of the FSHD mice provided a detailed characterization of a novel mouse model of a major human MD which is bound to be used to further study both the pathogenesis and the therapy of such important disease. Moreover, the observation that fundamental features of the dystrophic process are common to the FSHD mice and to the most studied murine models of MD strongly suggest they can be on a common path of different pathogenetic mechanisms.

References

Allen DG, Whitehead NP & Yeung EW (2005). Mechanisms of stretch-induced muscle damage in normal and dystrophic muscle: role of ionic changes. J Physiol 567, 723735. Bonaldo P, Braghetta P, Zanetti M, Piccolo S, Volpin D & Bressan GM (1998). Collagen VI deciency induces early onset myopathy in the mouse: an animal model for Bethlem myopathy. Hum Mol Genet 7, 21352140. Bottinelli R, Betto R, Schiafno S & Reggiani C (1994). Unloaded shortening velocity and myosin heavy chain and alkali light chain isoform composition in rat skeletal muscle bres. J Physiol 478, 341349. Bottinelli R, Canepari M, Pellegrino MA & Reggiani C (1996). Force-velocity properties of human skeletal muscle bres: myosin heavy chain isoform and temperature dependence. J Physiol 495, 573586. Bottinelli R, Schiafno S & Reggiani C (1991). Force-velocity relations and myosin heavy chain isoform compositions of skinned bres from rat skeletal muscle. J Physiol 437, 655672. Briguet A, Courdier-Fruh I, Foster M, Meier T & Magyar JP (2004). Histological parameters for the quantitative assessment of muscular dystrophy in the mdx-mouse. Neuromuscul Disord 14, 675682. Calado A, Tome FM, Brais B, Rouleau GA, Kuhn U, Wahle E & Carmo-Fonseca M (2000). Nuclear inclusions in oculopharyngeal muscular dystrophy consist of poly(A) binding protein 2 aggregates which sequester poly(A) RNA. Hum Mol Genet 9, 23212328. Casanova G & Jerusalem F (1979). Myopathology of myotonic dystrophy. A morphometric study. Acta Neuropathol (Berl) 45, 231240.

Coirault C, Lambert F, Marchand-Adam S, Attal P, Chemla D & Lecarpentier Y (1999). Myosin molecular motor dysfunction in dystrophic mouse diaphragm. Am J Physiol Cell Physiol 277, C1170C1176. Consolino CM & Brooks SV (2004). Susceptibility to sarcomere injury induced by single stretches of maximally activated muscles of mdx mice. J Appl Physiol 96, 633638. DAntona G, Lanfranconi F, Pellegrino MA, Brocca L, Adami R, Rossi R, Moro G, Miotti D, Canepari M & Bottinelli R (2006). Skeletal muscle hypertrophy and structure and function of skeletal muscle bres in male body builders. J Physiol 570, 611627. DAntona G, Pellegrino MA, Adami R, Rossi R, Carlizzi CN, Canepari M, Saltin B & Bottinelli R (2003). The effect of ageing and immobilization on structure and function of human skeletal muscle bres. J Physiol 552, 499511. Deconinck N & Dan B (2007). Pathophysiology of duchenne muscular dystrophy: current hypotheses. Pediatric Neurol 36, 17. Denti MA, Rosa A, DAntona G, Sthandier O, De Angelis FG, Nicoletti C, Allocca M, Pansarasa O, Parente V, Musaro A, Auricchio A, Bottinelli R & Bozzoni I (2006). Body-wide gene therapy of Duchenne muscular dystrophy in the mdx mouse model. Proc Natl Acad Sci U S A 103, 37583763. Durbeej M & Campbell KP (2002). Muscular dystrophies involving the dystrophin-glycoprotein complex: an overview of current mouse models. Curr Opin Genet Dev 12, 349361. Elia M, Carter A, Bacon S, Winearls CG & Smith R (1981). Clinical usefulness of urinary 3-methylhistidine excretion in indicating muscle protein breakdown. Br Med J (Clin Res Ed) 282, 351354. Ferrari G, Cusella-De Angelis G, Coletta M, Paolucci E, Stornaiuolo A, Cossu G & Mavilio F (1998). Muscle regeneration by bone marrow-derived myogenic progenitors. Science 279, 15281530. Fink RH, Stephenson DG & Williams DA (1986). Calcium and strontium activation of single skinned muscle bres of normal and dystrophic mice. J Physiol 373, 513525. Fink RH, Stephenson DG & Williams DA (1990). Physiological properties of skinned bres from normal and dystrophic (Duchenne) human muscle activated by Ca2+ and Sr2+. J Physiol 420, 337353. Fougerousse F, Gonin P, Durand M, Richard I & Raymackers JM (2003). Force impairment in calpain 3-decient mice is not correlated with mechanical disruption. Muscle Nerve 27, 616623. Gabellini D, DAntona G, Moggio M, Prelle A, Zecca C, Adami R, Angeletti B, Ciscato P, Pellegrino MA, Bottinelli R, Green MR & Tupler R (2006). Facioscapulohumeral muscular dystrophy in mice overexpressing FRG1. Nature 439, 973977. Gabellini D, Green MR & Tupler R (2002). Inappropriate gene activation in FSHD: a repressor complex binds a chromosomal repeat deleted in dystrophic muscle. Cell 110, 339348. Gillis JM (1999). Understanding dystrophinopathies: an inventory of the structural and functional consequences of the absence of dystrophin in muscles of the mdx mouse. J Muscle Res Cell Motil 20, 605625.

Godt RE & Maughan DW (1977). Swelling of skinned muscle bers of the frog. Experimental observations. Biophys J 19, 103116. Gregorevic P, Plant DR, Stupka N & Lynch GS (2004). Changes in contractile activation characteristics of rat fast and slow skeletal muscle bres during regeneration. J Physiol 558, 549560. Harridge SD, Bottinelli R, Canepari M, Pellegrino MA, Reggiani C, Esbjornsson M & Saltin B (1996). Whole-muscle and single-bre contractile properties and myosin heavy chain isoforms in humans. Pugers Arch 432, 913920. Hook P, Sriramoju V & Larsson L (2001). Effects of aging on actin sliding speed on myosin from single skeletal muscle cells of mice, rats, and humans. Am J Physiol Cell Physiol 280, C782C788. Horowits R, Dalakas MC & Podolsky RJ (1990). Single skinned muscle bers in Duchenne muscular dystrophy generate normal force. Ann Neurol 27, 636641. Irwin WA, Bergamin N, Sabatelli P, Reggiani C, Megighian A, Merlini L, Braghetta P, Columbaro M, Volpin D, Bressan GM, Bernardi P & Bonaldo P (2003). Mitochondrial dysfunction and apoptosis in myopathic mice with collagen VI deciency. Nat Genet 35, 367371. Klesert TR, Cho DH, Clark JI, Maylie J, Adelman J, Snider L, Yuen EC, Soriano P & Tapscott SJ (2000). Mice decient in Six5 develop cataracts: implications for myotonic dystrophy. Nat Genet 25, 105109. Krivickas LS, Ansved T, Suh D & Frontera WR (2000). Contractile properties of single muscle bers in myotonic dystrophy. Muscle Nerve 23, 529537. Laemmli UK. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680685. Lynch GS, Rafael JA, Chamberlain JS & Faulkner JA (2000). Contraction-induced injury to single permeabilized muscle bers from mdx, transgenic mdx, and control mice. Am J Physiol Cell Physiol 279, C1290C1294. Mankodi A, Logigian E, Callahan L, McClain C, White R, Henderson D, Krym M & Thornton CA (2000). Myotonic dystrophy in transgenic mice expressing an expanded CUG repeat. Science 289, 17691773. McKeran RO, Halliday D & Purkiss P (1977). Increased myobrillar protein catabolism in Duchenne muscular dystrophy measured by 3-methylhistidine excretion in the urine. J Neurol Neurosurg Psychiatry 40, 979981. Moens P, Baatsen PH & Marechal G (1993). Increased susceptibility of EDL muscles from mdx mice to damage induced by contractions with stretch. J Muscle Res Cell Motil 14, 446451. Muller J, Vayssiere N, Royuela M, Leger ME, Muller A, Bacou F, Pons F, Hugon G & Mornet D (2001). Comparative evolution of muscular dystrophy in diaphragm, gastrocnemius and masseter muscles from old male mdx mice. J Muscle Res Cell Motil 22, 133139. Muntoni F, Brockington M, Torelli S & Brown SC (2004). Defective glycosylation in congenital muscular dystrophies. Curr Opin Neurol 17, 205209.

Nguyen HH, Jayasinha V, Xia B, Hoyte K & Martin PT (2002). Overexpression of the cytotoxic T cell GalNAc transferase in skeletal muscle inhibits muscular dystrophy in mdx mice. Proc Natl Acad Sci U S A 99, 56165621. Pellegrino MA, Canepari M, Rossi R, DAntona G, Reggiani C & Bottinelli R (2003). Orthologous myosin isoforms and scaling of shortening velocity with body size in mouse, rat, rabbit and human muscles. J Physiol 546, 677689. Petrof BJ, Stedman HH, Shrager JB, Eby J, Sweeney HL & Kelly AM (1993). Adaptations in myosin heavy chain expression and contractile function in dystrophic mouse diaphragm. Am J Physiol Cell Physiol 265, C834C841. Ramamurthy B, Hook P, Jones AD & Larsson L (2001). Changes in myosin structure and function in response to glycation. FASEB J 15, 24152422. Ranum LP & Day JW (2004). Myotonic dystrophy. RNA pathogenesis comes into focus. Am J Human Genet 74, 793804. Rappsilber J, Ryder U, Lamond AI & Mann M (2002). Largescale proteomic analysis of the human spliceosome. Genome Res 12, 12311245. Raymackers JM, Debaix H, Colson-Van Schoor M, De Backer F, Tajeddine N, Schwaller B, Gailly P & Gillis JM (2003). Consequence of parvalbumin deciency in the mdx mouse: histological, biochemical and mechanical phenotype of a new double mutant. Neuromuscul Disord 13, 376387. Richard I, Roudaut C, Marchand S, Baghdiguian S, Herasse M, Stockholm D, Ono Y, Suel L, Bourg N, Sorimachi H, Lefranc G, Fardeau M, Sebille A & Beckmann JS (2000). Loss of calpain 3 proteolytic activity leads to muscular dystrophy and to apoptosis-associated IB/nuclear factor B pathway perturbation in mice. J Cell Biol 151, 15831590. Sampaolesi M, Torrente Y, Innocenzi A, Tonlorenzi R, DAntona G, Pellegrino MA, Barresi R, Bresolin N, De Angelis MG, Campbell KP, Bottinelli R & Cossu G (2003). Cell therapy of -sarcoglycan null dystrophic mice through intra-arterial delivery of mesoangioblasts. Science 301, 487492. Sarkar PS, Appukuttan B, Han J, Ito Y, Ai C, Tsai W, Chai Y, Stout JT & Reddy S (2000). Heterozygous loss of Six5 in mice is sufcient to cause ocular cataracts. Nat Genet 25, 110114. Seznec H, Agbulut O, Sergeant N, Savouret C, Ghestem A, Tabti N, Willer JC, Ourth L, Duros C, Brisson E, Fouquet C, Butler-Browne G, Delacourte A, Junien C & Gourdon G (2001). Mice transgenic for the human myotonic dystrophy region with expanded CTG repeats display muscular and brain abnormalities. Hum Mol Genet 10, 27172726. Takagi A, Kojima S, Ida M & Araki M (1992). Increased leakage of calcium ion from the sarcoplasmic reticulum of the mdx mouse. J Neurol Sci 110, 160164. Torrente Y, Belicchi M, Sampaolesi M, Pisati F, Meregalli M, DAntona G, Tonlorenzi R, Porretti L, Gavina M, Mamchaoui K, Pellegrino MA, Furling D, Mouly V, Butler-Browne GS, Bottinelli R, Cossu G & Bresolin N (2004). Human circulating AC133+ stem cells restore dystrophin expression and ameliorate function in dystrophic skeletal muscle. J Clin Invest 114, 182195. Warnes DM, Tomas FM & Ballard FJ (1981). Increased rates of myobrillar protein breakdown in muscle-wasting diseases. Muscle Nerve 4, 6266.

Watchko JF, ODay TL & Hoffman EP (2002). Functional characteristics of dystrophic skeletal muscle: insights from animal models. J Appl Physiol 93, 407417. Webster C, Silberstein L, Hays AP & Blau HM (1988). Fast muscle bers are preferentially affected in Duchenne muscular dystrophy. Cell 52, 503513. Williams DA, Head SI, Lynch GS & Stephenson DG (1993). Contractile properties of skinned muscle bres from young and adult normal and dystrophic (mdx) mice. J Physiol 460, 5167. Yeung EW, Head SI & Allen DG (2003). Gadolinium reduces short-term stretch-induced muscle damage in isolated mdx mouse muscle bres. J Physiol 552, 449458.
Yeung EW, Whitehead NP, Suchyna TM, Gottlieb PA, Sachs F & Allen DG (2005). Effects of stretch-activated channel blockers on [Ca2+ ]i and muscle damage in the mdx mouse. J Physiol 562, 367380.
Acknowledgements This work has been supported by grants from the Italian Ministry of University and Research (PRIN 2005) and from Cariplo Foundation, Italy. The authors wish to thank Dr Raffaella Adami for help in single bre experiments.