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Human Molecular Genetics Advance Access originally published online on August 22, 2005
Human Molecular Genetics 2005 14(19):2801-2811; doi:10.1093/hmg/ddi313
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oupjournals.org

Mdm muscular dystrophy: interactions with calpain 3 and a novel functional role for titin's N2A domain

Kimberly A. Huebsch1, Elena Kudryashova2, Christine M. Wooley1, Roger B. Sher1, Kevin L. Seburn1, Melissa J. Spencer2 and Gregory A. Cox1,*

1The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609, USA and 2Department of Neurology and Pediatrics, Duchenne Muscular Dystrophy Research Center, University of California, Los Angeles, CA 90095, USA

* To whom correspondence should be addressed. Fax: +1 2072886073; Email:gac{at}jax.org

Received June 30, 2005; Accepted August 10, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 Statistical analysis
 Gait analysis
 REFERENCES
 
Human tibial muscular dystrophy and limb-girdle muscular dystrophy 2J are caused by mutations in the giant sarcomeric protein titin (TTN) adjacent to a binding site for the muscle-specific protease calpain 3 (CAPN3). Muscular dystrophy with myositis (mdm) is a recessive mouse mutation with severe and progressive muscular degeneration caused by a deletion in the N2A domain of titin (TTN-N2A{Delta}83), disrupting a putative binding site for CAPN3. To determine whether the muscular dystrophy in mutant mdm mice is caused by misregulation of CAPN3 activity, genetic crosses with CAPN3 overexpressing transgenic (C3Tg) and CAPN3 knockout (C3KO) mice were generated. Here, we report that overexpression of CAPN3 exacerbates the mdm disease, leading to a shorter life span and more severe muscular dystrophy. However, in a direct genetic test of CAPN3's role as a mediator of mdm pathology, C3KO;mdm double mutant mice showed no change in the progression or severity of disease indicating that aberrant CAPN3 activity is not a primary mechanism in this disease. To determine whether we could detect a functional deficit in titin in a non-disease state, we examined the treadmill locomotion of heterozygous +/mdm mice and detected a significant increase in stride time with a concomitant increase in stance time. Interestingly, these altered gait parameters were completely corrected by CAPN3 overexpression in transgenic C3Tg;+/mdm mice, supporting a CAPN3-dependent role for the N2A domain of TTN in the dynamics of muscle contraction.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 Statistical analysis
 Gait analysis
 REFERENCES
 
The mdm mutation occurred spontaneously on the C57BL/6J background at The Jackson Laboratory in 1982 (1Go) and has been identified as a complex rearrangement that involves the insertion of a 5' truncated LINE retrotransposon and a genomic deletion of 781 bp within the N2A region of the titin (Ttn) gene that results in an in-frame 83 amino acid deletion (TTN-N2A{Delta}83) (2Go). Homozygous mdm mutant mice have a severe and progressive degeneration of distal and proximal skeletal muscles that is evident by 2–3 weeks of age. Affected mice develop a rigid gait, a severe kyphosis due to axial skeletal muscle involvement and typically do not survive beyond 2 months of age when on an inbred background. Histological studies indicate that degeneration is specific to skeletal muscles with no obvious cardiomyopathy or impairment of the central or peripheral nervous system. Skeletal muscles of both fore and hind limbs have a severe dystrophic phenotype including the presence of central nuclei and variation in fiber size indicating multiple rounds of degeneration and regeneration.

The mouse Ttn gene spans 280 kb, includes over 360 exons (similar to the human TTN gene) (3Go), and encodes the largest known mammalian protein with a molecular weight exceeding 3 MDa. Extending from the Z-line to the M-line (a full half-sarcomere) (4Go), titin is thought to have two major functions in the sarcomere. First, titin directs sarcomere assembly by binding to and localizing a number of sarcomeric and cytoskeletal proteins, thus forming a scaffold to align thick and thin filaments in proper register and at the correct interfilament distance (5Go). A second titin function is to provide muscle with elasticity by folding and unfolding of the PEVK (rich in Pro, Glu, Val and Lys) region. The majority of titin (~90%) is composed of a repetitive structure containing numerous copies of immunoglobulin-like (Ig) and fibronectin-like (FN3) domains. The remaining 10% of titin consists of non-repetitive sequences including the PEVK domain and the titin kinase domain. Recent studies have identified a titin kinase domain-associated signaling complex which functions in response to mechanical stretch to regulate muscle gene transcription (6Go). In addition, titin contains binding sites for several different proteins including members of the muscle-specific RING-finger (MURF) family of signaling proteins, telethonin (Tcap), a structural protein linking titin to the Z-disc (7Go–10Go) and at least two binding sites for calpain 3, one located in the N2A region overlapping with the mdm mutation site (TTN-N2A{Delta}83), whereas the other is located near the C-terminus of titin (11Go–13Go). Mutations adjacent to the C-terminal calpain 3 binding site of titin cause tibial muscular dystrophy (TMD) and limb-girdle muscular dystrophy type 2J (LGMD2J) in humans (14Go–16Go). Although several functional domains of TTN have been inferred from homology to known proteins or by direct protein–protein interaction studies, the enormous size of the titin molecule has prevented a direct demonstration of function for most of these putative domains in an experimental system. The TTN-N2A{Delta}83 deletion in mdm mutant mice provides a novel model system to explore the function of this critical domain in normal and dystrophic skeletal muscles.

Calpain 3 (Capn3, previously p94) is a skeletal muscle-specific isoform of the calpain cysteine protease family (reviewed in 17Go) and was identified by positional cloning as the gene responsible for limb-girdle muscular dystrophy 2A (LGMD2A) (16Go). LGMD2A is an autosomal recessive human muscular dystrophy characterized by muscle wasting, cell death and decreased calpain 3 activity and expression (18Go). Calpain 3 undergoes extensive autolysis and is thought to be stabilized through its interaction with titin (11Go). Yeast two-hybrid analysis revealed that calpain 3 associates with the N2A region of titin, and immunofluorescent staining of normal human skeletal muscle confirmed the localization of calpain 3 in the N2A region of myofibrils (11Go,17Go,19Go,20Go). Transgenic mice overexpressing a catalytically inactive form of CAPN3 display a mild muscular dystrophy phenotype suggesting that a specific proteolytic function of CAPN3 is required for normal muscle maintenance (21Go). In addition, Capn3 null (C3KO) mice have defects in sarcomere structure and reductions in muscle mass and fiber size, which highlight an important role for calpain 3 in muscle differentiation and growth (22Go). Secondary reductions in CAPN3 expression have also been associated with a number of other human muscular dystrophies [LGMD2B (Miyoshi Myopathy), LGMD2J and TMD]. Muscle extracts from mutant mdm mice also show significant reductions in CAPN3 protein levels compared with that from wild-type controls (2Go,15Go,23Go). Owing to the loss of a putative CAPN3 binding site in the N2A domain of TTN and the reduced levels of CAPN3 observed in mdm skeletal muscles, we and others have hypothesized that calpain 3 is critical to the mdm disease mechanism (2Go,13Go). The unique IS2 domain of calpain 3 is required both for interaction with titin and for its autolytic activity, suggesting that its interaction with titin stabilizes the protease (11Go,24Go). Binding and stabilization of CAPN3 are disrupted by the mdm mutation in Ttn; therefore, the remaining CAPN3 may initiate the dystrophy via the unregulated proteolysis of its normal or novel substrates.

Consistent with this idea, we show that overexpression of CAPN3 in mdm mutant mice exacerbates the disease, resulting in a shorter life span and more severe muscular dystrophy. However, using loss-of-function crosses (C3KO;mdm), we demonstrate that CAPN3 is not required for the initiation or progression of muscular dystrophy in mdm mice suggesting that a critical TTN function is lacking in the mutant TTN-N2A{Delta}83 molecule. In an effort to uncover such a functional change in titin in a non-pathological state, we undertook a study of treadmill locomotion in heterozygous (+/mdm) mice and have identified significant differences in standard gait indices compared with their wild-type (+/+) littermates. Overexpressing CAPN3 corrected the alterations of gait in +/mdm mice indicating that these deficits are CAPN3 dependent. Our analysis of heterozygous +/mdm mutant mice provides the first evidence for a physiological effect of the TTN-N2A{Delta}83 deletion on a complex motor phenotype in the absence of any overt disease.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 Statistical analysis
 Gait analysis
 REFERENCES
 
The mdm mutation does not affect expression of TTN
The Ttnmdm mutation has a very subtle effect on the full-length titin mRNA, deleting only four exons (249 bp of coding sequence) from a full-length transcript of >100 kb (2Go). A minor second mRNA transcript, detected by RT–PCR, arising from the mdm mutation results in a truncation of the titin coding region by splicing in-frame with a LINE-1 retrotransposon (2Go). Although the Ttnmdm mRNA isoforms have been described, very little is known about the proteins they encode. If translated, the major mRNA isoform would result in a titin protein of nearly full-length missing only 83 amino acids in the N2A domain (TTN-N2A{Delta}83), whereas the minor mRNA isoform would be expected to encode a truncated TTN protein up to exon 110 (~1.1 MDa). To determine whether the mdm muscular dystrophy results from altered stability of the TTN-N2A{Delta}83 molecule or expression of a truncated ~1.1 MDa protein, titin protein expression was examined in mouse skeletal muscle by western blot analysis using an N-terminal (T12) titin antibody. As shown in Figure 1, the 83 amino acid difference between wild-type and mdm TTN-N2A{Delta}83 is not detectable in a protein of this size (>3 MDa). No detectable difference in the level of TTN expression is evident in mdm/mdm, +/mdm or wild-type skeletal muscle extracts (Fig. 1). Additionally, with no band evident at ~1.1 MDa, there is no indication that a truncated form of titin is expressed in +/mdm or mdm/mdm skeletal muscle (Fig. 1B). Because TTN is expressed at normal levels in homozygous mdm skeletal muscle and no abnormal truncated protein is expressed, it follows that the 83 amino acid deletion in the N2A domain of titin contains a critical functional domain or binding site.



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  		Figure 1. Analysis of titin expression in mdm skeletal muscle. (A) Silver staining of muscle extracts on 2% acrylamide gel strengthened with 0.5% agarose. (B) Indirect immunoblot of muscle extracts from C57BL/6J (B6) (WT), B6-+/mdm and B6-mdm/mdm mice probed with N-terminal titin-specific monoclonal antibody (T12) followed by HRP-conjugated secondary antibody. Titin T1 band represents intact TTN molecule; other muscle proteins are labeled, a degradation product of T1 (titin T2), nebulin (N) and myosin heavy chain (MHC).

 
Overexpression of calpain 3 exacerbates mdm muscular dystrophy
CAPN3 binds to the N2A region of titin in yeast two-hybrid experiments and this binding is abolished by the mdm mutation (13Go). If aberrant proteolysis of normal or novel substrates by CAPN3 underlies the muscular dystrophy in mdm mice, we would expect overexpression of CAPN3 to exacerbate the disease. To test this hypothesis, transgenic mice overexpressing CAPN3 (C3Tg) under the control of the human skeletal actin promoter (25Go) were crossed with +/mdm mice and C3Tg;mdm mice were generated. Western blotting of skeletal muscle extracts from C3Tg;mdm and C3Tg mice showed the robust overexpression of CAPN3 by the transgene in both C3Tg;mdm and C3Tg skeletal muscle (Fig. 2A). C3Tg;mdm mice displayed a similar, but much more accelerated disease when compared with non-transgenic mdm mice. C3Tg;mdm mice were smaller and less robust than non-Tg mdm littermates and exhibited a significantly shorter life span (34.8±5.2 versus 69.6±3.9 days, P<0.0001) (Fig. 2B). Most C3Tg;mdm mice failed to survive beyond 4 weeks (Fig. 2B), and body weight analysis at several time points revealed that C3Tg;mdm animals consistently weighed less than their non-transgenic mdm counterparts. This difference was significant at 3–4 weeks of age (4.48±1.05 versus 6.03±0.89 g, P=0.0036) (Fig. 2C).



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  		Figure 2. Analysis of C3Tg;mdm mutant mice. (A) Immunoblot of skeletal muscle extracts from B6 (WT), mdm, C3KO;mdm, C3Tg;mdm and C3Tg mice probed with calpain specific primary antibody (NCL-CALP-12A2). Calpain 3 is 94 kDa. The Ab cross-reacts with the ubiquitous calpains 1 and 2 at 70–80 kDa (lower band in all lanes). Equal amounts of total protein were loaded into lanes 1–3, 66% less was loaded in lanes 4 and 5. (B) Kaplan–Meier survival analysis of C3Tg;mdm mice and non-Tg;mdm littermates. Non-Tg;mdm survive significantly longer (mean 69.6±3.9 days) (n=19) compared with C3Tg expressing mdm mutant mice (mean 34.8±5.2 days) (n=12); P<0.0001. (C) Body weight analysis of mdm homozygotes and C3Tg;mdm mutant mice. Body weights were obtained once a week for each individual, and data for weeks 1 and 2 (weeks 1–2) and for weeks 3 and 4 (weeks 3–4) were pooled. C3Tg;mdm weeks 1–2 (n=5), mdm (n=8), C3Tg;mdm weeks 3–4 (n=8), mdm (n=18). At the 3–4 week time point, C3Tg;mdm mice weigh significantly less than their non transgenic mdm littermates; P=0.0036.

 
Hind limb muscles from all genotype combinations (C3Tg, C3Tg;+/mdm, C3Tg;mdm/mdm and non-Tg littermates) were examined. As previously reported (25Go), C3Tg muscle was unaffected except for the presence of infrequent central nuclei (Fig. 3A and D). Similarly, muscle from C3Tg;+/mdm and non-Tg;+/mdm displayed only rare fibers containing central nuclei (data not shown). Cross-sections through the midpoint of soleus muscles dissected from 17-day-old C3Tg;+/+, non-Tg;mdm/mdm and C3Tg;mdm/mdm mice reveal the relative decrease in skeletal muscle mass due to each genotype (Fig. 3A–C). The C3Tg muscle (Fig. 3A) is equivalent in size to wild-type mouse muscle of the same age (data not shown). Although mdm soleus muscle is considerably smaller than control or C3Tg muscle (Fig. 3B), it does not display overt signs of disease at this age (Fig. 3E). In contrast, soleus muscles of mdm animals overexpressing the CAPN3 transgene (C3Tg;mdm) were significantly smaller and have severely abnormal morphology compared with non-transgenic mdm animals (Fig. 3C and F compared with 3B and E). Close examination reveals the presence of numerous necrotic myofibers and a large percentage of muscle fibers containing central nuclei (Fig. 3F). Together, these results confirm that the dystrophy is exacerbated in C3Tg;mdm mice suggesting that overexpression of calpain 3 significantly accelerates the mdm disease process. Damage caused by overexpression of CAPN3 in a degenerating muscle may be independent of the mdm disease mechanism. Alternatively, these results are consistent with an active role for CAPN3 in causing the mdm dystrophy due to the loss of its binding site on titin and subsequent misregulation.



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  		Figure 3. C3Tg;mdm mutant mouse skeletal muscle morphology. Representative hematoxylin and eosin stained 5 µm cross-sections of soleus muscle from P17 C3Tg, B6-mdm and C3Tg;mdm mice. (A–C) Cross-section through center (largest diameter portion) of soleus muscle at 10x magnification. (D–F) 40x magnification of same muscles. (A and D) C3Tg, (B and E) mdm, (C and F) C3Tg;mdm. Scale bar in (C) represents 200 µm and in (F) 50 µm.

 
The mdm phenotype is unchanged in C3KO;mdm mutant mice
To directly test whether aberrant activation of CAPN3 is responsible for the mdm phenotype, calpain 3 null mdm (C3KO;mdm) double mutant mice were generated. Indirect immunoblotting of skeletal muscle confirmed the absence of CAPN3 protein in these double mutant mice (Fig. 2A). By comparison, muscle extracts from both C57BL/6J (B6) and B6-mdm mice show endogenous levels of CAPN3 similar to that previously described (Fig. 2) (2Go,23Go). The lower cross-reacting band shows ubiquitously expressed CAPN1 and 2, unaffected by the removal of CAPN3, visible in all lanes (Fig. 2A). The resulting C3KO;mdm mice were phenotypically identical to mdm mice (Fig. 4A). C3KO;mdm mice, like mdm homozygotes, develop a severe kyphosis, a rigid gait and have progressive muscle degeneration. The absence of CAPN3 in mdm mice caused no significant difference in body weight at any time point examined (Fig. 4B) or in their life span when compared with mdm mice wild-type at the Capn3 locus (Fig. 4C). Histological studies showed that skeletal muscles of mutant mdm (Capn3+/+) mice have a severe dystrophic phenotype including the presence of central nuclei and variation in fiber size at 8 weeks of age (26Go) (Fig. 5A). Although C3KO mice have been shown to replicate features of the human LGMD2A phenotype with small areas of focal necrosis surrounded by otherwise unaffected skeletal muscle (22Go), C3KO;mdm double mutant muscle pathology was indistinguishable from that found in homozygous mdm mice (Fig. 5C). To quantify this result, cross-sections of soleus muscle from each genotype were examined, and the number of muscle fibers containing central nuclei was counted. Homozygous mdm mice displayed central nuclei in >35% of fibers of the soleus muscle, whereas 3% of C3KO mouse soleus muscle fibers contained central nuclei (Fig. 5D). Soleus muscle of the C3KO;mdm double mutant mice contained central nuclei in greater than 30% of muscle fibers which was not significantly different from mdm mutant mice (P=0.093) (Fig. 5D).



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  		Figure 4. C3KO;mdm mice are phenotypically similar to mdm mice. (A) Photograph of 5-week-old C3+/+;mdm/mdm (mdm), C3–/–;mdm/mdm (C3KO;mdm) and C57BL/6J (WT) mice. mdm and WT were littermates. (B) Growth curve showing no significant difference in body weight between mdm and C3KO;mdm mice at time points from 1 week to 13 weeks and above (each point represents at least eight measurements); P≥0.131. (C) Kaplan–Meier survival analysis of C3KO;mdm mice and mdm littermates. There is no significant life span difference between C3KO;mdm mice (mean life span 79.9±31.2 days) (n=42) and mdm mice (mean life span 93±23.4 days) (n=10); P=0.15.

 


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  		Figure 5. C3KO;mdm mouse skeletal muscle morphology. C3KO;mdm mice display the same muscular dystrophy phenotype as mdm mice. (A–C) Representative hematoxylin and eosin stained 5 µm cross-sections of 8-week-old soleus muscle from (A) mdm, (B) C3KO and (C) C3KO;mdm. Arrows indicate a few of the central nuclei seen in (A) mdm and (C) C3KO;mdm muscle. Scale bar represents 50 µm. (D) Graph represents the average percent of central nuclei observed in soleus muscle cross-sections. Each bar, n=3.

 
Lack of skeletal muscle pathology in heterozygous +/mdm mice
The finding that aberrant CAPN3 activation is not the primary mechanism contributing to mdm muscular dystrophy leads us to explore alternative mechanisms for the TTN-N2A{Delta}83 mutation mediated disease pathogenesis. Previous studies have suggested the existence of a signaling complex that couples the titin-N2A domain with MARPs [muscle ankyrin repeat proteins, Ankrd1 (CARP), Ankrd2 and Ankrd23 (DARP)] that are known to regulate muscle gene expression in response to mechanical stress (13Go,27Go). Upregulation of MARPs in muscle disease and after injury or exercise in normal muscle suggests a role in muscle stress response pathways leading to muscle hypertrophy (27Go,28Go). Homozygous mdm mouse skeletal muscle fibers remain small in diameter at postnatal day 14 (P14), at which time normal mouse skeletal muscle has undergone developmental hypertrophy, suggesting that hypertrophy may be impaired in mdm skeletal muscle (see Fig. 3E for P17 mdm muscle and Fig. 5A for 8 week mdm muscle).

We hypothesized that heterozygous +/mdm mice, expressing a mix of wild-type TTN and mutant TTN-N2A{Delta}83, might reveal subtle deficits that would provide insight into the pathogenesis observed in mdm homozygotes. Therefore we tested whether the muscles of mdm heterozygotes differed from wild-type in their response to synergist overload. To do this, the medial gastrocnemius (MG) was overloaded by unilaterally denervating the other ankle extensors [lateral gastrocnemius (LG), plantaris and soleus] of the left hind limb. After 4 weeks, the MG muscles were removed from both the overloaded and control legs and examined. Across all animals (+/+ and +/mdm), the overloaded MG was 10–50% larger than the MG from the contralateral control MG [69.3±17.8 mg (overloaded MG) versus 50.7±7.0 mg (control MG), P=0.0321] and denervated muscles showed severe atrophy (data not shown). The increased weight of the overloaded MG muscle relative to its contralateral control was similar for both +/+and +/mdm (28.0±18.7% increase for +/+ versus 20.3±12.3% increase for +/mdm, P=0.20) and histological examination of cross-sections of overloaded MG from two wild-type and two +/mdm mice showed no evidence of muscle damage (data not shown). These findings confirm that the presence of 50% mutant TTN-N2A{Delta}83 does not interfere with signaling necessary for promoting the typical activity-induced adaptations, nor do mdm heterozygous animals show any significant indications of muscle damage in normal or overloaded states.

A functional role for TTN-N2A during locomotion
As a molecular spring, the I-band region of titin is thought to provide the passive tension during sarcomere stretch and subsequently restore the muscle to resting length after contraction (29Go). Additionally, titin has been proposed to have a functional role in the dynamics of muscle contraction (30Go). Titin has been shown to bind Ca2+ in its PEVK domain and consequently change its conformation, suggesting that titin's elasticity changes in response to Ca2+ on a contraction-by-contraction basis (31Go) and a portion of the PEVK domain (N-terminal 56 amino acids) is deleted by the TTN-N2A{Delta}83 mutation. During walking, there is continuous contraction/relaxation cycling of locomotory muscles that involves the coordinated interaction of passive and active muscle properties (32Go). Analysis of treadmill locomotion in the mouse has proven to be a sensitive assay of neuromuscular function in the absence of significant pathology (33Go), and we hypothesized that if the mdm mutation affected titin's ability to function as molecular spring, mdm heterozygotes might show altered gait dynamics. To test this hypothesis, data were collected from 8-week-old +/mdm and +/+ littermates and analyzed for differences in standard gait indices including stride, stance and swing time. Heterozygous B6-+/mdm mice, which have no apparent muscle pathology, were found to have altered gait when compared with B6-+/+ controls (Fig. 6). Statistical analysis (ANOVA) revealed a main effect of genotype averaged across front and rear paws. In all cases, measures showed the same tendency for both paws, but the differences were not always significant for front and rear paws separately. When compared with wild-type, the heterozygous +/mdm mice had a longer stride time (Fig. 6A) (P=0.03) and an accompanying increase in the stance time (Fig. 6B) (P<0.001). Together, these results demonstrate that +/mdm mice use a subtly modified strategy for treadmill walking when compared with their wild-type counterparts at the same speed, suggesting a previously unrecognized functional role for TTN-N2A during the normal gait cycle of the mouse. Our finding that overexpression of CAPN3 exacerbates mdm muscular dystrophy led us to ask whether CAPN3 overexpression alters the +/mdm phenotype. Gait data were collected from CAPN3 overexpressing heterozygous mdm mice (C3Tg;+/mdm) and their non-transgenic littermates at 8 weeks of age. Transgenic C3Tg;+/mdm mice had a shorter stride time (Fig. 6A) (P=0.01) and an accompanying decrease in the stance time (Fig. 6B) (P<0.01) when compared with heterozygous +/mdm mice. C3Tg; +/mdm and +/+ mice were not statistically different from each other (Fig. 6) (P≥0.6) indicating that gait deficits found in heterozygous mdm (+/mdm) mice can be restored by overexpression of CAPN3.



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  		Figure 6. Gait analysis of +/mdm. Box and Whisker plot comparisons of (A) mean stride and (B) mean stance times of front and rear paws (averaged) for C57BL/6J (+/+), +/mdm and C3Tg;+/mdm mice. Both stride and stance times are significantly different in +/mdm when compared with +/+ mice (P=0.03 for stride and P<0.001 for stance), as well as in +/mdm when compared with C3Tg;+/mdm (P=0.01 for stride and P<0.01 for stance). Stride and stance times are not significantly different between +/+ and C3Tg;+/mdm mice (P=0.6 for stride and P=0.9 for stance). Error bars are ±1 standard deviation and boxes are ±1 standard error. B6-+/+ (n=10), +/mdm (n=10), C3Tg;+/mdm (n=5).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 Statistical analysis
 Gait analysis
 REFERENCES
 
Previous studies showing that CAPN3 levels are reduced in mdm skeletal muscles and that CAPN3 binding to titin's N2A domain is abolished by the TTN-N2A{Delta}83 mutation led to the hypothesis that CAPN3 was critical to the mdm disease mechanism (2Go,11Go,13Go,23Go). In this study, we set out to determine whether aberrant activation of CAPN3 underlies the muscular dystrophy in mdm mice. As predicted, overexpression of CAPN3 in mdm mutant mice exacerbates the disease. However, in a direct test of our hypothesis using loss-of-function crosses (C3KO;mdm), we demonstrate that CAPN3 is not required for the initiation or progression of muscular dystrophy in mdm mice. This finding suggests that additional TTN-N2A binding partners, besides CAPN3, may exist or that a critical structural, dynamic or signaling function is lacking in the mutant TTN-N2A{Delta}83 molecule. To investigate a potential functional change in titin, we analyzed heterozygous (+/mdm) mice in routine activity (locomotion) and for their response to muscle stress (muscle overload) and have identified significant differences in standard gait indices including stride, stance and swing time compared with their wild-type (+/+) littermates. Further, wild-type gait parameters were restored to heterozygous (+/mdm) mice by overexpression of CAPN3. This finding supports a CAPN3-dependent role for titin in the dynamics of muscle contraction. Although our analysis of mutant mdm mice has eliminated a direct role for CAPN3 in the underlying dystrophy, it provides the first evidence for a physiological effect of the TTN-N2A{Delta}83 deletion on a complex motor phenotype in the absence of any overt disease phenotype.

Mutations in titin cause human TMD, LGMD2J and hereditary myopathy with early respiratory failure (14Go,15Go,34Go). As part of the sarcomeric contractile complex, titin does not fit into the well-characterized models of muscular dystrophy with defects in proteins of the dystrophin-associated glycoprotein complex (DGC) resulting in sarcolemmal instability (reviewed in 35Go). Recently discovered mutations in telethonin, myotilin (Ttid) and calpain 3 as well as in titin have suggested the existence of a titin-associated family of muscular dystrophies (14Go,36Go–38Go) unrelated to DGC deficits. Examination of mdm skeletal muscle showed no signs of sarcolemmal damage by uptake of Evans blue dye (data not shown); therefore, the mechanism(s) of disease, in mdm as with others in this second group, remains largely unknown.

The deletion of a putative binding site for CAPN3 in mdm mice suggested a disease mechanism linking the N2A domain of TTN to this muscle-specific protease. Although the majority of its in vivo substrates are unknown, CAPN3 can cleave many different substrates in vitro (39Go). Unable to bind to the N2A domain of titin, CAPN3 has the potential for aberrant activity causing damage to muscle cells and accounting for the disease pathology observed in mdm muscle. If aberrant CAPN3 activity was responsible for the mdm disease, we expected that increased CAPN3 expression should alter the onset or severity of disease. By crossing mdm mice with a transgenic line overexpressing full-length CAPN3 (~60-fold) (25Go), we found that the disease was indeed exacerbated. However, further experiments (C3KO;mdm crosses) are not consistent with the aberrant activity hypothesis; therefore, an alternate explanation for the exacerbated disease in C3Tg;mdm mice is required. The TTN-N2A/CAPN3 interaction in C3Tg mice with wild-type TTN may protect muscles from damage caused by CAPN3 overexpression, resulting in an extremely mild phenotype with infrequent central nuclei. In the absence of the protective TTN-N2A/CAPN3 interaction, the C3Tg;mdm mice exhibit a muscle disease with an earlier onset and more rapid progression. A second possibility is that the phenotype is simply an additive effect of two independent disease mechanisms, CAPN3-dependent muscle damage and mdm muscular dystrophy together resulting in the accelerated C3Tg;mdm disease.

By genetic crosses, we have removed CAPN3 from mdm mutant mice and have found their disease unchanged, indicating that aberrant activity of CAPN3, if it occurs, is not an underlying cause for the muscle degeneration. Although our results suggest that the CAPN3 protease is not actively causing the disease, the TTN-N2A{Delta}83 mutation may have eliminated critical site-specific functions in addition to those that require CAPN3 activity. Potential CAPN3 substrates in close proximity to the TTN-N2A domain include the MARP family of sarcomere-associated/nuclear proteins whose cytokine-like expression is induced following injury, stretch, denervation or during recovery after metabolic challenge (40Go–43Go). Localizing to both the I-band of the sarcomere and the nucleus, MARPs [Ankrd1 (CARP), Ankrd2 and Ankrd23 (DARP)] are thought to provide a link between mechanical stretch and gene expression (27Go). The MARP family members bind a unique sequence in titin-N2A between Ig80 and Ig81 domains, and although this binding is not expected to be eliminated by the mdm mutation (13Go,27Go), our data would be consistent with an alteration in the activity of this putative titin-N2A dependent stretch sensor complex. The N2A domain of titin is an ideal location for this proposed stretch sensor complex, as it lies between two elastic regions, the Ig repeat domain and the PEVK domain. The small myofiber diameters observed at postnatal day 14 mdm skeletal muscles are consistent with an impaired muscle hypertrophy response. However, our finding that +/mdm skeletal muscle is able to hypertrophy to the same extent as wild-type (+/+) in overload experiments indicates that either the titin-N2A dependent stretch sensor complex is not affected by the mdm mutation or half the number of signaling complexes are sufficient for normal muscle hypertrophy responses.

Analysis of gait parameters revealed a difference in locomotion between wild-type (+/+) and heterozygous mdm (+/mdm) animals. Heterozygous mdm mice exhibited a longer stride time with the difference entirely contributed by the stance portion of the stride (Fig. 6). Previous studies have drawn a cross-species correlation between stride frequency and titin isoform expression (30Go). Differentially expressed titin isoforms vary in the lengths of the elastic elements, Ig domains and/or PEVK domains, shorter isoforms being stiffer (greater passive tension) than longer isoforms (44Go,45Go). Animals with a greater stride frequency express shorter titin isoforms (30Go) indicating that the properties of titin contribute to differences in gait dynamics. The longer stride time of +/mdm mice may indicate a reduction in passive tension properties caused by the mdm mutation in TTN. Previously, mechanical properties of mdm muscle fibers were reported as indistinguishable from wild-type (13Go). Our data indicate that the analysis of mechanical properties in vivo and in muscle with no pathology (+/mdm) may yield different results than in vitro experiments with isolated muscle fibers. Interestingly, putative calcium-binding sites have been identified in the PEVK region of TTN (46Go), 56 amino acids of which are deleted as a result of the mdm mutation. It has further been shown that calcium binding changes the secondary structure of this portion of the titin molecule (31Go) potentially causing changes in titin elasticity during contraction/relaxation cycling in response to calcium flux.

Our finding that mdm heterozygotes have an altered gait supports the notion of a dynamic functional role for titin in modulating muscle contraction during normal treadmill walking. In addition, we find overexpression of CAPN3 in heterozygous (+/mdm) mice restores gait parameters to wild-type levels. Although the precise functional role of titin and the TTN-N2A{Delta}83 mutation during gait and its relationship with other contractile proteins and processes remain unknown, it appears that this role is CAPN3 dependent. Our gait data suggest that CAPN3 is rate-limiting in its interactions with TTN-N2A. Overexpression of CAPN3 may ensure the occupation of all remaining TTN-N2A sites and the proper functioning of the titin-N2A dependent stretch sensor complex in heterozygous (+/mdm) mouse muscle. Alternatively, overexpression of CAPN3 may compensate for gait deficiencies of +/mdm mice through a TTN-N2A independent pathway. The fact that the mdm mutation affects only a portion of the PEVK region and that the effect on gait is observed in the presence of only 50% mutant titin implies a highly sensitive role in regulating normal muscle contraction. It also suggests that the functional effect of the mdm mutation in the homozygotes, which have no normal titin, would be profound and could initiate the observed pathological processes. Finally, it remains possible that the mdm muscular dystrophy involves another, yet unknown, protein or pathway whose function is disrupted by the TTN-N2A{Delta}83 mutation. The results of this study clearly demonstrate that neither the reduction of CAPN3 nor its aberrant activity is responsible for the mdm muscle degeneration.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
Statistical analysis
 Gait analysis
 REFERENCES
 
Mice
Incipient congenic C57BL/6J-Ttnmdm/Cx mice (B6.B6C3Fe-mdm) were generated by backcrossing the B6C3Fe-a/a-Ttnmdm/J mutation to C57BL/6J (B6) mice for eight generations. B6;129-Capn3–/–tm1Spc (C3KO) and STOCK-Tg(ACTA1-Capn3)7.2Spc (C3Tg) mice obtained from the UCLA Duchenne Muscular Dystrophy Research Center were produced as previously described (22Go,25Go). C3KO mice were backcrossed to B6 for three generations prior to intercrossing to generate B6;129-Capn3–/– (N3F1) mice for analysis, and wt C3Tg line 7.2 mice were derived from C57BL/6JxBalb/c parents that were backcrossed to C57/BL10 (B10) for at least three generations before importation to The Jackson Laboratory where they were maintained by backcrossing to B6 for at least three generations. All comparisons were made with age-matched littermates within the same inbred or hybrid background strains. Mice were maintained in humidity- and temperature-controlled rooms with a 12:12 dark:light cycle. They were given free access to HCl-acidified water and an NIH-mouse/rat diet with 6% fat content (5K54, PMI Feeds, Inc., St Louis, MO, USA). All procedures performed on the animals were reviewed and approved by our institutional animal care and use committee.

Antibodies
Antibodies used for western blotting were calpain 3 specific monoclonal NCL-CALP-12A2 (Novocastra Laboratories, Newcastle upon Tyne, UK), N-terminal specific titin monoclonal antibody T12, generous gift of Dr Peter van der Ven, University of Potsdam, Germany, and HRP-conjugated anti-mouse IgG (Chemicon, Temecula, CA, USA) or (Jackson ImmunoResearch, West Grove, PA, USA).

Muscle protein lysate preparation and western blotting
For titin electrophoresis, samples were prepared according to (47Go). Briefly, tibialis anterior muscles from wild-type, heterozygous and mdm-mice were homogenized in 40 volumes of sample buffer containing 8 M urea, 2 M thiourea, 3% sodium dodecyl sulfate (SDS), 75 mM dithiothreitol (DTT), 50 mM Tris, pH 6.8 heated at 60°C for 5 min in a water bath, vortexed and centrifuged for 5 min at 13 000g. For calpain 3 electrophoresis, hind limb skeletal muscle was snap-frozen and ground into a fine powder in liquid nitrogen using a mortar and pestle. Muscle powder was solubilized with calpain 3 buffer containing 80 mM Tris, pH 6.8, 10% glycerol, 2% SDS, 100 mM DTT and protease inhibitor cocktail (Sigma-Aldrich, St Louis, MO, USA) at a concentration of 1 ml per 250 mg muscle. Suspensions were vortexed, boiled 1 min and centrifuged for 5 min at 13 000g.

For titin electrophoresis, 10 µg of total protein in a volume of 3–5 µl was loaded per well in 2% polyacrylamide gel strengthened with agarose (SeaKem LE) in Fairbank's buffer system as described (48Go) with slight modifications. To prevent the gel from sliding off the plate cell, 0.5 cm of plug gel (15%) was poured, 10% glycerol (final concentration) was added to the resolving gel and 10 mM of 2-mercaptoethanol was added to the electrophoresis buffer. Gels were stained with Silver Stain Plus kit (Bio-Rad, Laboratories, Hercules, CA, USA) or transferred for 2 h at 100 V to nitrocellulose in transfer buffer containing 192 mM glycine, 25 mM Tris, pH 8.3, 20% methanol and immunoblotted with N-terminal specific titin antibody, T12, and appropriate secondary antibody for analysis by enhanced chemiluminescence (ECL-Plus) as recommended by the manufacturer (Amersham Pharmacia, Piscataway, NJ, USA). For calpain 3 electrophoresis, 150 µg (B6, mdm and C3KO;mdm) or 50 µg (C3Tg;mdm and C3Tg) of protein was combined with an equal volume of 2x Laemmli sample buffer [160 mM Tris–HCl (pH 6.8), 4% SDS, 30% glycerol, 200 mM DTT and 0.02% bromophenol blue] and separated by SDS–PAGE on a 4–15% polyacrylamide gel (Bio-Rad Laboratories), transferred for 30 min at 100 V to Immun-BlotTM PVDF membranes (Bio-Rad Laboratories) and immunoblotted with calpain 3 specific mouse monoclonal antibody, 12A2 followed by appropriate secondary antibody. Bands were visualized with western blotting luminol reagent (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Evans blue injection
Evans blue dye (EBD) was dissolved in phosphate-buffered saline (PBS) (0.15 M NaCl, 10 mM phosphate buffer, pH 7.4) at a concentration of 10 mg/ml and was injected into the peritoneal cavity of mice (0.1 ml per 10 g body weight) as described (49Go). Twelve to fifteen hours after injection, mice were sacrificed. Hind limb skeletal muscle was cyropreserved, sectioned and examined for uptake of dye. When examined by fluorescence microscopy, EBD staining is a bright red emission.

Muscle histology
Skeletal muscles were dissected from affected mdm and unaffected littermate control mice after they were deeply anesthetized with avertin (1.25% tribromoethanol/amyl alcohol) by intraperitoneal injection (0.02 ml/g) and transcardially perfused with PBS, followed by Bouin's fixative. Light microscopic histological examination of muscle sections was carried out on a Nikon E600 upright microscope and SPOT RT color digital camera (Diagnostic Instruments, Inc.), following hematoxylin and eosin staining of Bouin's fixed paraffin-embedded 5 µm sections prepared according to standard histological procedures.

Generation of C3–/–;mdm mice
B6;129-Capn3–/– mice (N3) were bred with B6-+/mdm heterozygotes to generate B6;129-Capn3+/–; +/mdm offspring that were identified by PCR genotyping of the mdm mutation as previously described (2Go). B6;129-Capn3+/–,+/mdm F1 mice were intercrossed; however, as both the calpain 3 and titin genes are located on chromosome 2 separated by 44 Mb, a recombination event was required to obtain Capn3–/–; +/mdm mice. Intercrosses resulted in the expected 25% double homozygous (B6;129-Capn3–/–, mdm/mdm) offspring that were identified by PCR genotyping for the mdm mutation and the absence of the wild-type Ttn and Capn3 alleles as previously described (2Go,22Go).

Generation of C3Tg;mdm mice
C3Tg mice were bred with B6-+/mdm heterozygotes. C3Tg;+/mdm offspring were identified by genotyping for the mdm mutation and the C3Tg as previously described (2Go,25Go). C3Tg;+/mdm mice were backcrossed with B6-+/mdm mice to produce C3Tg;mdm/mdm offspring, which were identified by PCR genotyping for the C3Tg as previously described (25Go).


   Statistical analysis
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 Statistical analysis
Gait analysis
 REFERENCES
 
Life span and body weight data are expressed as mean±one standard deviation. Statistical comparisons used Student's t-test for unequal samples (two-tailed) unless otherwise noted. A probability value of ≤0.05 was used as a limit for declaring statistical significance.


   Gait analysis
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 Statistical analysis
 Gait analysis
REFERENCES
 
A device for recording and analyzing standard gait parameters in mice was validated in a previous study (33Go). Briefly, animals are placed on a clear treadmill with a digital video camera fixed below the walking surface. The treadmill is started and mice are recorded on video for 10–15 s while walking at 23 cm/s. Analysis software (Treadscan, Clever Sys, Inc.) is then used to determine when individual paws are in contact with the treadmill and allows us to obtain standard gait parameters. The software derives three gait measures directly for each foot: (1) stride time—time between two consecutive foot contacts with the treadmill; (2) stance time—the portion of the stride time that the foot is in contact with the treadmill surface and (3) swing time—the portion of the stride time that the foot is in the air. Representative values for each parameter were calculated using the average of consecutive strides (10Go–15Go) for each of the four paws. For statistical analysis, the right and left paws were averaged to give values for the front and rear paws for each animal. Data were collected from C57BL/6J+/mdm, C3Tg;+/mdm and +/+ littermates (+/mdm, n=10; C3Tg;+/mdm, n=5 and +/+, n=10) at 8 weeks of age. A two-way ANOVA (genotypexpaw) was performed for each measure. Tukey's honest significant difference test was used to compare individual mean values. A probability value of ≤0.05 was used as a limit for declaring statistical significance.

Surgical overload
Under avertin anesthesia (0.25 mg/g), a 1 cm skin incision was made along the midline of the dorsal aspect of the hind limb at the level of the popliteal fossa. The skin was opened and a similar incision was made through the overlying hamstrings musculature to expose the tibial nerve at its entry point into the triceps surae muscles. Individual muscle nerves innervating the LG, soleus and plantaris muscles were exposed, freed and then sectioned and ligated with 8.0 silk. Ligated nerve ends were deflected away from their target muscles to further prevent reinnervation. This procedure left the MG as the only intact ankle extensor.


   ACKNOWLEDGEMENTS
 
We are grateful to Drs Robert Burgess and Wayne Frankel (TJL) for critical review of this manuscript and Mr David Schroeder for excellent technical support. Antibody to titin N-terminal domain was generously provided by Dr Peter van der Ven (University of Potsdam, Germany). This work was supported by NIH grant R01AR049043 to G.A.C., K.A.H. was supported by a fellowship from NIH T32 HD07065-25 and scientific services at The Jackson Laboratory are supported in part by NCI-CA34196 (Cancer Center Support Grant).

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


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 Statistical analysis
 Gait analysis
 REFERENCES
 

  1. Lane, P.W. (1985) Muscular dystrophy with myositis (mdm). Mouse News Lett., 73, 18.

  2. Garvey, S.M., Rajan, C., Lerner, A.P., Frankel, W.N. and Cox, G.A. (2002) The muscular dystrophy with myositis (mdm) mouse mutation disrupts a skeletal muscle-specific domain of titin. Genomics, 79, 146–149.[CrossRef][ISI][Medline]

  3. Bang, M.L., Centner, T., Fornoff, F., Geach, A.J., Gotthardt, M., McNabb, M., Witt, C.C., Labeit, D., Gregorio, C.C., Granzier, H. et al. (2001) The complete gene sequence of titin, expression of an unusual approximately 700-kDa titin isoform, and its interaction with obscurin identify a novel Z-line to I-band linking system. Circ. Res., 89, 1065–1072.[Abstract/Free Full Text]

  4. Furst, D.O., Osborn, M., Nave, R. and Weber, K. (1988) The organization of titin filaments in the half-sarcomere revealed by monoclonal antibodies in immunoelectron microscopy: a map of ten non-repetitive epitopes starting at the Z-line extends close to the M-line. J. Cell Biol., 106, 1563–1572.[Abstract/Free Full Text]

  5. Trinick, J. (1996) Titin as a scaffold and spring. Curr. Biol., 6, 258–260.[CrossRef][ISI][Medline]

  6. Lange, S., Xiang, F., Yakovenko, A., Vihola, A., Hackman, P., Rostkova, E., Kristensen, J., Brandmeier, B., Franzen, G., Hedberg, B. et al. (2005) The kinase domain of titin controls muscle gene expression and protein turnover. Science, 308, 1599–1603.[Abstract/Free Full Text]

  7. Centner, T., Yano, J., Kimura, E., McElhinny, A.S., Pelin, K., Witt, C.C., Bang, M.L., Trombitas, K., Granzier, H., Gregorio, C.C. et al. (2001) Identification of muscle specific ring finger proteins as potential regulators of the titin kinase domain. J. Mol. Biol., 306, 717–726.[CrossRef][ISI][Medline]

  8. McElhinny, A.S., Kakinuma, K., Sorimachi, H., Labeit, S. and Gregorio, C.C. (2002) Muscle-specific RING finger-1 interacts with titin to regulate sarcomeric M-line and thick filament structure and may have nuclear functions via its interaction with glucocorticoid modulatory element binding protein-1. J. Cell Biol., 157, 125–136.[Abstract/Free Full Text]

  9. McElhinny, A.S., Perry, C.N., Witt, C.C., Labeit, S. and Gregorio, C.C. (2004) Muscle-specific RING finger-2 (MURF-2) is important for microtubule, intermediate filament and sarcomeric M-line maintenance in striated muscle development. J. Cell Sci., 117, 3175–3188.[Abstract/Free Full Text]

  10. Furukawa, T., Ono, Y., Tsuchiya, H., Katayama, Y., Bang, M.L., Labeit, D., Labeit, S., Inagaki, N. and Gregorio, C.C. (2001) Specific interaction of the potassium channel beta-subunit minK with the sarcomeric protein T-cap suggests a T-tubule-myofibril linking system. J. Mol. Biol., 313, 775–784.[CrossRef][ISI][Medline]

  11. Sorimachi, H., Kinbara, K., Kimura, S., Takahashi, M., Ishiura, S., Sasagawa, N., Sorimachi, N., Shimada, H., Tagawa, K., Maruyama, K. et al. (1995) Muscle-specific calpain, p94, responsible for limb girdle muscular dystrophy type 2A, associates with connectin through IS2, a p94-specific sequence. J. Biol. Chem., 270, 31158–31162.[Abstract/Free Full Text]

  12. Kinbara, K., Sorimachi, H., Ishiura, S. and Suzuki, K. (1997) Muscle-specific calpain, p94, interacts with the extreme C-terminal region of connectin, a unique region flanked by two immunoglobulin C2 motifs. Arch. Biochem. Biophys., 342, 99–107.[CrossRef][ISI][Medline]

  13. Witt, C.C., Ono, Y., Puschmann, E., McNabb, M., Wu, Y., Gotthardt, M., Witt, S.H., Haak, M., Labeit, D., Gregorio, C.C. et al. (2004) Induction and myofibrillar targeting of CARP, and suppression of the Nkx2.5 pathway in the MDM mouse with impaired titin-based signaling. J. Mol. Biol., 336, 145–154.[CrossRef][ISI][Medline]

  14. Hackman, P., Vihola, A., Haravuori, H., Marchand, S., Sarparanta, J., De Seze, J., Labeit, S., Witt, C., Peltonen, L., Richard, I. et al. (2002) Tibial muscular dystrophy is a titinopathy caused by mutations in TTN, the gene encoding the giant skeletal-muscle protein titin. Am. J. Hum. Genet., 71, 492–500.[CrossRef][ISI][Medline]

  15. Bushby, K.M. and Beckmann, J.S. (2003) The 105th ENMC sponsored workshop: pathogenesis in the non-sarcoglycan limb-girdle muscular dystrophies, Naarden, April 12–14, 2002. Neuromuscul. Disord., 13, 80–90.[CrossRef][Medline]

  16. Richard, I., Broux, O., Allamand, V., Fougerousse, F., Chiannilkulchai, N., Bourg, N., Brenguier, L., Devaud, C., Pasturaud, P., Roudaut, C. et al. (1995) Mutations in the proteolytic enzyme calpain 3 cause limb-girdle muscular dystrophy type 2A. Cell, 81, 27–40.[CrossRef][ISI][Medline]

  17. Sorimachi, H., Ono, Y. and Suzuki, K. (2000) Skeletal muscle-specific calpain, p94, and connectin/titin: their physiological functions and relationship to limb-girdle muscular dystrophy type 2A. Adv. Exp. Med. Biol., 481, 383–395; discussion 387–395.

  18. Nishino, I. and Ozawa, E. (2002) Muscular dystrophies. Curr. Opin. Neurol., 15, 539–544.[CrossRef][ISI][Medline]

  19. Herasse, M., Ono, Y., Fougerousse, F., Kimura, E., Stockholm, D., Beley, C., Montarras, D., Pinset, C., Sorimachi, H., Suzuki, K. et al. (1999) Expression and functional characteristics of calpain 3 isoforms generated through tissue-specific transcriptional and posttranscriptional events. Mol. Cell. Biol., 19, 4047–4055.[Abstract/Free Full Text]

  20. Keira, Y., Noguchi, S., Minami, N., Hayashi, Y.K. and Nishino, I. (2003) Localization of calpain 3 in human skeletal muscle and its alteration in limb-girdle muscular dystrophy 2A muscle. J. Biochem. (Tokyo), 133, 659–664.[Abstract/Free Full Text]

  21. Tagawa, K., Taya, C., Hayashi, Y., Nakagawa, M., Ono, Y., Fukuda, R., Karasuyama, H., Toyama-Sorimachi, N., Katsui, Y., Hata, S. et al. (2000) Myopathy phenotype of transgenic mice expressing active site-mutated inactive p94 skeletal muscle-specific calpain, the gene product responsible for limb girdle muscular dystrophy type 2A. Hum. Mol. Genet., 9, 1393–1402.[Abstract/Free Full Text]

  22. Kramerova, I., Kudryashova, E., Tidball, J.G. and Spencer, M.J. (2004) Null mutation of calpain 3 (p94) in mice causes abnormal sarcomere formation in vivo and in vitro. Hum. Mol. Genet., 13, 1373–1388.

  23. Haravuori, H., Vihola, A., Straub, V., Auranen, M., Richard, I., Marchand, S., Voit, T., Labeit, S., Somer, H., Peltonen, L. et al. (2001) Secondary calpain3 deficiency in 2q-linked muscular dystrophy: titin is the candidate gene. Neurology, 56, 869–877.[Abstract/Free Full Text]

  24. Sorimachi, H., Toyama-Sorimachi, N., Saido, T.C., Kawasaki, H., Sugita, H., Miyasaka, M., Arahata, K., Ishiura, S. and Suzuki, K. (1993) Muscle-specific calpain, p94, is degraded by autolysis immediately after translation, resulting in disappearance from muscle. J. Biol. Chem., 268, 10593–10605.[Abstract/Free Full Text]

  25. Spencer, M.J., Guyon, J.R., Sorimachi, H., Potts, A., Richard, I., Herasse, M., Chamberlain, J., Dalkilic, I., Kunkel, L.M. and Beckmann, J.S. (2002) Stable expression of calpain 3 from a muscle transgene in vivo: immature muscle in transgenic mice suggests a role for calpain 3 in muscle maturation. Proc. Natl Acad. Sci. USA, 99, 8874–8879.[Abstract/Free Full Text]

  26. Heimann, P., Menke, A., Rothkegel, B. and Jockusch, H. (1996) Overshooting production of satellite cells in murine skeletal muscle affected by the mutation ‘muscular dystrophy with myositis’ (mdm, Chr 2). Cell Tissue Res., 283, 435–441.[CrossRef][ISI][Medline]

  27. Miller, M.K., Bang, M.L., Witt, C.C., Labeit, D., Trombitas, C., Watanabe, K., Granzier, H., McElhinny, A.S., Gregorio, C.C. and Labeit, S. (2003) The muscle ankyrin repeat proteins: CARP, ankrd2/Arpp and DARP as a family of titin filament-based stress response molecules. J. Mol. Biol., 333, 951–964.[CrossRef][ISI][Medline]

  28. Kojic, S., Medeot, E., Guccione, E., Krmac, H., Zara, I., Martinelli, V., Valle, G. and Faulkner, G. (2004) The Ankrd2 protein, a link between the sarcomere and the nucleus in skeletal muscle. J. Mol. Biol., 339, 313–325.[CrossRef][ISI][Medline]

  29. Helmes, M., Trombitas, K. and Granzier, H. (1996) Titin develops restoring force in rat cardiac myocytes. Circ. Res., 79, 619–626.[Abstract/Free Full Text]

  30. Lindstedt, S.L., Reich, T.E., Keim, P. and LaStayo, P.C. (2002) Do muscles function as adaptable locomotor springs? J. Exp. Biol., 205, 2211–2216.[Abstract/Free Full Text]

  31. Tatsumi, R., Maeda, K., Hattori, A. and Takahashi, K. (2001) Calcium binding to an elastic portion of connectin/titin filaments. J. Muscle Res. Cell Motil., 22, 149–162.[CrossRef][ISI][Medline]

  32. Ettema, G.J. (1996) Elastic and length-force characteristics of the gastrocnemius of the hopping mouse (Notomys alexis) and the rat (Rattus norvegicus). J. Exp. Biol., 199, 1277–1285.[Abstract]

  33. Wooley, C.M., Sher, R.B., Kale, A., Frankel, W.N., Cox, G.A. and Seburn, K.L. (2005) Gait analysis detects early changes in transgenic SOD1(G93A) mice. Muscle Nerve, 32, 43–50.[CrossRef][ISI][Medline]

  34. Edstrom, L., Thornell, L.E., Albo, J., Landin, S. and Samuelsson, M. (1990) Myopathy with respiratory failure and typical myofibrillar lesions. J. Neurol. Sci., 96, 211–228.[CrossRef][ISI][Medline]

  35. Michele, D.E. and Campbell, K.P. (2003) Dystrophin–glycoprotein complex: post-translational processing and dystroglycan function. J. Biol. Chem., 278, 15457–15460.[Free Full Text]

  36. Hauser, M.A., Horrigan, S.K., Salmikangas, P., Torian, U.M., Viles, K.D., Dancel, R., Tim, R.W., Taivainen, A., Bartoloni, L., Gilchrist, J.M. et al. (2000) Myotilin is mutated in limb girdle muscular dystrophy 1A. Hum. Mol. Genet., 9, 2141–2147.[Abstract/Free Full Text]

  37. Moreira, E.S., Wiltshire, T.J., Faulkner, G., Nilforoushan, A., Vainzof, M., Suzuki, O.T., Valle, G., Reeves, R., Zatz, M., Passos-Bueno, M.R. et al. (2000) Limb-girdle muscular dystrophy type 2G is caused by mutations in the gene encoding the sarcomeric protein telethonin. Nat. Genet., 24, 163–166.[CrossRef][ISI][Medline]

  38. Richard, I., Roudaut, C., Marchand, S., Baghdiguian, S., Herasse, M., Stockholm, D., Ono, Y., Suel, L., Bourg, N., Sorimachi, H. et al. (2000) Loss of calpain 3 proteolytic activity leads to muscular dystrophy and to apoptosis-associated IkappaBalpha/nuclear factor kappaB pathway perturbation in mice. J. Cell Biol., 151, 1583–1590.[Abstract/Free Full Text]

  39. Goll, D.E., Thompson, V.F., Li, H., Wei, W. and Cong, J. (2003) The calpain system. Physiol. Rev., 83, 731–801.