Muscular dystrophies are a clinically and genetically heterogeneous group of disorders that show myofiber degeneration and regeneration. Identification of animal models of muscular dystrophy has been instrumental in research on the pathogenesis, pathophysiology, and treatment of these disorders. We review our understanding of the functional status of dystrophic skeletal muscle from selected animal models with a focus on1) the mdx mouse model of Duchenne muscular dystrophy, 2) the Bio 14.6 δ-sarcoglycan-deficient hamster model of limb-girdle muscular dystrophy, and 3) transgenic null mutant murine lines of sarcoglycan (α, β, δ, and γ) deficiencies. Although biochemical data from these models suggest that the dystrophin-sarcoglycan-dystroglycan-laminin network is critical for structural integrity of the myofiber plasma membrane, emerging studies of muscle physiology suggest a more complex picture, with specific functional deficits varying considerably from muscle to muscle and model to model. It is likely that changes in muscle structure and function, downstream of the specific, primary biochemical deficiency, may alter muscle contractile properties.
- mdx mouse
muscular dystrophies are a group of human and animal disorders that show myofiber degeneration and regeneration, typically associated with progressive muscle weakness. Clinically and genetically, they are a heterogeneous group of inherited diseases. Mutations in several individual genes are now known to underlie the pathogenesis of different types of muscular dystrophy (Table 1) (37, 45). Some of these gene mutations involve proteins expressed throughout the body, yet appear to preferentially affect skeletal muscle. These disorders include defects in nuclear envelope and mRNA metabolism. Specifically, defects in two membrane components of the nuclear envelope cause different forms of Emery-Dreifus muscular dystrophy: 1) emerin (X-linked recessive Emery-Dreifuss muscular dystrophy) and2) lamin A/C (autosomal dominant Emery-Dreifuss muscular dystrophy) (8). Three genes involved in generalized mRNA metabolism cause specific dystrophies, again despite the fact that the biochemical deficiency should be expressed throughout the body. Mutations in poly(A) binding protein 2 cause oculopharyngeal muscular dystrophy (87), whereas expression of long trinucleotide repeats in specific mRNAs disrupts mRNA splicing and regulation and leads to two different forms of myotonic dystrophy (55). The mechanisms underlying the muscle-specific phenotype of these generically expressed proteins are not understood, and this question remains a challenge for researchers.
Other types of muscular dystrophies are caused by genes expressed specifically in muscle and involve myofiber plasma membrane function (dysferlin, calpain III, calveolin) or structural support (see below). Three proteins, dysferlin, calpain III, and calveolin, appear to be involved in plasma membrane maintenance and/or traffic and cause Miyoshi limb-girdle muscular dystrophy (LGMD) 2B, LGMD-2A, and LGMD-1C, respectively (59, 65, 81). A mouse model for dysferlin deficiency (SJL mice) has been used as a model for both heart failure and autoimmune disease, depending on the background strain harboring the mutation (3). The limited knowledge of the role of these proteins in muscle cell biology has hindered the understanding of the pathophysiology of these types of muscular dystrophies, and no physiological studies of muscle function have been reported.
The most common and devastating types of muscular dystrophies are also those that are most fully characterized; each involves a defect of the muscle membrane cytoskeleton. There are thought to be at least two membrane cytoskeleton systems: 1) the vinculin-integrin-laminin network and 2) the dystrophin-dystroglycan-laminin network (45). These two networks appear to be at least in part functionally redundant, and this may be why patients with mutations in any one of these components survive to birth, yet evidence muscle abnormalities during postnatal development (11, 43). The one component shared by both networks is laminin. Laminins are a major component of the basal lamina, and the functional protein is a heterotrimer containing alpha, beta, and gamma subunits. There are different genes for each of the subunit types, and these show tissue- and cell-type-specific expression. Muscle laminin is composed of alpha-2, beta-1, and gamma-1 proteins, with this trimer often called “merosin.” Mutations in the alpha-2 gene cause a very severe form of muscular dystrophy that shows a neonatal or even prenatal onset (congenital muscular dystrophy) (31, 74). The loss of this muscle protein in patients is associated with large-scale degeneration of myofibers at or around the time of birth, with subsequent failure of muscle regeneration and profound weakness. A mouse model for this disorder has been known for many years [dy/dy mice (96)], as well as recently characterized murine laminin alpha-2 chain null mutants, all of which similarly show early lethality (66). One would expect that both the dystrophin-dystroglycan-laminin and vinculin-integrin-laminin networks would be defective in these patients and mice, and this may explain the severe nature of this disorder.
The most common and extensively studied muscular dystrophies are those that involve mutations of the dystrophin-dystroglycan-laminin network. Considerable effort has been expended on understanding the physiology and cellular biology of this network, and the remainder of this review discusses the proteins specifically affecting the dystrophin-based membrane cytoskeleton. The organization of the dystrophin-based membrane cytoskeleton of muscle fibers is shown in Fig.1 and demonstrates the relationship between dystrophin and the other dystrophin-associated proteins within the cytoskeletal oligomeric protein complex. The dystrophin-associated proteins include the dystroglycan (α-dystroglycan and β-dystroglycan), sarcoglycan (SG; α-, β-, γ-, δ-SG), and cytoplasmic (syntrophin) subcomplexes. The dystroglycan complex consists of α-dystroglycan, which associates with the basal-lamina protein merosin in the extracellular matrix, and β-dystroglycan, a transsarcolemmal protein that binds α-dystroglycan and dystrophin. The SG complex consists of four transsarcolemmal proteins, α-, β-, γ-, and δ-SG, that associate with each other and likely function as a single unit, with δ-SG serving as the link to β-dystroglycan of the dystroglycan complex. Within the cytoplasmic subcomplex, the COOH-terminal region of dystrophin is bound to syntrophin, whereas the amino-terminus of dystrophin binds F-actin.
Abnormalities of dystrophin are the most common cause of muscular dystrophy (27, 45), accounting for both the Duchenne and Becker phenotypes. Duchenne muscular dystrophy (DMD) is a devastating muscular dystrophy and the most common inherited childhood-lethal disorder of humans worldwide (45). DMD is caused by mutations in the dystrophin gene that precludes the production of stable dystrophin molecules and results in sarcolemmal instability and contraction-induced myofiber necrosis. Dystrophin is highly conserved through vertebrate evolution, the dystrophin gene is X-linked in placental mammals, and dystrophin deficiency appears to be completely specific for DMD (46, 48). Thus any animal that manifests dystrophin deficiency as an X-linked inherited trait is a candidate model of DMD. Among other causes of muscular dystrophy are1) Becker muscular dystrophy, a disorder known to be allelic (due to a different mutation in the same gene) to DMD (54), and 2) genetic defects of the SG subcomplex (α, β, γ, δ) of the myofiber cytoskeleton (sarcoglycanopathies), a common cause of LGMD-2C, -2D, -2E, and -2F (1, 27, 37, 40, 86).
Identification of animal models for DMD and the sarcoglycanopathies has been instrumental in research on the pathogenesis, pathophysiology, and treatment of these disorders. Indeed, the past 15 yr have been witness to a virtual explosion of knowledge regarding the molecular pathology of DMD, Becker muscular dystrophy, and LGMD in humans and their mammalian counterparts (45). This knowledge, in turn, is being used to develop novel approaches to the treatment of these disorders, including gene therapy (47, 93, 95), myoblast transfer (71), and new pharmacological interventions (38). There remains, however, a gap between our knowledge of the molecular pathology of muscular dystrophy and downstream biomechanical events. Given that most muscular dystrophies are associated with primary defects of the muscle membrane cytoskeleton and characterized phenotypically by muscle weakness, a host of potential functional targets exist beyond those involved in the cascade leading to muscle cell death (45, 78). Targets include the metabolic pathways involved in intracellular calcium regulation (t tubule system, sarcoplasmic reticulum, and excitation-contraction coupling), myosin molecular motor function, and bioenergetics (mitochondria and phosphocreatine-creatine kinase energy shuttle), among others. An expanded knowledge of these secondary consequences of muscular dystrophy will enhance our understanding of these disorders and dystrophic skeletal muscle dysfunction. Moreover, the aforementioned novel therapeutic approaches will ultimately be judged by their ability to enhance dystrophic skeletal muscle function. To date, such therapies have been almost exclusively applied and tested in animal models. Below, we discuss our current understanding of the functional status of dystrophic skeletal muscle from selected animal models used in the study of muscular dystrophy with a specific focus on 1) the mdx mouse model of DMD,2) the Bio 14.6 δ-SG-deficient hamster model of LGMD, and3) transgenic null mutant murine lines of SG (α, β, δ, and γ) deficiencies. In addition to characterizing the force-generating capacity of dystrophic muscle in the isometric and miometric (shortening) modes, dystrophic muscle function assessed during repetitive lengthening activations, a paradigm of mechanical stress, is detailed (23, 36, 75). The latter measurement is thought to be particularly important because evidence suggests that a function of the dystrophin-based membrane skeleton is to protect against stress-induced muscle damage (75).
ANIMAL MODELS OF DMD: MDX MOUSE
The most widely used animal model of muscular dystrophy is themdx mouse, the first animal model of DMD identified (9, 45, 48). This mouse line occurred spontaneously on a C57BL/10 background and was fortuitously identified during a screen for red blood cell defects in a C57BL/10 colony (9). Serum creatine kinase levels were strikingly elevated in this line, suggesting a dystrophic process. Dystrophin deficiency in themdx muscle was subsequently identified (46) and shown to result from a stop codon mutation in exon 23(89) of the dystrophin gene. More recently, a series of new alleles of the mdx strain were induced by mutagenesis; all demonstrate a phenotype consistent with the original mdxallele (52).
Isometric contractile characteristics.
Most studies of mdx mouse muscle have been performed on limb and diaphragm muscle in the isometric mode. These studies generally evidence twitch contraction kinetics that approximate those of age-matched C57BL/10 control muscle (29, 72, 76, 80). Some studies have reported a slowing in twitch contraction and half-relaxation times associated with an increase in the proportion of type I fibers (72, 76), whereas others have shown the opposite (83). Absolute peak twitch force (Pt) approximates that observed in C57BL/10 control muscle (77) whereas specific twitch force (Pt/CSA), i.e., Pt normalized for an estimate of muscle cross-sectional area (CSA) is consistently decreased in mdx muscle relative to control (76, 77). Similarly, absolute peak tetanic force (Po) is comparable to or exceeds that observed in C57BL/10 muscle (32, 60), but specific peak tetanic force (Po/CSA), i.e., Po normalized for CSA, is consistently lower in mdx mouse vs. C57BL/10 muscle (7, 20, 21, 29, 60, 61, 63, 77, 83). Fatigue resistance during repetitive isometric contractions is either at control levels or greater (76, 83), the latter response presumably related to a shift in myosin heavy chain phenotype to slower isoforms with greater oxidative capacity and lower myofibrillar ATPase activity (76).
The lower Pt/CSA and Po/CSA in mdxmuscle likely reflects an increasing contribution of degenerating and/or necrotic fibers to mdx muscle CSA (32) compared with control C57BL/10 muscle. Indeed, DMD in humans is characterized by increased bulk in selected muscles that, early in the disease process, results from true hypertrophy of muscle fibers (45). This hypertrophy in DMD patients later evolves into pseudohypertrophy as the muscle is gradually replaced by first fibrotic tissue and then fat (45). Skeletal muscle frommdx mice generally shows increased mass (77,83), although this can be later in onset and variable across specific muscle and age groups (72, 73). Thus cycles of muscle fiber degeneration typically seen as early as 3–5 wk of life in mdx mice are apparently balanced by fiber regeneration, leading to hypertrophic changes followed later in life in selected muscles by atrophy (73). Muscle hypertrophy inmdx limb muscle is effective in maintaining control values of absolute force (60). In contrast to mdx limb muscle, the mdx diaphragm demonstrates more progressive cycles of fiber degneneration and fiber loss throughout the lifespan of the mdx mouse. The proportion of viable fibers in diaphragm decreases from ∼70% in 4- to 6-mo-old animals to 40% in 24-mo-old animals (63, 76). Thus one difference betweenmdx limb muscle and mdx diaphragm is thatmdx diaphagm evinces greater and more progressive decrements in Po/CSA with age (29). In addition to injured or necrotic fibers, other potential mechanism(s) that could contribute to a reduced Po/CSA in mdx muscle includes adverse changes in excitation-contraction coupling, reduced calcium sensitivity (altered thin-filament regulation), lower contractile protein concentration, altered cross-bridge cycling kinetics, and/or lower average force per cross bridge. These possible mechanisms merit further investigation because there are limited, and at times conflicting, data on such phenomenon in mdx muscle (15, 35, 36).
Dynamic shortening activations.
A more limited number of studies have characterized the dynamic shortening properties, i.e., force velocity, power, and work functions of mdx muscle. These studies consistently report a downward shifted force-velocity relationship and diminished maximum power and work output in mdx muscle (15, 20, 21, 32, 60, 61, 63,91). Unfortunately, there are limited data with respect to the mechanism(s) that may underlie these functional deficits. Investigators have reported a shift in myosin heavy chain expression to slower isoforms (76) and abnormalities in cross-bridge properties characterized by both a reduction in total number of cross bridges and the force produced per cross bridge in mdx muscle to account for these deficits (15). The latter, in turn, may be related to defective energy transport in mdx muscle (6) and/or altered calcium handling due to chronic calcium overload (35, 47, 79). The observed changes in myosin heavy chain isoform composition (and presumably actomyosin ATPase activity), however, do not appear to be of sufficient degree to fully account for the observed reduced force per cross bridge inmdx muscle (15). Changes in cross-bridge cycling kinetics could contribute to the impaired dynamic shortening properties of mdx muscle (15). In this regard, Coirault et al. (15) report that both the duration of the cross-bridge cycle and the rate constant for cross-bridge detachment are significantly lower in mdx mice compared with C57BL/10 controls. These authors conclude that impaired dynamic shortening inmdx muscle is a result of myosin molecular motor dysfunction (15). Additional study is necessary to confirm and further define the mechanism(s) of impaired dynamic shortening activation in dystrophic muscle.
Response to repetitive lengthening activations.
Several studies have assessed the contractile response ofmdx muscle to repetitive dynamic lengthening activations (i.e., “eccentric contractions”), a paradigm of mechanical stress (23, 44, 75). The rationale for such study resides in1) reports demonstrating that skeletal muscle injury is greatest after exercise that involves lengthening of activated muscle (greater than that which follows intense isometric or shortening contractions) and 2) evidence suggesting that a function of the dystrophin-based membrane cytoskeleton is to protect against stressed-induced muscle damage (75). As predicted, several reports demonstrate that mdx muscle does demonstrate an enhanced susceptibility to injury during repetitive lengthening activation, as assessed by both contraction-induced sarcolemmal rupture and force deficits (7, 22, 23, 36, 44, 67, 75). Indeed, the literature would suggest that enhanced susceptibility to lengthening-activation injury, as assessed by force deficit, is a hallmark of dystrophin-deficient muscle function (36, 75).
Until recently, it has been unclear why mdx muscle is so susceptible to lengthening-activation force deficits. Previous study has shown that mdx diaphragm evidences enhanced passive stiffness relative to controls (90, 91). Whether this characteristic predisposes or protects against lengthening-activation injury remains undocumented. Recent reports taken together, however, confirm that the locus of contraction-induced injury in mdxmuscle resides in the sarcolemma (62) and suggest that reduced elasticity of mdx muscle itself may predispose muscle to an enhanced susceptibility of lengthening-activation force decrements. Specifically, the amount of negative work done during the lengthening phase of activation has been identified as an important factor in modulating the extent of lengthening-activation force deficits (51). Moreover, investigators have reported that the amount of negative work performed by mdx diaphragm increases dramatically with increases in muscle length in association with its enhanced passive stiffness (91), significantly more so than control muscle. These findings suggest that reduced muscle elasticity may predispose mdx muscle to injury during lengthening activations. This area of study merits further investigation, particularly in light of data (see below) suggesting that dystrophic muscle from the closely biochemically related sarcoglycanopathies do not evidence similar heightened susceptibility to lengthening-activation force deficits (39).
Other animal models of DMD.
Other animal models of DMD have been identified, although a relative paucity of functional studies on their respective limb or respiratory musculature exist. These include a canine X-linked or golden retriever muscular dystrophy (17, 18, 24, 56) that show dystrophin deficiency as an X-linked trait (17, 18) associated with a splicing mutation in exon 8 (88). Dystrophin-deficient dogs show a rapidly progressive course of muscle weakness, wasting, and contracture, with onset at ∼3 mo of age and death occurring anywhere from the neonatal period to adulthood (17, 56). Independent alleles have been identified in a series of breeds, including rottweilers, wire-hair fox terriers, shelties, and German short-haired pointers (45). Varying degrees of muscle involvement and marked temporal differences in the dystrophic phenotype across individual dogs have limited the utility of this model for functional studies. In addition, at least two independent isolates of dystrophinopathy have also been identified in domestic cats (12, 34, 94). Dystrophin-deficient cats show marked muscular hypertrophy that becomes so severe that they succumb to dehydration because of lingual hypertrophy or to starvation because of crural diaphragm hypertrophy (33, 34). Cats also demonstrate considerable skeletal muscle stiffness and cardiac involvement but never lose mass and thus are better able to conserve muscle force-generating capacity.
The “appropriateness” of these animal dystrophinopathies for providing insights regarding human DMD has generated considerable debate within the scientific community. Clearly, the clinical phenotype of each animal model differs from the human disease and from each other. Nevertheless, all of the aforementioned models have loss-of-function mutations in the same highly conserved dystrophin gene, so they are certainly genetically homologous. They all lack the highly conserved dystrophin protein in muscle tissue, so they are biochemically homologous. Muscle tissue itself is exceedingly highly conserved through evolution, and, in fact, all animal models share many of the histological features of human DMD, including fiber size variation and the degeneration/regeneration of myofibers. All have striking elevations in serum creatine kinase. The phenotypic differerences reside in the progressive aspects of the disease, particularly with respect to connective tissue proliferation in muscle, and failure of regeneration over time. All dystrophin-deficient species show marked hypertrophy at a young age, but, in dogs and humans, the hypertrophy changes to loss of muscle (wasting) and loss of strength. There is general consensus that the dystrophin-deficient mouse, dog, and cat are outstanding genetic and biochemical models for DMD despite phenotypic differences. The fact that they show differences in phenotype is valuable in that they provide insight into the secondary consequences of primary dystrophinopathy. However, it can be more difficult to use these animals as therapeutic models since the downstream consequences of dystrophin deficiency may vary considerably from species to species.
The autosomal-recessive LGMDs are a heterogeneous group of muscular diseases characterized by progressive muscle weakness and, in severe cases, death in the second or third decade of life (1). The more severe phenotypes are often caused by mutations in the SG genes α (LGMD-2D), β (LGMD-2E), δ (LGMD-2F), and γ (LGMD-2C) (4, 5, 27, 28, 53, 58, 69, 86). A primary deficiency of any single SG leads to partial or complete absence of all other sarcoglycans on the sarcolemma, suggesting that sarcoglycans are mutually dependent on each other for assembly and stability in the plasma membrane (28, 70, 84, 92). Phenotypically, the SG deficiencies are indistinguishable from the dystrophin deficiencies (27). However, dystrophinopathies show secondary deficiencies of all SG proteins, whereas sarcoglycanopathies usually show normal levels of dystrophin (42,57). LGMDs, although less common than DMD, represent an important group in which to study the dystrophic process. Until the recent discovery of a δ-SG mutation in the Bio 14.6 inbred dystrophic hamster (70, 84) and the generation of α- (26), β- (2, 30), δ- (41), and γ-SG (42, 85) null mutant transgenic mouse lines, study of LGMDs has been hindered by the lack of an animal model.
BIO 14.6 HAMSTER MODEL OF δ-SG DEFICIENCY
The inbred Bio 14.6 hamster was identified in 1962 as a recessively inherited dystrophic myopathy model that affected both cardiac and skeletal muscle (50). The skeletal muscle of Bio 14.6 hamster displays classical signs of muscular dystrophy, including necrosis, central nucleation, and variable size of muscle fibers (10, 49, 50, 57, 92). The genetic cause for the dystrophic disease in the Bio 14.6 hamster was recently identified as a loss-of-function mutation of the δ-SG gene (70, 84). δ-SG is a 35-kDa transmembrane glycoprotein component of the dystrophin-based membrane cytoskeleton of muscle fibers. It is part of a functional tetrameric complex that includes α-, β-, γ-, and δ-SG proteins in equal stoichiometry. As with their human counterpart, primary loss of δ-SG in Bio 14.6 hamster also results in the secondary deficiency of the other skeletal muscle SGs (α, β, and γ) (49, 57, 92). The shared muscle histopathology and protein biochemistry between Bio 14.6 hamster and human patients with LGMD-2F renders this animal as an excellent model to study the functional consequences of δ-SG deficiency. The clinical picture, however, deviates considerably between affected humans and hamsters, with the δ-SG deficient hamster showing muscle hypertrophy as a notable finding and early death from cardiac failure. Humans with the same defect generally show more limited cardiac involvement (64). Although the cardiac features of the Bio 14.6 hamster have been extensively studied, there is relatively limited data available on skeletal muscle function (10, 68).
Isometric contractile characteristics.
Bio 14.6 hamster dystrophic muscle evidence twitch-contraction kinetics that approximate those of age-matched F1B control muscle (10,68). Absolute peak Pt is generally comparable to that observed in F1B hindlimb muscle (10, 68, 95), whereas Pt/CSA is decreased in Bio 14.6 dystrophic hamster muscle relative to control (10, 95). Similarly, absolute Po is comparable to that of F1B hamster, whereas Po/CSA is consistently lower in Bio 14.6 dystrophic hamster vs. F1B muscle (10, 95). The lower Pt/CSA and Po/CSA in Bio 14.6 hamster muscle may reflect a combination of a greater muscle mass and an increasing contribution of degenerating and/or necrotic fibers to dystrophic muscle CSA compared with control F1B muscle.
There are no reported data on Bio 14.6 hamster dystrophic-shortening activations and only unpublished observations (T. L. O'Day, personal communication) regarding the response of δ-SG-deficient Bio 14.6 muscle to repetitive lengthening activations. The latter is of interest, however, in that the Bio 14.6 muscle does not demonstrate increased susceptibility to force deficit when compared with F1B control muscle (Fig. 2). This finding runs counter to the notion that dystrophic muscle always shows enhanced susceptibility to lengthening-activation injury and suggests that nonmechanical events may be operative in the pathogenesis of the dystrophic process in SG-deficient states (see below) (39). It is of interest, in this regard, that Bio 14.6 hamster does not show the decreased passive stiffness observed in themdx mouse but in fact the opposite as recently reported by Coirault et al. (16). The later study demonstrates increased muscle compliance in the δ-SG-deficient hamster, results that contrast sharply with those found in mdx muscle, suggesting that muscle viscoelastic properties are critically dependent on the presence or absence of specific cytoskeletal proteins and the myopathic animal model considered (see below) (16). Relevant to these observations is the fact that dystrophin strongly binds intracellular actin filaments at multiple sites along the protein (82). The connection between the intracellular actin cytoskeleton and the dystrophin-based membrane cytoskeleton is still intact in SG deficiency but is lost in dystrophin deficiency.
γ-SG TRANSGENIC MURINE NULL MUTANT
Recent years have seen the genesis of SG-deficient transgenic null mutant lines. Table 2 shows the mechanical characteristics of these null mutant lines and how they compare with those observed in the Bio 14.6 δ-SG-deficient hamster and dystrophin-deficient mdx mouse. γ-SG null mutant transgenic mice (gsg −/−) (39, 42, 85) show contractile properties in vitro that do not differ from their wild-type counterparts (39). Specifically, muscle [extensor digitorum longus (EDL)] from gsg −/− demonstrates comparable muscle mass, CSA, Pt, Po, and Po/CSA as muscles from wild-type counterparts. Importantly,gsg −/− EDL also showed force output decline and Procion orange uptake (a marker of sarcolemmal integrity) at levels comparable to control muscle during repetitive lengthening activations. These findings suggest that γ-SG deficiency does not enhance the susceptibility of gsg −/− muscle to lengthening-activation injury. The aforementioned functional response to repetitive lengthening activations is comparable to that seen in the Bio 14.6 δ-SG-deficient hamster and suggests that nonmechanical events may be important in the pathophysiology of muscular dystrophy within the context of SG deficiency (39). As pointed out by Hack and colleagues (39, 40), these findings further suggest that the loss of SGs may contribute to muscle degeneration in both the dystrophin- and SG-associated muscular dystrophies.
δ-SG MURINE NULL MUTANT LINE
δ-SG null mutant transgenic mice (dsg −/−) have also been generated (41). This model is analogous to the Bio 14.6 δ-SG-deficient hamster, but dsg −/− skeletal muscle shows some differences in isolated muscle mechanics when compared with the Bio 14.6 hamster and other transgenic SG-deficient null mutant lines. As with both the Bio 14.6 hamster and gsg−/− null mutant, dsg −/− mice demonstrate classic histological findings of dystrophy, including cell death, muscle regeneration, inflammation, and fibrosis as well as reduced survival (41). Dsg −/− muscle mass, Pt, Po, and Po/CSA, however, do not differ from wild-type muscle (41). Interestingly, dsg −/− muscle does show an enhanced susceptibility to lengthening-activation force deficits (41). These findings suggest δ- and γ-SG deficiency have different molecular consequences and resultant functional impacts that may also be species specific. Hack et al. (39) have proposed that the absence of lengthening-activation deficits in gsg −/− muscle suggests a nonmechanical role for γ-SG in myocyte survival. The role of δ-SG remains less clear given the species-specific response of δ-SG deficiency on muscle mechanics, both in terms of force generation and in susceptibility to lengthening-activation force deficits. As stated by Hack and colleagues (41), however, these functional differences underscore the important nature of the SG complex and the need to understand the role of each component in the pathogenesis of muscular dystrophy. In this regard, recent work suggests that δ-SG deficiency can adversely impact vascular smooth muscle function, induce ischemic injury in skeletal muscle, and exacerbate muscular dystrophy (19).
α-SG NULL MUTANT MICE
α-SG-deficient null mutant transgenic mice have also been generated by targeted disruption of the α-SG gene (26). These mice show the complete absence of α-SG transcript and protein, a complete loss of the SG complex and sarcospan, and demonstrate a progressive muscular dystrophy (26) similar to its human homologue, LGMD-2D (27). α-SG −/− mice demonstrate significant muscle hypertrophy and muscle-specific changes in contractile properties. Hypertrophic slow-twitch weight-bearing soleus muscle demonstrates Po values that are greater than control with a resultant specific force that is not different from control. In contrast, the hypertrophic fast-twitch EDL (26) and tibialis anterior (25) muscles demonstrate Povalues that are comparable to control levels and a resultant Po/CSA that is significantly lower than control. Fast- and slow-twitch α-SG-deficient skeletal muscle also evidences disparate passive stiffness; soleus muscle shows normal resistance to passive stretch, whereas the EDL shows reduced elasticity when compared with control EDL (26). Moreover, α-SG-deficient tibialis anterior muscle does not demonstrate enhanced susceptibility to lengthening-activation force deficits (T. L. O'Day, personal observation). A recent preliminary report on α-SG-deficient diaphragm demonstrates decreased Po/CSA under both uniaxial and biaxial loading compared with control muscle (13). Moreover, in contrast to control muscle, biaxial loading was not associated with an increase in maximal diaphragm force, indicating an inability of α-SG −/− diaphragm sarcolemma to transmit forces in the transverse fiber direction (13). This finding suggests that the SG complex is important in transmitting force in the transverse direction to the long axis of muscle. Taken together, these data suggest that α-SG is an important component of the SG-sarcospan complex and that the functional consequences of the hypertrophic response in this model are muscle specific. The mechanisms responsible for the hypertrophic response and its muscle-specific nature merit further study.
β-SG-DEFICIENT NULL MUTANT MICE
β-SG-deficient mice have also been generated by targeted mutagenesis (2, 30). Skeletal muscle from β-SG-deficient mice, like their α- and δ-SG-deficient transgenic null mutant counterparts, demonstrates increased muscle mass (2) and reduced Po/CSA (30). There are no data on the passive stiffness of β-SG-deficient muscle nor on the response of such muscles to repetitive lengthening activations.
Mutations in components of the dystrophin-SG-dystroglycan-laminin network lead to devastating and common muscular dystrophies in humans. The current models suggest that this network is critical for structural integrity of the myofiber plasma membrane. Although considerable data, such as dye-exclusion studies, support this model, emerging studies of muscle physiology in the animal models suggest a more complex picture, with specific functional deficits varying considerably from muscle to muscle and model to model. It is likely that changes of muscle structure and function, downstream of the specific primary biochemical deficiency, may alter muscle contractile properties (45, 47,78). A full understanding of the apparently variable consequences of single biochemical defects on muscle contractile function may require genome-wide approaches to understanding the response of muscle to injury and compensatory molecular mechanisms invoked by the muscle. Expression profiling and proteomic approaches will likely soon provide us with a more complete picture (14).
This work was supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR-45925, the Parent Project USA, the Mario Lemieux Centers for Patient Care and Research, and the 25 Club of Magee-Women's Hospital.
Address for reprint requests and other correspondence: J. F. Watchko, Division of Neonatology and Developmental Biology, Dept. of Pediatrics, Magee-Women's Hospital, 300 Halket St., Pittsburgh, PA 15213 (E-mail:).
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