Muscular dystrophy in dysferlin-deficient mouse models

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Neuromuscular Disorders 23 (2013) 377–387 www.elsevier.com/locate/nmd

Review

Muscular dystrophy in dysferlin-deficient mouse models Mark A. Hornsey a, Steven H. Laval a,⇑, Rita Barresi b, Hanns Lochmu¨ller a, Kate Bushby a a

Newcastle University, Institute of Genetic Medicine, International Centre for Life, Central Parkway, Newcastle-upon-Tyne NE1 3BZ, United Kingdom b NCG Diagnostic and Advisory Service for Rare Neuromuscular Diseases, Muscle Immunoanalysis Unit, Dental Hospital, Newcastle-upon-Tyne, United Kingdom Received 5 November 2012; received in revised form 9 January 2013; accepted 5 February 2013

Abstract Mutations in the dysferlin gene result in the development of a range of early adult-onset, progressive muscular dystrophies, collectively known as the dysferlinopathies. There is currently no effective treatment for these disorders. Several spontaneous and engineered alleles at the mouse dysferlin locus have been isolated and these dysferlin-deficient mouse strains are providing valuable insights into the role dysferlin plays in skeletal muscle physiology, heart function, and the regulation of the innate immune system. In addition, mouse models of dysferlinopathy are now widely used to test novel therapeutic strategies. Each dysferlin-deficient mouse strain has been characterised to varying degrees using a variety of histological and functional assays, occasionally producing results inconsistent with other strains. Here, we review each mouse model and physiological changes in various systems which accompany their muscle disease with emphasis on the how the disease process develops in different mouse models of dysferlinopathy. This review highlights the urgent requirement for standardised assays and outcome measures that will unify and coordinate research efforts throughout the field, procedures that are necessary if potential therapies are to be tested efficiently and effectively. Ó 2013 Elsevier B.V. All rights reserved. Keywords: Dysferlin; Muscular dystrophy; Mouse models

1. Review Mutations in the dysferlin gene, DYSF, result in the development of a number of progressive muscular dystrophies known collectively as the dysferlinopathies [1–3], which generally present with a characteristic pattern of muscle involvement. Affected individuals often have a history of excellent athletic performance, before experiencing their first symptoms mainly around early adulthood. Patients have high serum creatine kinase (CK) levels, on average more than forty times the norm, and present with distinct patterns of weakness with predominantly proximal or distal onset, that defines the disorder as either, limb girdle muscular dystrophy type 2B (LGMD 2B), Miyoshi myopathy or distal myopathy with anterior tibial onset [1,4–7]. As the disease progresses, it increasingly involves both the proximal and distal ⇑ Corresponding author.

E-mail address: [email protected] (S.H. Laval). 0960-8966/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.nmd.2013.02.004

musculature, and therefore these diagnostic categories are generally viewed as one disorder with a wide spectrum of clinical onset [8–12]. Sequence analysis of the dysferlin gene reveals no obvious mutational hotspots and no obvious genotype–phenotype correlation [13]. Affected individuals have a markedly reduced or complete loss of the dysferlin protein in skeletal muscle, which shows classic signs of progressive dystrophy [14,15]. Biopsies from dysferlinopathy patients have a higher percentage of immature fibres than other muscular dystrophy controls [16], together with a very prominent mononuclear cell infiltrate in muscle, and consequently, the disease in these patients is often misdiagnosed as polymyositis [11,17,18]. Disease progression is relatively slow when compared with other muscular dystrophies; nevertheless, patients are eventually confined to a wheelchair. There is currently no effective treatment. Dysferlin is a member of the “ferlin” family as it shares homology with the Caenorhabditis elegans gene fer-1 [1].

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FER-1 is involved in calcium-dependent, vesicle fusion within the developing spermatid and mutation of the gene results in infertility [19]. Dysferlin has seven Ca2+binding motifs known as C2 domains that have significant homology to similar domains found in protein kinase C and the synaptotagmin family. C2 domains bind proteins and phospholipids in a Ca2+ dependent manner and the synaptotagmins are known regulators of vesicle fusion [20–26]. Several groups are now defining dysferlin’s protein structure [27,28] and elucidating its phospholipid binding specificities [23,29]. Dysferlin is a type II transmembrane protein that localises to the periphery of muscle fibres [30]. A large body of evidence suggests that dysferlin is an important regulator of vesicle fusion at the sarcolemma, and has an essential role in muscle membrane repair [31–36]. However, dysferlin functions clearly extend beyond sarcolemma repair with possible roles in vesicle trafficking, endocytosis, membrane receptor recycling, membrane turnover [37,38], muscle regeneration and T-tubule formation [16,39], focal adhesion [40] and ATP-dependent intercellular signalling [41]. Dysferlin is also expressed in cells of the innate immune system, and there is evidence that it modulates macrophage function [42], regulates cytokine release [16] and controls complement activation [43–45]. There is also significant expression found within endothelial cells, syncytiotrophoblasts of the human placenta and podocytes of the kidney [46–50]. Studying animal models has provided tremendous insight into the pathological process behind many disorders, including muscular dystrophy, while providing a necessary platform for the testing of new therapies. Mice are highly complex organisms with a muscle structure very similar to humans. The human and mouse dysferlin genes share >90% amino acid sequence homology [51], and there are a number of dysferlindeficient mice (naturally occurring and constructed) that are being used as models of dysferlinopathy [52]. Here we review the data on these models, the advantages and limitations of each for therapeutic testing, and their suitability in view of future research directions.

There is one report of histological alterations at P1 in the hind limb muscle of A/J mice backcrossed onto the 129SvJ background. These alterations were “less evident” at 8 weeks of age, suggesting a possible delay in maturation [37]. 3. Spontaneous mutation – SJL/J mice The SJL/J mouse is another naturally occurring model of dysferlinopathy, and also develops a mild, progressive myopathy [14]. A splice-site mutation in the dysferlin gene results in a 171 bp in-frame RNA deletion that removes 57 amino acids and most of the C2–E domain [51]. Dysferlin expression is reduced to 15% of wild type levels and serum CK levels are elevated (Table 1). A progressive dystrophy develops with minimal involvement at 2 and 4 months (small numbers of centrally nucleated myofibres), more common necrotic fibres at 6 months, followed by progressively advanced dystrophic features, including fat deposition, appearing at 10 months [53–57]. Most degenerative fibres in the muscles of SJL/J mice were of the fast-twitch type [56], while selective loss of fast-twitch/type 2 fibres has been observed in patients [58]. SJL/J muscle has a prominent macrophage and CD4+ infiltrate that is also noted in patient biopsies. Increasing cell infiltrates in both SJL/J and A/J mice were observed from 2 months to one year of age and consisted of approximately 60% macrophages (Mac-1+ and/or Mac-3+) and 30% T cells; CD4+ cells more abundant than CD8+ [54,56]. A study directly comparing the dystrophy in muscles of A/J and SJL/J confirmed the earlier onset in SJL/J mice compared with A/J and a more rapid rate of progression [56]. Only the diaphragm is spared with mild lesions appearing in the SJL/J mouse and none in the A/J. However, force measurement studies by a second group showed 50% functional deterioration of the A/J diaphragm at 10 and 36 weeks of age. Histology at 10 weeks appeared normal with pathological alterations only apparent in the older animals [59]. 4. A/J and SJL/J mice on defined genetic backgrounds

2. Spontaneous mutation – A/J mice One of the most widely used mouse models of dysferlinopathy was discovered at the Jackson Laboratory. Mice on the A/J inbred background develop a progressive muscular dystrophy, the result of an ETn retrotransposon insertion within intron 4 of the dysferlin gene (Table 1). Histological dystrophic features appear at 4–5 months of age, with the proximal muscles more affected than distal ones. Evidence of compromised sarcolemma integrity was demonstrated by elevated serum CK, increased Evan’s blue dye (EBD) uptake and ultra-structural abnormalities at the sarcolemma. Disease progression is slow, except in the abdominal muscles, which show a more rapid rate of muscle wasting [53].

A/J and SJL/J mice possess a number of features not typically observed in patients. A/J mice are poor breeders that exhibit a progressive loss of hearing, a high incidence of lung adenomas and are deficient in complement C5. SJL/J mice have a susceptibility to autoimmune diseases and a high incidence of reticulum cell sarcomas (see the JAXÒ database for comprehensive lists). Aggressive behaviour has been reported in SJL/J mice [53], however there is no evidence of similar traits in other dysferlin-deficient mice. Genetic background can have a profound effect on the observed phenotype of any given gene-targeted allele [60], and the susceptibilities noted above are possibly the result of unknown genetic modulators and not the dysferlin-deficiency itself. To help

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Table 1 Characteristics of the main dysferlin-deficient mouse models. JAXÒ designation (stock number)

Background Mutation

Effect on protein

CK

A/J (646)

Inbred A/J

Spontaneous ETn retrotransposon insertion in intron 4

No dysferlin expression

5-fold 4– higher 5 months than controls

B6.A-Dysfprmd/GeneJ (aka BLA/J) (12767)

C57BL/6J

No dysferlin expression

No data

SJL/J (686)

Wildderived strain of Swiss mice

Spontaneous ETn retrotransposon insertion in intron 4 Splice site mutation in exon 45

15% of wild type levels

4-fold 3– higher 4 weeks than controls

B10.SJL-Dysfim/AwaJ (aka C57/BL10.SJLDysf) (11128)

C57BL/10

Splice site mutation in exon 45

15% of wild type levels

2-fold 3– higher 4 weeks than controls

tm1Kcam

129-Dysf

/J (6830)

B6.129-Dysftm1Kcam/J (13149)

Dysf

/

(Ho et al., 2004)

129

C57BL/6

Mixed 129SvJ and C57BL/6J

Neomycin resistance gene replacement removes last three coding exons including the transmembrane domain Neomycin resistance gene replacement removes last three coding exons including the transmembrane domain Neomycin resistance gene replacement removes exon 45

Onset Active (muscle myopathy histology)

2 months

Muscles Ultra-structural most affected abnormalities

Comments

8– 12 months

Quadriceps femoris

Progression in [53,56,133] abdominal muscles / similar to Dysf . Increased frequency of rhabdomyosarcomas over 20 months of age

Reported as similar to A/J 6– 8 months. Fat deposition begins at 10 months

Psoas major

Thickening and focal duplication of the basal lamina. Subsarcolemma vesicle accumulation No data

Distal bias to muscle histopathology

[61]

Site specific, selective loss of fast twitch fibres. Noted aggressive behaviour

[14,53–56]

6– 8 months

Quadriceps femoris, triceps

Thickening and focal duplication of the basal lamina. Subsarcolemma vesicle accumulation T-tubule abnormalities in soleus muscle (8 week old)

No dysferlin expression

6-fold 2 months higher than controls

8 months

No dysferlin expression

No data

2 months

8 months

No dysferlin expression

6-fold 2 months higher than controls

5– 6 months

identify the genuine causative effect of dysferlin-deficiency, both strains have been backcrossed onto more defined genetic backgrounds. Lostal et al. [61] backcrossed A/J mice onto the C57BL/ 6 background, designated B6.A-Dysfprmd/GeneJ (Table 1). These “BLA/J” mice are no longer C5 complementdeficient, which otherwise leaves the A/J mouse susceptible to infections. Dystrophic features in the A/J and BLA/J mice were reported to be very similar, although a small increase in central myonuclei was detected in some muscles at one month of age, slightly earlier than the original line. Psoas major and gastrocnemius muscles were the most severely affected followed by the tibialis anterior (TA) and then quadriceps. Interestingly, this is the only model where initial dystrophic involvement appears more distal than proximal. SJL/J mice were backcrossed onto the C57BL/10 background [14]. Despite initial claims of a more severe phenotype [62], the dystrophy in these B10.SJL-Dysfim/ AwaJ mice (Table 1) appears to be very similar to the

Quadriceps femoris and triceps brachii

Brachii and abdominals Iliopsoas and quadriceps

No reported differences from the original129Dysftm1Kcam/ J knockout Quadriceps femoris and triceps brachii

References

Similar progression to [13,14,16,62] SJL/J. No evidence of overt aggression

Sarcolemma disruption and subsarcolemma vesicle accumulation

Minimal damage to muscle following downhill running

[32]

No data

No reported differences from the original129Dysftm1Kcam/J knockout

[43]

Thickening and Histopathology focal duplication similar to SJL/J of the basal lamina. Subsarcolemma vesicle accumulation

[53]

observed pathology in SJL/J. Dysferlin protein levels were also similar, 20–30% of control levels in neonatal muscle and 10% in adults (unpublished), compared with 15% in adult SJL/J muscle [14]. CK levels are also elevated [16], contrary to previous reports documenting normal serum CK but elevated pyruvate kinase levels [62]. A simple model of compromised sarcolemma integrity leading to the non-specific release of myofibre contents does not predict the release of one enzyme over another; however, no other group has reported normal CK levels in dysferlin-deficient mice. 5. Targeted dysferlin knockouts The residual levels of dysferlin protein detected in SJL/J skeletal muscle raised concerns that these mice were not accurate models of dysferlinopathy; as the majority of patients have extremely reduced to no detectable dysferlin protein [15]. In order to study muscle function in the complete absence of dysferlin, a number of knockout mutants have been specifically engineered.

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The C2–E domain was targeted with a neomycin insert, completely abolishing dysferlin expression [53]. As expected, these Dysf / mice developed progressive muscle pathology, with the earliest signs observed at 2 months, primarily in the proximal and abdominal muscles. Pathology was not observed in distal muscles (gastrocnemius, soleus and TA) until 5 months of age (Table 1). CK levels were elevated and ultra-structural abnormalities were reported, similar to alterations studied in human biopsy and the SJL/J and A/J mice [53,63,64]. The authors concluded that the pathology in the knockout closely resembles that observed in SJL/J. A second knockout, 129-Dysftm1Kcam/J, has been engineered, where an insertion event removes the last three coding exons [32]. There is a complete loss of dysferlin protein and progressive muscular dystrophy which begins at 2 months of age and affects muscles differentially. Many myofibres showed signs of increased permeability by EBD uptake, however there was no increase in EBD-positive cells following exercise, in marked contrast to the observations in dystrophin-deficient mdx mice (a model of Duchenne muscular dystrophy), where structural weakness results in compromised sarcolemma integrity [65]. This knockout has also been backcrossed onto the C57BL/6 background and are designated B6.129-Dysftm1Kcam/J [43]. There are no reports of significant differences with the original knockout line (Table 1). In summary, the histological changes observed in SJL/J and knockout mice are comparable, displaying many of the features seen in patients, while A/J and BLA/J mice exhibit a milder phenotype with later onset and slower disease progression. Proximal muscles are initially more affected than distal ones, except in the BLA/J mouse, which has a distal bias; pathology spreads in all models to include both proximal and distal musculature. Despite residual dysferlin expression in SJL/J, the dystrophic phenotype is more severe than the null A/J. The residual protein fails to target the sarcolemma and is likely non-functional (unpublished observations; SHL). These strains retain their individual dystrophic phenotypes when backcrossed onto similar genetic backgrounds, suggesting that other genetic factors do not have a major impact on disease initiation and progression. 6. Behavioural and functional studies A wide variety of techniques have been used to study muscle function and force generation. These range from relatively simple tests such as balancing on a rotating rod, to more sensitive and highly technical in situ force measurement protocols [66–68]. Unfortunately, there are few comparative studies were any one functional assay has been performed on a number of dysferlin-deficient strains. The most comprehensive study of behavioural and functional status in a dysferlin-deficient mouse was published in 2010 [67]. SJL/J mice between the ages of 9 and 25 weeks were assessed for a wide variety of parameters, with many of the results demonstrating

muscle dysfunction. Animals weighed less and normalised grip strength was greater than controls. There was a decrease in open field measurements but no difference in rotarod performance (there is also evidence of reduced activity in BLA/J mice, JAXÒ website). In vitro force measurement of extensor digitorum longus (EDL) and soleus at 25 weeks showed that specific force of both muscles was significantly reduced. Although one could argue that C57BL/6 is not the most suitable control, this work, with such rigorous, wide ranging functional analysis may be a model for future strain characterisation, providing valuable baseline measurements that will be essential for future therapeutic studies. 7. Measurement of contractile properties There is a significant body of data from investigators measuring the contractile properties of dysferlin-deficient muscle. Two basic methods have been utilised, in vitro (sometimes described as ex vivo), where the muscle, or isolated individual myofibres, to be analysed are dissected before stimulation [43,69–72], and in situ, where stimulation and force measurement are performed while the muscle remains in its original position, retaining its blood and nerve supply [16,66,73,74]. The muscle is activated and held at a constant length (an isometric contraction) and the force measured; an optimal length will produce a maximum force. Repetitive muscle stimulation of sufficiently short duration results in a summation of force to a plateau, and thus maximum isometric tetanic force is calculated. This tetanic force is normalised to muscle weight or cross-sectional area to produce the specific force. Normalised force measurements in dysferlin-deficient mice have been reported to be higher [75], lower [16], and no different, than controls [43,76]. Comparison is difficult as a variety of different strains, ages and muscles were used, however, the prevailing view is that the contractile apparatus and force generative capacity of skeletal muscle is unaffected by dysferlin-deficiency, particularly at pre-symptomatic disease stages (Table 2). The dysferlin-deficient diaphragm may be an interesting exception, as force generated by this tissue was only 50% of control values [59]. It is likely that the diaphragm of a caged mouse has the largest work load of any muscle and, as the authors suggest, may serve as an indicator of susceptibility to activity-related muscle disease, providing us with a better model of the disease progression in human limb muscles. Pathology in the diaphragm of mdx mice is also more severe than the observed dystrophy in limb muscles, more closely resembling the disease progression in Duchenne patients [69,77]. 8. Force deficit following injury Using the techniques described above, skeletal muscle can be stretched and susceptibility to contraction-induced

M.A. Hornsey et al. / Neuromuscular Disorders 23 (2013) 377–387

injury can be assessed in a very precise and consistent manner. Maximally activated muscle is subjected to a number of lengthening contractions before force deficit and force recovery is measured [78]. A number of protocols have been reported (Table 2). In the main, there is no significant difference in force deficit following lengthening contractions and the contractile apparatus thus appears intact and functional [16,43,75]. This is in marked contrast to dystrophin-deficient muscle, which is highly susceptible to similar injuries [66], indicative of the very different mechanisms of pathology [79]. 9. Injury models, force recovery and muscle regeneration The cellular and molecular events that take place following muscle injury are well documented [80]. However, the rate at which these regenerative events take place in different inbred strains varies, with fast healers such as LG/J recovering almost four times faster from an ear punch than slow healers like BALB/cByJ and SJL/J; A/J is in the middle of this regenerative list [81]. SJL/J muscle has a well documented increased regenerative capacity following muscle crush and isograft injuries, but this superior regenerative capacity is only in comparison with the very slow healing Balb/c mice [82–84]. Data on the regenerative capacity of other dysferlin-deficient strains is limited, although numerous injury protocols have been used, usually in the assessment of therapeutic regimes. Treadmill running and snake venoms are the two most commonly reported [13,16,32,43]. Susceptibility to damage and long-term recovery of dysferlin-deficient muscle following lengthening

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contractions has also been studied. Elegant experiments have allowed investigators to study the susceptibility to damage and the recovery from injury as separate events [75,76,85]. The ankle dorsiflexor muscles of irradiated and non-irradiated A/J mice were subjected to either large or small stretches in the presence of fluorescent dextran (Table 2). Many dysferlin-deficient myofibres subjected to large-strain injury underwent necrosis followed by myogenesis, unlike wild type myofibres that were able to repair without significant inflammation, necrosis or myogenesis. After small-strain injuries, both groups appeared to react similarly to the initial damage (necrosis and replacement by myogenesis) however, force recovery by dysferlin-deficient muscle was delayed. Regeneration in B10.SJL-Dysfim/AwaJ mice is also delayed. Muscle injected with the snake venom notexin, showed reduced neutrophil recruitment, impaired cytokine release, a delay in the removal of necrotic fibres, and a delay in force recovery when compared with C57BL/10 controls [16]. The authors suggest that dysfunctional vesicle fusion events at the sarcolemma disrupt cytokine release and thus communication with infiltrating immune cells, delaying necrotic fibre removal and regenerative events. Due to the slow progression and mild phenotype displayed in dysferlin-deficient mice, protocols that mimic a lifetime of muscle injury and accelerate the dystrophic phenotype will be useful in the development of new therapies. Dysferlin-deficient muscle subjected to repeated cycles of degeneration and regeneration displayed a dystrophic phenotype whereas controls showed complete recovery [16]. However, no delay in necrotic fibre removal

Table 2 Summary of force generation data in dysferlin-deficient mouse models. Strain

Controls

Age/sex

In situ/ Analysed muscle in vitro

A/J

A/WySnJ, 12– In situ C57BL/6 14 weeks/ Male

TA – following contraction of the ankle dorsiflexors

Torque normalised to muscle 15 lengthening weight higher in dysferlincontractions to yield deficient muscles 40% force reduction

A/J

A/WySnJ

TA – following contraction of the ankle dorsiflexors

No difference in A/J and A/ WySnJ contractile torque

12– In situ 16 weeks/ Male

EDL

A/J

A/WySnJ, 10 and C57BL/6, 36 weeks mdx

In vitro

Soleus

Diaphragm

BL10.SJL-Dysfjm/ AwaJ

C57BL/ 10mdx

12– In situ 14 weeks/ Male

TA

B6.129Dysftm1Kcam/J

C57BL/6

2 and 8 months

EDL

a

Lo, Optimal fibre length.

In vitro

Pre-injury force generation

Specific force slightly lower than wild type controls at 10 weeks of age No signficant differences in soleus specific force

Stretch injury

15 repetitive largestrain lengthening contractions 150 repetitive smallstrain lengthening contractions Fqqive lengthening contractions (10% of a Lo) Five lengthening contractions (17.5% of a Lo) Five lengthening contractions (10% of a Lo) Two lengthening contractions (40% of a Lo)

50% reduction in diaphragm specific force at 10 and 36 weeks Specific force slightly lower than controls pre-injury. Maximum isometric twitch and tetanic forces no different from C57BL/10 No difference in specific force Seven lengthening at 2 and 8 months compared contractions (30% of a Lo) to controls

Comments

Reference

A/J and control muscle equally [75] susceptible to lengthening injury. A/J slower to recover to pre-injury levels; requires myogenesis A/J and control muscle equally [76] susceptible to lengthening injury. A/J recovery requires myogenesis. Controls do not Recovery in both A/J and controls requires myogenesis. Slower rate of recovery in A/J EDL – no difference in force deficit compared with controls following lengthening contractions Force drop in 36 week soleus less [59] than 10 week old muscle Force drop in 36 week diaphragm greater than 10 week old diaphragm No difference in force recovery [16] following lengthening contractions

No difference in force deficits

[43]

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was observed following barium chloride injections into dysferlin knockout mice (unpublished observations; MAH). Whether this is the result of the different strains used, or fundamental differences in the nature of the injury when using venom or barium chloride is unknown. Dysferlin-deficient mice have been crossed with strains engineered to express the firefly luciferase gene, controlled by the Pax7 promoter and activated by tamoxifen [28,86]. Pax7-specific expression allows the tracking of muscle satellite cells and regeneration [87,88]. Preliminary results suggest that a lack of dysferlin increases the rate of regeneration in muscle [28]. 10. Cardiac involvement In mouse cardiomyocytes, dysferlin is localised to the intercalated disks, with some residual expression in the sarcoplasm [89]. There is evidence of very mild cardiomyopathy in A/J mice at ten months of age [89]. In the 129-Dysftm1Kcam/J knockout mice, cardiac pathology is extremely mild and only detectable in 56 and 80 week old animals. Further deterioration was observed in 54 week old mice stressed by downhill running, and more severe cardiac dysfunction was recorded in dysferlin/ dystrophin double knockouts [90]. Pathology was more severe than either dysferlin- or dystrophin-deficiency alone. The data suggests that dysferlin plays, at most, a very minimal role in the heart function, and the murine models reflect very closely the human condition. There is limited evidence of cardiac dysfunction in dysferlinopathy patients, although only a small number of affected individuals have been rigorously assessed for signs of cardiomyopathy [91,92]. As the incidence of heart disease in the general population is relatively high, it is difficult to establish any correlation of dysferlin-deficiency with heart disease [93]. A larger set of prospective patient data is likely to become available through a 3-year natural history study that is being conducted by an international consortium in collaboration with the Jain Foundation [94]. 11. Dysferlin over-expression in skeletal muscle Several groups have studied the effects of dysferlin overexpression on muscle physiology. Using muscle-specific promoters, dysferlin levels up to 8 times higher than endogenous appear to have no long term, adverse effect on muscle structure or function [43,95,96]. Mice expressing very high levels (36- or 176-fold above endogenous), showed signs of non-necrotic myopathy, with reduced fibre diameter and a selective loss of fast-twitch fibre types. The very highest levels of over-expression resulted in mice with kyphosis, reduced weight and grip strength, atrophy of the hind limb musculature, and signs of fibrosis and calcification in the heart. CK levels were normal suggesting that sarcolemma integrity was not impaired and that the mechanisms of pathology in dysferlin-deficient and over-expressing lines are different [96]. Is the dysferlin protein cytotoxic when

expressed at very high levels? Massive protein overexpression per se is not necessarily detrimental and is therefore protein dependent. Murine dystrophin expressed at very high levels in mdx mice rescues the disease phenotype [97]. However, c-sarcoglycan, when over-expressed in muscle, forms cytoplasmic aggregates and fails to reach the sarcolemma inducing dystrophy [98]. Mice over-expressing caveolin-3 (Cav-3) develop muscular dystrophy as Cav-3 and dystrophin compete for binding to the same site on bdystroglycan, consequently disrupting the formation of the DGC [99–102]. Dysferlin is not an integral member of the Dystrophin–Glycoprotein Complex (DGC) [32], however, it may form part of another complex of proteins forming at or near the sarcolemma. The exact nature of this “dysferlin complex”, the associated proteins (either spatially or temporally), indeed its very existence, is still a matter of debate. Dysferlin binds AHNAK, calpain-3, caveolin-3, mitsugumin 53 and the annexins A1 and A2 [35,100,101,103– 106], and this list of reported binding partners is increasing [40,107,108]. If such a complex does exist then altering dysferlin stoichiometry may have deleterious consequences. 12. Dysferlin and the immune system Muscle biopsies from dysferlin-deficient patients exhibit a prominent mononuclear cell infiltrate. In an attempt to reduce inflammation and prevent muscle degeneration, a recent clinical trial administered the glucocorticoid Deflazacort to patients with dysferlinopathy. Glucocorticosteroids have been used with consistent benefit in boys with Duchenne, although the benefit may not be due solely to their anti-inflammatory effects [109]. Unfortunately, the Deflazacort trial was halted, as a significant number of patients suffered loss of muscle strength, or did not benefit [110]. This result suggests that dysferlinopathy has a unique immune system involvement, and many groups are now studying the role dysferlin may play in the innate immune and complement systems [16,42,43,45,111]. A number of cell types of the innate immune system express dysferlin, including neutrophils and CD14+ monocytes [112–115], however the role these cell types play, if any, in either initiating or exacerbating disease pathology remains uncertain. Can muscle-specific expression of dysferlin rescue the pathology observed in dysferlin-deficient mice? To address this question, A/J mice were crossed with the Dysf-TG line (over-expressing 3-fold levels of dysferlin in myofibres). Histology in 8 and 11 month old A/JxDysf-TG mice and C57BL/6 controls was similar, as was macrophage infiltration, force generation and CK levels [95]. Rescuing of the phenotype by muscle-specific dysferlin expression, suggests that the primary defect in dysferlinopathy involves the loss of dysferlin in myofibres and that any dysfunction of the immune system does not appear to initiate the pathological process. However, macrophage phagocytic function is certainly affected by a lack of dysferlin [42] and work in our lab is further defining which monocyte sub-

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types express dysferlin and what effect dysferlin-deficiency might have on their differentiation and function. Complement proteins also play a significant role in the development of dysferlinopathy [43–45]. Activation of the complement system initiates a proteolytic cascade, producing proteins that induce pro-inflammatory responses and mark pathogens, cells or foreign particles for clearance by cell lysis or phagocytosis [116]. To prevent inappropriate activation, the system is held in-check by a number of inhibitory proteins, one of which is the DecayAccelerating Factor/CD55. This inhibitory factor is downregulated in the skeletal muscles of dysferlin-deficient mice and patients, possibly making muscle cells more susceptible to the potentially damaging actions of complement proteins [44,45]. Complement factors C1qA and CFB are up-regulated in dysferlin-null line [43]. To further investigate the role complement plays in dysferlinopathy, a dysferlin-null mouse has been crossed with a strain deficient in the central complement component C3; necessary for complement activation [43,117]. Dysferlin/C3 knockout mice have a markedly reduced dystrophic pathology with more uniform myofibre diameters and a greatly reduced number of centronucleated myofibres. Dystrophic phenotype in these mice was not completely rescued, and viewed in the context of the A/JxDysf-TG mice, suggests that myopathy is probably initiated at the level of the muscle fibre, while disease pathology may be exacerbated by immune dysfunction. The same group generated a dysferlin/C5 double knockout. C5 is another complement component, the

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cleavage products of which (C5a and C5b) are potent mediators of inflammation and chemotaxis. C5b initiates the formation of the membrane attack complex (MAC), a pore structure that inserts into target cell membranes causing lysis. Disease pathology in these dysferlin/C5 knockouts was indistinguishable from the dysferlin-nulls [43], providing evidence that cell lysis by the MAC is not central to the development of muscle pathology in these animals. 13. Other dysferlin-deficient models For a number of recently generated dysferlin-deficient mice there is, at this point, only limited data [118–122]. There are also a number of dysferlin-deficient models in non-murine species – see Table 3 [19,123–125]. 14. Conclusions Even this brief review of the various dysferlinopathy mouse models emphasises the variety of models and their variable states of comparison. Different researchers have used a spectrum of tests to evaluate the response of these models to genetic background [43,90,95], different injury models [16,32,33,75] and therapeutic interventions [59,67,119,126]. Unfortunately, it is not possible to state with any clarity which model system or analysis method compares optimally with the human disease state, and where strains have been directly compared, significant differences have been found, even between closely related strains [53,85].

Table 3 Recently described mouse models and non-murine models of dysferlin-deficiency. DysferlinDeficient Model

Description

Muscular dystrophy

Comments

References

BLA/J crossed with aSCID mouse (SCID/ BLAJ) SJL/J crossed with b2microglobulin null mouse C57BL/6JChr6A/J/NaJ (JAXÒ stock number 4384) Zebrafish

Dysferlin-deficient mouse with severe reduction in mature T and B lymphocytes. Innate immune system intact Elimination of MHC class I expression, with dysferlindeficiency

Muscular dystrophy similar to BLA/J. Similar number of central myonuclei and fibrosis in quadriceps at 5–6 months of age

Suggests lymphocytes play no significant role in dystrophy progression

[119]

Myopathy similar to SJL/J (histology of 8– 9 month old quadriceps, TA, gastrocnemius and soleus). Higher numbers of infiltrating Mac-1+ cells than SJL/J. No data

Suggests MHC I does not have key role in development of dysferlin-deficient dystrophy

[120]

Dysferlin-deficiency resulted in muscle abnormalities

Wild type Drosophila express dysferlin in muscle, brain and eye. Possible role in stabilising muscle structure Mutants infertile as fer-1 necessary for fusion of membranous organelles to sperm plasma membrane Mutation affects male and female fertility

Chromosome substitution strain. A/J chromosome 6 (containing dysferlin mutation) on C57BL/6J background Antisense oliogonucleotide morpholino dysferlin knockdown

C. elegans

Mutations of dysferlin homolog fer-1

Dysferlin expressed in body wall muscle cells. Loss of fer-1 does not cause gross sarcomere disorganisation

Drosophila melanogaster

Mutations of the single Drosophila ferlin gene, misfire(mfr)

No data

a

SCID, severe combined immunodeficiency.

[121]

[124]

[19,125]

[123]

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Dysferlin-deficient mice, although far from a perfect model of the human disorder, have provided, and will continue to provide, valuable insight into dysferlin function and the processes that lead to muscular dystrophy. It is clear that dysferlin has a range of functions within the myofibre (and in other tissues and cell types) and consequently the key challenge will be the defining of primary pathogenic mechanisms from compensating secondary effects. Like mdx mice, dysferlin-deficient animals have a mild phenotype, and the slow progression of disease makes functional studies and pre-clinical trials time-consuming and costly. A number of potentially therapeutic treatments have recently been published, including the use of adeno-associated viral vectors [61], antioxidants [126] exon-skipping strategies [127,128], rho-kinase inhibitors [67], stem cell transplantation [119] and the nonsense suppression drug PTC124 [129,130]. Unfortunately, these studies have been conducted on a variety of strains, utilising a variety of functional assays, and making a considered assessment of their potential clinical benefit is difficult. This highlights the urgent need for standardization, particularly in the area of therapeutic testing, and a consensus is required on the dysferlin-deficient strains to be used, the availability of characterisation data, the appropriate controls, the techniques employed, and the outcomes and experimental endpoints that are measured. We have noted considerable inter-individual variation within our dysferlin-deficient colonies, particularly in response to treadmill running and notexin injury. The many factors that lead to biological variation in mdx mice and the issues that need to be considered when standardising breeding methods and analytical techniques have been highlighted [131]. The TREAT-NMD Neuromuscular Network has a list of unifying protocols, standardized operating procedures (SOPs), currently maintained for the mdx mouse, the Golden Retriever Muscular Dystrophy (GRMD) dog (models of Duchenne), the spinal muscular atrophy (SMA) mouse and congenital muscular dystrophy (CMD) mouse [132]. Such protocols improve the comparability of studies and maximize efficiency, and their use is now actively sought by funding bodies and journal editors. We believe a similar series of SOPs should be set up in the field of dysferlin research, assisting researchers in their efforts to translate preclinical therapeutic strategies from the lab to the clinic.

Abbreviations Cav-3, caveolin-3; CK, creatine kinase; CMD, congenital muscular dystrophy; DGC, dystrophin– glycoprotein complex; EBD, Evan’s blue dye; EDL, extensor digitorum longus; GRMD, golden retriever muscular dystrophy; JAXÒ, The Jackson Laboratory; LGMD, limb girdle muscular dystrophy; MAC,

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