Transgenic mice expressing mutant caveolin-3 show severe myopathy associated with increased nNOS activity

Caveolin-3 is the muscle-specific isoform of the caveolin protein family, which is a major component of caveolae, small membrane invaginations found in most cell types. Caveolins play important roles in the formation of caveola membranes, acting as scaffolding proteins to organize and concentrate lipid-modified signaling molecules, and modulate a signaling pathway. For instance, caveolin-3 interacts with neuronal nitric oxide synthase (nNOS) and inhibits its catalytic activity. Recently, specific mutations in the caveolin-3 gene, including the Pro104Leu missense mutation, have been shown to cause an autosomal dominant limb-girdle muscular dystrophy (LGMD1C), which is characterized by the deficiency of caveolin-3 in the sarcolemma. However, the molecular mechanism by which these mutations cause the deficiency of caveolin-3 and muscle cell degeneration remains elusive. Here we generated transgenic mice expressing the Pro104Leu mutant caveolin-3. They showed severe myopathy accompanied by the deficiency of caveolin-3 in the sarcolemma, indicating a dominant negative effect of mutant caveolin-3. Interestingly, we also found a great increase of nNOS activity in their skeletal muscle, which, we propose, may play a role in muscle fiber degeneration in caveolin-3 deficiency.     

Overexpression of P104L mutant caveolin-3 in mice develops hypertrophic cardiomyopathy with enhanced contractility in association with increased endothelial nitric oxide synthase activity

The effect of endogenous nitric oxide synthase (NOS) on cardiac contractility and architecture has been a matter of debate. A role for NOS in cardiac hypertrophy has recently been demonstrated by studies which have shown hypertrophic cardiomyopathy (HCM) with altered contractility in constitutive NOS (cNOS) knockout mice. Caveolin-3, a strong inhibitor of all NOS isoforms, is expressed in sarcolemmal caveolae microdomains and binds to cNOS in vivo: endothelial nitric oxide synthase (eNOS) in cardiac myocytes and neuronal nitric oxide synthase (nNOS) in skeletal myocytes. The current study characterized the biochemical and cardiac parameters of P104L mutant caveolin-3 transgenic mice, a model of an autosomal dominant limb-girdle muscular dystrophy (LGMD1C). Transgenic mouse hearts demonstrated HCM, enhanced basal contractility, decreased left ventricular end diastolic diameter, and loss and cytoplasmic mislocalization of caveolin-3 protein. Surprisingly, cardiac muscle showed activation of eNOS catalytic activity without increased expression of all NOS isoforms. These data suggest that a moderate increase in eNOS activity associated with loss of caveolin-3 results in HCM.     

Fukutin-related protein mutations that cause congenital muscular dystrophy result in ER-retention of the mutant protein in cultured cells

Mutations in the gene encoding fukutin-related protein (FKRP) cause a spectrum of diseases including congenital muscular dystrophy type 1C (MDC1C), limb girdle muscular dystrophy 2I (LGMD2I) and congenital muscular dystrophies (CMDs) with brain malformations and mental retardation. Although these diseases are associated with abnormal dystroglycan processing, the cellular consequences of the idiosyncratic FKRP mutations have not been determined. Here we show, in cultured cells, that FKRP mutants associated with the more severe disease phenotypes (S221R, A455D, P448L) are retained in the endoplasmic reticulum (ER), whereas the wild-type protein and the mutant L276I that causes LGMD2I are found predominantly in the Golgi apparatus. The ER-retained proteins have a shorter half-life than the wild-type FKRP and are preferentially degraded by the proteasome. Furthermore, calnexin binds preferentially to the ER-retained mutants suggesting that it may participate in the quality control pathway for FKRP. These data provide the first evidence that the ER-retention of mutant FKRP may play a role in the pathogenesis of CMD and potentially explain why the allelic disorder LGMD2I is milder, because the mutated protein is able to reach the Golgi apparatus.     

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

A defect of the gene for p94 (calpain 3), a skeletal muscle-specific calpain, is responsible for limb girdle muscular dystrophy type 2A (LGMD2A), or 'calpainopathy', which is an autosomal recessive and progressive neuromuscular disorder. To study the relationships between the physiological functions of p94 and the etiology of LGMD2A, we created transgenic mice that express an inactive mutant of p94, in which the active site Cys129 is replaced by Ser (p94:C129S). Three lines of transgenic mice expressing p94:C129S mRNA at various levels showed significantly decreased grip strength. Sections of soleus and extensor digitorum longus (EDL) muscles of the aged transgenic mice showed increased numbers of lobulated and split fibers, respectively, which are often observed in limb girdle muscular dystrophy muscles. Centrally placed nuclei were also frequently found in the EDL muscle of the transgenic mice, whereas wild-type mice of the same age had almost none. There was more p94 protein produced in aged transgenic mice muscles and it showed significantly less autolytic degradation activity than that of wild-type mice. Although no necrotic-regenerative fibers were observed, the age and p94:C129S expression dependence of the phenotypes strongly suggest that accumulation of p94:C129S protein causes these myopathy phenotypes. The p94:C129S transgenic mice could provide us with crucial information on the molecular mech-anism of LGMD2A.     

Caveolin-3 deficiency causes muscle degeneration in mice

Caveolin-3 is a muscle-specific protein integrated in the caveolae, which are small invaginations of the plasma membrane. Mutations of the caveolin-3 gene, localized at 3p25, have been reported to be involved in the pathogenesis of limb-girdle muscular dystrophy (LGMD1C or caveolinopathy) with mild clinical symptoms, inherited through an autosomal dominant form of genetic transmission. To elucidate the pathogenetic mechanism, we developed caveolin-3-deficient mice for use as animal models of caveolinopathy. Caveolin-3 mRNA and its protein were absent in homozygous mutant mice. In heterozygous mutant mice, both the mRNA and its protein were normal in size, but their amounts were reduced by about half. The density of caveolae in skeletal muscle plasma membrane was roughly proportional to the amount of caveolin-3. In homozygous mutant mice, muscle degeneration was recognized in soleus muscle at 8 weeks of age and in the diaphragm from 8 to 30 weeks, although there was no difference in growth and movement between wild-type and mutant mice. No apparent muscle degeneration was observed in heterozygous mutant mice, indicating that pathological changes caused by caveolin-3 gene disruption were inherited through the recessive form of genetic transmission.     

Asymptomatic carriers for homozygous novel mutations in the FKRP gene: the other end of the spectrum

Autosomal recessive limb-girdle muscular dystrophy linked to 19q13.3 (LGMD2I) was recently related to mutations in the fukutin-related protein gene (FKRP) gene. Pathogenic changes in the same gene were detected in congenital muscular dystrophy patients (MDC1C), a severe disorder. We have screened 86 LGMD genealogies to assess the frequency and distribution of mutations in the FKRP gene in Brazilian LGMD patients. We found 13 Brazilian genealogies, including 20 individuals with mutations in the FKRP gene, and identified nine novel pathogenic changes. The commonest C826A European mutation was found in 30% (9/26) of the mutated LGMD2I alleles. One affected patient homozygous for the FKRP (C826A) mutation also carries a missense R125H change in one allele of the caveolin-3 gene (responsible for LGMD1C muscular dystrophy). Two of her normal sibs were found to be double heterozygotes. In two unrelated LGMD2I families, homozygous for novel missense mutations, we identified four asymptomatic carriers, all older than 20 years. Genotype-phenotype correlation studies in the present study as well as in patients from different populations suggests that the spectrum of variability associated with mutations in the FKRP gene seems to be wider than in other forms of LGMD. It also reinforces the observations that pathogenic mutations are not always determinant of an abnormal phenotype, suggesting the possibility of other mechanisms modulating the severity of the phenotype that opens new avenues for therapeutic approaches.     

Transgenic overexpression of caveolin-3 in the heart induces a cardiomyopathic phenotype

Caveolins are structural protein components of caveolar membrane domains. Caveolin-3, a muscle-specific member of the caveolin family, is expressed in skeletal muscle tissue and in the heart. The multiple roles that caveolin-3 plays in cellular physiology are becoming more apparent. We have shown that lack of caveolin-3 expression in skeletal muscle resembles limb-girdle muscular dystrophy-1C. In contrast, we have demonstrated that overexpression of caveolin-3 in skeletal muscle tissue promotes defects similar to those seen in Duchenne muscular dystrophy (DMD). Thus, a tight regulation of caveolin-3 expression is fundamental for normal muscle functions. Since caveolin-3 is also endogenously expressed in cardiac myocytes, and cardiomyopathies are observed in DMD patients, we looked at the effects of overexpression of caveolin-3 on cardiac structure and function by characterizing caveolin-3 transgenic mice. Our results indicate that overexpression of caveolin-3 causes severe cardiac tissue degeneration, fibrosis and a reduction in cardiac functions. We also show that dystrophin and its associated glycoproteins are down-regulated in caveolin-3 transgenic heart. In addition, we demonstrate that the activity of nitric oxide synthase (NOS) is down-regulated by high levels of caveolin-3 in the heart. Taken together, these results indicate that overexpression of caveolin-3 is sufficient to induce severe cardiomyopathy. In addition, these findings suggest that caveolin-3 transgenic mice may represent a valid mouse model for studying the molecular mechanisms underlying cardiomyopathies associated with Duchenne muscular dystrophy.     

Expression of the muscular dystrophy-associated caveolin-3P104L mutant in adult mouse skeletal muscle specifically alters the Ca2+ channel function of the dihydropyridine receptor

Caveolins are plasma-membrane-associated proteins potentially involved in a variety of signalling pathways. Different mutations in CAV3, the gene encoding for the muscle-specific isoform caveolin-3 (Cav-3), lead to muscle diseases, but the underlying molecular mechanisms remain largely unknown. Here, we explored the functional consequences of a Cav-3 mutation (P104L) inducing the 1C type limb-girdle muscular dystrophy (LGMD 1C) in human on intracellular Ca²⁺ regulation of adult skeletal muscle fibres. A YFP-tagged human Cav-3^P104L mutant was expressed in vivo in muscle fibres from mouse. Western blot analysis revealed that expression of this mutant led to an ∼80% drop of the level of endogenous Cav-3. The L-type Ca²⁺ current density was found largely reduced in fibres expressing the Cav-3^P104L mutant, with no change in the voltage dependence of activation and inactivation. Interestingly, the maximal density of intramembrane charge movement was unaltered in the Cav-3^P104L-expressing fibres, suggesting no change in the total amount of functional voltage-sensing dihydropyridine receptors (DHPRs). Also, there was no obvious alteration in the properties of voltage-activated Ca²⁺ transients in the Cav-3^P104L-expressing fibres. Although the actual role of the Ca²⁺ channel function of the DHPR is not clearly established in adult skeletal muscle, its specific alteration by the Cav-3^P104L mutant suggests that it may be involved in the physiopathology of LGMD 1C. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Loss of caveolin-3 induced by the dystrophy-associated P104L mutation impairs L-type calcium channel function in mouse skeletal muscle cells

Caveolins are membrane scaffolding proteins that associate with and regulate a variety of signalling proteins, including ion channels. A deficiency in caveolin-3 (Cav-3), the major striated muscle isoform, is responsible for skeletal muscle disorders, such as limb-girdle muscular dystrophy 1C (LGMD 1C). The molecular mechanisms leading to the muscle wasting that characterizes this pathology are poorly understood. Here we show that a loss of Cav-3 induced by the expression of the LGMD 1C-associated mutant P104L (Cav-3^P104L) provokes a reduction by half of the maximal conductance of the voltage-dependent L-type Ca²⁺ channel in mouse primary cultured myotubes and fetal skeletal muscle fibres. Confocal immunomiscrocopy indicated a colocalization of Cav-3 and Ca_v1.1, the pore-forming subunit of the L-type Ca²⁺ channel, at the surface membrane and in the developing T-tubule network in control myotubes and fetal fibres. In myotubes expressing Cav-3^P104L, the loss of Cav-3 was accompanied by a 66% reduction in Ca_v1.1 mean labelling intensity. Our results suggest that Cav-3 is involved in L-type Ca²⁺ channel membrane function and localization in skeletal muscle cells and that an alteration of L-type Ca²⁺ channels could be involved in the physiopathological mechanisms of caveolinopathies.     

Molecular and muscle pathology in a series of caveolinopathy patients

Mutations in the caveolin-3 gene (CAV3) cause limb girdle muscular dystrophy (LGMD) type 1C (LGMD1C) and other muscle phenotypes. We screened 663 patients with various phenotypes of unknown etiology, for caveolin-3 protein deficiency, and we identified eight unreported caveolin-deficient patients (from seven families) in whom four CAV3 mutations had been detected (two are unreported). Following our wide screening, we estimated that caveolinopathies are 1% of both unclassified LGMD and other phenotypes, and demonstrated that caveolin-3 protein deficiency is a highly sensitive and specific marker of primary caveolinopathy. This is the largest series of caveolinopathy families in whom the effect of gene mutations has been analyzed for protein level and phenotype. We showed that the same mutation could lead to heterogeneous clinical phenotypes and muscle histopathological changes. To study the role of the Golgi complex in the pathological pathway of misfolded caveolin-3 oligomers, we performed a histopathological study on muscle biopsies from caveolinopathy patients. We documented normal caveolin-3 immunolabeling at the plasmalemma in some regenerating fibers showing a proliferation of the Golgi complex. It is likely that caveolin-3 overexpression occurring in regenerating fibers (compared with caveolin-deficient adult fibers) may lead to an accumulation of misfolded oligomers in the Golgi and to its consequent proliferation.     

Dissociation of the dystroglycan complex in caveolin-3-deficient limb girdle muscular dystrophy

Limb girdle muscular dystrophy is a group of clinically and genetically heterogeneous disorders inherited in an autosomal recessive or dominant mode. Caveolin-3, the muscle-specific member of the caveolin gene family, is implicated in the pathogenesis of autosomal dominant limb girdle muscular dystrophy 1C. Here we report on a 4-year-old girl presenting with myalgia and muscle cramps due to a caveolin-3 deficiency in her dystrophic skeletal muscle as a result of a heterozygous 136G-->A substitution in the caveolin-3 gene. The novel sporadic missense mutation in the caveolin signature sequence of the caveolin-3 gene changes an alanine to a threonine (A46T) and prevents the localization of caveolin-3 to the plasma membrane in a dominant negative fashion. Caveolin-3 has been suggested to interact with the dystrophin-glycoprotein complex, which in striated muscle fibers links the cytoskeleton to the extracellular matrix and with neuronal nitric oxide synthase. Similar to dystrophin-deficient Duchenne muscular dystrophy, a secondary decrease in neuronal nitric oxide synthase and alpha-dystroglycan expression was detected in the caveolin-3-deficient patient. These results implicate an important function of the caveolin signature sequence and common mechanisms in the pathogenesis of dystrophin-glycoprotein complex-associated muscular dystrophies with caveolin-3-deficient limb girdle muscular dystrophy.     

The distribution and characterization of skeletal muscle lesions in dysferlin-deficient SJL and A/J mice

The pathogenesis of limb-girdle muscular dystrophy type 2B (LGMD2B) dysferlinopathy remains to be investigated. The distribution and characterization of skeletal muscle lesions were examined in two different LGMD2B mouse models, SJL and A/J mice (at 10 and 35 weeks old), in association with the endoplasmic reticulum (ER) stress. SJL mice showed an earlier age of onset and a faster progression of skeletal muscle lesions as compared with those of A/J mice; the sensitivity difference to muscular dystrophic lesions between SJL and A/J mice was observed in the lumbar muscles (particularly, lumbar longissimus and sublumbar muscles); the lesions seen mainly in SJL mice at 35 weeks old consisted of degeneration, necrosis, fatty infiltration, variation in muscle fiber size and atrophy in muscle fibers. Enzyme-histochemically, the fast-twitch muscle fiber was predominant for the degenerative changes seen in the rectus femoris and lateral longissimus muscles of SJL mice. Immunohistochemically, the main reactive cell type observed in and around degenerative and/or necrotic muscle fibers was macrophages, demonstrable with an anti-F4/80 antibody. Because the analyses of spliced XBP1 mRNA, a marker of ER stress, did not show the increased expression, it was considered that ER stress did not affect the progression of skeletal muscle lesions in SJL mice with the advanced stage of dysferlinopathy.     

Biochemical and ultrastructural evidence of endoplasmic reticulum stress in LGMD2I

Limb girdle muscular dystrophy type 2I (LGMD2I) is due to mutations in the fukutin-related protein gene (FKRP), encoding a putative glycosyltransferase involved in alpha-dystroglycan processing. To further characterize the molecular pathogenesis of LGMD2I, we conducted a histological, immunohistochemical, ultrastructural and molecular analysis of ten muscle biopsies from patients with molecularly diagnosed LGMD2I. Hypoglycosylation of alpha-dystroglycan was observed in all FKRP-mutated patients. Muscle histopathology was consistent with either severe muscular dystrophy or myopathy with a mild inflammatory response consisting of up-regulation of class I major histocompatibility complex in skeletal muscle fibers and small foci of mononuclear cells. At the ultrastructural level, muscle fibers showed focal thinning of basal lamina and swollen endoplasmic reticulum cisternae with membrane re-arrangement. The pathways of the unfolded protein response (UPR; glucose-regulated protein 78 and CHOP) were significantly activated in LGMD2I muscle tissue. Our data suggest that the UPR response is activated in LGMD2I muscle biopsies, and the observed histopathological and ultrastructural alterations may be related to sarcoplasmic structures involved in FKRP and alpha-dystroglycan metabolism and malfunctioning.     

Mutations in the caveolin-3 gene: When are they pathogenic?

Limb-girdle muscular dystrophies (LGMD) are a heterogeneous group of genetic disorders usually with autosomal recessive (AR) inheritance and, less often, displaying autosomal dominant (AD) inheritance. Mutations in the caveolin-3 gene (CAV-3) associated with a reduction of protein expression cause AD-LGMD1C muscular dystrophy. Based on a previous study in the American and Brazilian population, it has been suggested that CAV-3 mutations might also cause AR-LGMD. Here we report the analysis of the CAV-3 gene in 61 additional Brazilian LGMD patients and 100 additional Brazilian normal controls. Two rare G55S and C71W missense changes previously detected only in LGMD patients (and not detected in 100 normal controls from the American population) were now found in normal Brazilian controls. In addition, we have identified a novel R125H missense change in one LGMD female patient that was also found in two of her unaffected siblings. These observations, together with the normal immunofluorescence caveolin pattern in the muscle biopsy from two patients with the G55W and R125H changes in the CAV-3 gene suggest that the G55S, C71W, and R125H polymorphisms, on their own, are not sufficient to produce the pathology.     

Two endoplasmic reticulum-associated degradation (ERAD) systems for the novel variant of the mutant dysferlin: ubiquitin/proteasome ERAD(I) and autophagy/lysosome ERAD(II)

Dysferlin is a type-II transmembrane protein and the causative gene of limb girdle muscular dystrophy type 2B and Miyoshi myopathy (LGMD2B/MM), in which specific loss of dysferlin labeling has been frequently observed. Recently, a novel mutant (L1341P) dysferlin has been shown to aggregate in the muscle of the patient. Little is known about the relationship between degradation of dysferlin and pathogenesis of LGMD2B/MM. Here, we examined the degradation of normal and mutant (L1341P) dysferlin. Wild-type (wt) dysferlin mainly localized to the ER/Golgi, associated with retrotranslocon, Sec61alpha, and VCP(p97), and was degraded by endoplasmic reticulum (ER)-associated degradation system (ERAD) composed of ubiquitin/proteasome. In contrast, mutant dysferlin spontaneously aggregated in the ER and induced eukaryotic translation initiation factor 2alpha (eIF2alpha) phosphorylation and LC3 conversion, a key step for autophagosome formation, and finally, ER stress cell death. Unlike proteasome inhibitor, E64d/pepstatin A, inhibitors of lysosomal proteases did not stimulate the accumulation of the wt-dysferlin, but stimulated aggregation of mutant dysferlin in the ER. Furthermore, deficiency of Atg5 and dephosphorylation of eIF2alpha, key molecules for LC3 conversion, also stimulated the mutant dysferlin aggregation in the ER. Rapamycin, which induces eIF2alpha phosphorylation-mediated LC3 conversion, inhibited mutant dysferlin aggregation in the ER. Thus, mutant dysferlin aggregates in the ER-stimulated autophagosome formation to engulf them via activation of ER stress-eIF2alpha phosphorylation pathway. We propose two ERAD models for dysferlin degradation, ubiquitin/proteasome ERAD(I) and autophagy/lysosome ERAD(II). Mutant dysferlin aggregates on the ER are degraded by the autophagy/lysosome ERAD(II), as an alternative to ERAD(I), when retrotranslocon/ERAD(I) system is impaired by these mutant aggregates.     

Identification of lamin A/C ( LMNA) gene mutations in Korean patients with autosomal dominant Emery-Dreifuss muscular dystrophy and limb-girdle muscular dystrophy 1B

Mutations in the LMNA gene encoding lamins A and C by alternative splicing have been found to cause at least four different kinds of genetic disorders: autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD2; MIM 181350); limb-girdle muscular dystrophy type 1B (LGMD1B; MIM 159001); dilated cardiomyopathy type 1A (CMD1A; MIM 115200); and familial partial lipodystrophy (FPLD; MIM 151660). Recently, we have studied two Korean patients with atrioventricular conduction defects. They had variable extents of muscular dystrophy; one patient was diagnosed with EDMD2 and the other with LGMD1B. We performed a mutation analysis of the LMNA gene by direct sequencing and found two different missense mutations: R249Q and R377L, in the EDMD2 and LGMD1B patient, respectively. The R249Q mutation is located within the central rod domain of the LMNA gene, and has been described in at least five unrelated sporadic EDMD2 patients. On the other hand, the R377L mutation, also located within the rod domain, is a novel mutation, although a histidine substitution instead of leucine (R377H) has been reported previously in an LGMD1B patient. To our knowledge, this is the first report of LMNA gene mutations in Korean patients with EDMD2 and LGMD1B. Received: November 19, 2001 / Accepted: February 8, 2002     

Transgenic mice expressing the myotilin T57I mutation unite the pathology associated with LGMD1A and MFM

Myotilin is a muscle-specific Z-disc protein with putative roles in myofibril assembly and structural upkeep of the sarcomere. Several myotilin point mutations have been described in patients with limb-girdle muscular dystrophy type 1A (LGMD1A), myofibrillar myopathy (MFM), spheroid body myopathy (SBM), three similar adult-onset, progressive and autosomal dominant muscular dystrophies. To further investigate myotilin's role in the pathogenesis of these muscle diseases, we have characterized three independent lines of transgenic mice expressing mutant (T57I) myotilin under the control of the human skeletal actin promoter. Similar to LGMD1A and MFM patients, these mice develop progressive myofibrillar pathology that includes Z-disc streaming, excess myofibrillar vacuolization and plaque-like myofibrillar aggregation. These aggregates become progressively larger and more numerous with age. We show that the mutant myotilin protein properly localizes to the Z-disc and also heavily populates the aggregates, along with several other Z-disc associated proteins. Whole muscle physiological analysis reveals that the extensor digitorum longus muscle of transgenic mice exhibits significantly reduced maximum specific isometric force compared with littermate controls. Intriguingly, the soleus and diaphragm muscles are spared of any abnormal myopathology and show no reductions in maximum specific force. These data provide evidence that myotilin mutations promote aggregate-dependent contractile dysfunction. In sum, we have established a promising patho-physiological mouse model that unifies the phenotypes of LGMD1A, MFM and SBM.     

Impairment of Caveolae Formation and T-System Disorganization in Human Muscular Dystrophy with Caveolin-3 Deficiency

Caveolin-3, a muscle specific caveolin-related protein, is the principal structural protein of caveolar membranes. We have recently identified an autosomal dominant form of limb girdle muscular dystrophy (LGMD-1C) that is due to caveolin-3 deficiency and caveolin-3 gene mutations. Here, we studied by electron microscopy, including freeze-fracture and lanthanum staining, the distribution of caveolae and the organization of the T-tubule system in caveolin-3 deficient human muscle fibers. We found a severe impairment of caveolae formation at the muscle cell surface, demonstrating that caveolin-3 is essential for the formation and organization of caveolae in muscle fibers. In addition, we also detected a striking disorganization of the T-system openings at the sub-sarcolemmal level in LGMD-1C muscle fibers. These observations provide new perspectives in our understanding of the role of caveolin-3 in muscle and of the pathogenesis of muscle weakness in caveolin-3 deficient muscle.     

Diagnosis and cell-based therapy for Duchenne muscular dystrophy in humans,mice, and zebrafish

The muscular dystrophies are a heterogeneous group of genetically caused muscle degenerative disorders. The Kunkel laboratory has had a longstanding research program into the pathogenesis and treatment of these diseases. Starting with our identification of dystrophin as the defective protein in Duchenne muscular dystrophy (DMD), we have continued our work on normal dystrophin function and how it is altered in muscular dystrophy. Our work has led to the identification of the defective genes in three forms of limb girdle muscular dystrophy (LGMD) and a better understanding of how muscle degenerates in many of the different dystrophies. The identification of mutations causing human forms of dystrophy has lead to improved diagnosis for patients with the disease. We are continuing to improve the molecular diagnosis of the dystrophies and have developed a high-throughput sequencing approach for the low-cost rapid diagnosis of all known forms of dystrophy. In addition, we are continuing to work on therapies using available animal models. Currently, there are a number of mouse models of the human dystrophies, the most notable being the mdx mouse with dystrophin deficiency. These mice are being used to test possible therapies, including stem-cell-based approaches. We have been able to systemically deliver human dystrophin to these mice via the arterial circulation and convert 8% of dystrophin-deficient fibers to fibers expressing human dystrophin. We are now expanding our research to identify new forms of LGMD by analyzing zebrafish models of muscular dystrophy. Currently, we have 14 different zebrafish mutants exhibiting various phenotypes of muscular dystrophy, including muscle weakness and inactivity. One of these mutants carries a stop codon mutation in dystrophin, and we have recently identified another carrying a mutation in titin. We are currently positionally cloning the disease-causative mutation in the remaining 12 mutant strains. We hope that one of these new mutant strains of fish will have a mutation in a gene not previously implicated in human muscular dystrophy. This gene would become a candidate gene to be analyzed in patients which do not carry a mutation in any of the known dystrophy-associated genes. By studying both disease pathology and investigating potential therapies, we hope to make a positive difference in the lives of people living with muscular dystrophy.