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.     

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.     

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.     

Caveolin-3 in muscular dystrophy

The dystrophin-glycoprotein complex (DGC) serves as a link between cytoplasmic actin, the membrane and the extracellular matrix of striated muscle. Genetic defects in genes encoding a subset of DGC proteins result in muscular dystrophy and a secondary decrease in other DGC proteins. Caveolae are dynamic structures that have been implicated in a number of functions including endocytosis, potocytosis and signal transduction. Caveolin (VIP-21) is thought to play a structural role in the formation of non-clathrin-coated vesicles in a number of different cell types. Caveolin-3, or M-caveolin, was identified as a muscle- specific form of the caveolin family. We show that caveolin-3 co- purifies with dystrophin, and that a fraction of caveolin-3 is a dystrophin-associated protein. We isolated the gene for human caveolin- 3 and mapped it to chromosome 3p25. We determined the genomic organization of human caveolin-3 and devised a screening strategy to look for mutations in caveolin-3 in patients with muscular dystrophy. Of 82 patients screened, two nucleotide changes were found that resulted in amino acid substitutions (G55S and C71W); these changes were not seen in a control population. The amino acid changes map to a functionally important domain in caveolin-3, suggesting that these are not benign polymorphisms and instead are disease-causing mutations.      

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.     

Caveolinopathies: from the biology of caveolin-3 to human diseases

In muscle tissue the protein caveolin-3 forms caveolae – flask-shaped invaginations localized on the cytoplasmic surface of the sarcolemmal membrane. Caveolae have a key role in the maintenance of plasma membrane integrity and in the processes of vesicular trafficking and signal transduction. Mutations in the caveolin-3 gene lead to skeletal muscle pathology through multiple pathogenetic mechanisms. Indeed, caveolin-3 deficiency is associated to sarcolemmal membrane alterations, disorganization of skeletal muscle T-tubule network and disruption of distinct cell-signaling pathways. To date, there have been 30 caveolin-3 mutations identified in the human population. Caveolin-3 defects lead to four distinct skeletal muscle disease phenotypes: limb girdle muscular dystrophy, rippling muscle disease, distal myopathy, and hyperCKemia. In addition, one caveolin-3 mutant has been described in a case of hypertrophic cardiomyopathy. Many patients show an overlap of these symptoms and the same mutation can be linked to different clinical phenotypes. This variability can be related to additional genetic or environmental factors. This review will address caveolin-3 biological functions in muscle cells and will describe the muscle and heart disease phenotypes associated with caveolin-3 mutations.     

Lamin A/C truncation in dilated cardiomyopathy with conduction disease

Background  

Mutations in the gene encoding the nuclear membrane protein lamin A/C have been associated with at least 7 distinct diseases including autosomal dominant dilated cardiomyopathy with conduction system disease, autosomal dominant and recessive Emery Dreifuss Muscular Dystrophy, limb girdle muscular dystrophy type 1B, autosomal recessive type 2 Charcot Marie Tooth, mandibuloacral dysplasia, familial partial lipodystrophy and Hutchinson-Gilford progeria.     

mdx muscle pathology is independent of nNOS perturbation

In skeletal muscle, neuronal nitric oxide synthase (nNOS) is anchored to the sarcolemma via the dystrophin-glycoprotein complex. When dystrophin is absent, as in Duchenne muscular dystrophy patients and in mdx mice, nNOS is mislocalized to the interior of the muscle fiber where it continues to produce nitric oxide. This has led to the hypothesis that free radical toxicity from mislocalized nNOS may contribute to mdx muscle pathology. To test this hypothesis directly, we generated mice devoid of both nNOS and dystrophin. Overall, the nNOS- dystrophin null mice maintained the dystrophic characteristics of mdx mice. We evaluated the mice for several features of the dystrophic phenotype, including membrane damage and muscle morphology. Removal of nNOS did not alter the extent of sarcolemma damage, which is a hallmark of the dystrophic phenotype. Furthermore, muscle from nNOS-dystrophin null mice maintain the histological features of mdx pathology. Our results demonstrate that relocalization of nNOS to the cytosol does not contribute significantly to mdx pathogenesis.      

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. © 2001 Wiley‐Liss, Inc.     

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.     

An inhibitor of transforming growth factor beta type I receptor ameliorates muscle atrophy in a mouse model of caveolin 3-deficient muscular dystrophy

Skeletal muscle expressing Pro104Leu mutant caveolin 3 (CAV3(P104L)) in mouse becomes atrophied and serves as a model of autosomal dominant limb-girdle muscular dystrophy 1C. We previously found that caveolin 3-deficient muscles showed activated intramuscular transforming growth factor beta (TGF-β) signals. However, the cellular mechanism by which loss of caveolin 3 leads to muscle atrophy is unknown. Recently, several small-molecule inhibitors of TGF-β type I receptor (TβRI) kinase have been developed as molecular-targeting drugs for cancer therapy by suppressing intracellular TGF-β1, -β2, and -β3 signaling. Here, we show that a TβRI kinase inhibitor, Ki26894, restores impaired myoblast differentiation in vitro caused by activin, myostatin, and TGF-β1, as well as CAV3(P104L). Oral administration of Ki26894 increased muscle mass and strength in vivo in wild-type mice, and improved muscle atrophy and weakness in the CAV3(P104L) mice. The inhibitor restored the number of satellite cells, the resident stem cells of adult skeletal muscle, with suppression of the increased phosphorylation of Smad2, an effector, and the upregulation of p21 (also known as Cdkn1a), a target gene of the TGF-β family members in muscle. These data indicate that both TGF-β-dependent reduction in satellite cells and impairment of myoblast differentiation contribute to the cellular mechanism underlying caveolin 3-deficient muscle atrophy. TβRI kinase inhibitors could antagonize the activation of intramuscular anti-myogenic TGF-β signals, thereby providing a novel therapeutic rationale for the alternative use of this type of anticancer drug in reversing muscle atrophy in various clinical settings.     

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.     

Caveolin-3 T78M and T78K missense mutations lead to different phenotypes in vivo and in vitro

Caveolins are the principal protein components of caveolae, invaginations of the plasma membrane involved in cell signaling and trafficking. Caveolin-3 (Cav-3) is the muscle-specific isoform of the caveolin family and mutations in the CAV3 gene lead to a large group of neuromuscular disorders. In unrelated patients, we identified two distinct CAV3 mutations involving the same codon 78. Patient 1, affected by dilated cardiomyopathy and limb girdle muscular dystrophy (LGMD)-1C, shows an autosomal recessive mutation converting threonine to methionine (T78M). Patient 2, affected by isolated familiar hyperCKemia, shows an autosomal dominant mutation converting threonine to lysine (T78K). Cav-3 wild type (WT) and Cav-3 mutations were transiently transfected into Cos-7 cells. Cav-3 WT and Cav-3 T78M mutant localized at the plasma membrane, whereas Cav-3 T78K was retained in a perinuclear compartment. Cav-3 T78K expression was decreased by 87% when compared with Cav-3 WT, whereas Cav-3 T78M protein levels were unchanged. To evaluate whether Cav-3 T78K and Cav-3 T78M mutants behaved with a dominant negative pattern, Cos-7 cells were cotransfected with green fluorescent protein (GFP)-Cav-3 WT in combination with either mutant or WT Cav-3. When cotransfected with Cav-3 WT or Cav-3 T78M, GFP-Cav-3 WT was localized at the plasma membrane, as expected. However, when cotransfected with Cav-3 T78K, GFP-Cav-3 WT was retained in a perinuclear compartment, and its protein levels were reduced by 60%, suggesting a dominant negative action. Accordingly, Cav-3 protein levels in muscles from a biopsy of patient 2 (T78K mutation) were reduced by 80%. In conclusion, CAV3 T78M and T78K mutations lead to distinct disorders showing different clinical features and inheritance, and displaying distinct phenotypes in vitro.     

Lamin A N-terminal phosphorylation is associated with myoblast activation: impairment in Emery-Dreifuss muscular dystrophy

Background: Skeletal muscle disorders associated with mutations of lamin A/C gene include autosomal Emery–Dreifuss muscular dystrophy and limb girdle muscular dystrophy 1B. The pathogenic mechanism underlying these diseases is unknown. Recent data suggest an impairment of signalling mechanisms as a possible cause of muscle malfunction. A molecular complex in muscle cells formed by lamin A/C, emerin, and nuclear actin has been identified. The stability of this protein complex appears to be related to phosphorylation mechanisms.

Objective: To analyse lamin A/C phosphorylation in control and laminopathic muscle cells.

Methods: Lamin A/C N-terminal phosphorylation was determined in cultured mouse myoblasts using a specific antibody. Insulin treatment of serum starved myoblast cultures was carried out to evaluate involvement of insulin signalling in the phosphorylation pathway. Screening of four Emery–Dreifuss and one limb girdle muscular dystrophy 1B cases was undertaken to investigate lamin A/C phosphorylation in both cultured myoblasts and mature muscle fibres.

Results: Phosphorylation of lamin A was observed during myoblast differentiation or proliferation, along with reduced lamin A/C phosphorylation in quiescent myoblasts. Lamin A N-terminus phosphorylation was induced by an insulin stimulus, which conversely did not affect lamin C phosphorylation. Lamin A/C was also hyperphosphorylated in mature muscle, mostly in regenerating fibres. Lamin A/C phosphorylation was strikingly reduced in laminopathic myoblasts and muscle fibres, while it was preserved in interstitial fibroblasts.

Conclusions: Altered lamin A/C interplay with a muscle specific phosphorylation partner might be involved in the pathogenic mechanism of Emery–Dreifuss muscular dystrophy and limb girdle muscular dystrophy 1B.

     

Extreme phenotypic diversity and nonpenetrance in families with the LMNA gene mutation R644C

Mutations in the LMNA gene result in diverse phenotypes including Emery Dreifuss muscular dystrophy, limb girdle muscular dystrophy, dilated cardiomyopathy with conduction system disease, Dunnigan type familial partial lipodystrophy, mandibulo acral dysplasia, Hutchinson Gilford progeria syndrome, restrictive dermopathy and autosomal recessive Charcot Marie Tooth type 2. The c.1930C > T (R644C) missense mutation has previously been reported in eight unrelated patients with variable features including left ventricular hypertrophy, limb girdle muscle weakness, dilated cardiomyopathy and atypical progeria. Here we report on the details of nine additional patients in eight families with this mutation. Patients 1 and 2 presented with lipodystrophy and insulin resistance, Patient 1 having in addition focal segmental glomerulosclerosis. Patient 3 presented with motor neuropathy, Patient 4 with arthrogryposis and dilated cardiomyopathy with left ventricular non-compaction, Patient 5 with severe scoliosis and contractures, Patient 6 with limb girdle weakness and Patient 7 with hepatic steatosis and insulin resistance. Patients 8 and 9 are brothers with proximal weakness and contractures. Nonpenetrance was observed frequently in first degree relatives. This report provides further evidence of the extreme phenotypic diversity and low penetrance associated with the R644C mutation. Possible explanations for these observations are discussed.     

Clinical studies in familial VCP myopathy associated with Paget disease of bone and frontotemporal dementia

Inclusion body myopathy with Paget disease of the bone (PDB) and/or frontotemporal dementia (IBMPFD, OMIM 167320), is a progressive autosomal dominant disorder caused by mutations in the Valousin-containing protein (VCP, p97 or CDC48) gene. IBMPFD can be difficult to diagnose. We assembled data on a large set of families to illustrate the number and type of misdiagnoses that occurred. Clinical analysis of 49 affected individuals in nine families indicated that 42 (87%) of individuals had muscle disease. The majority were erroneously diagnosed with limb girdle muscular dystrophy (LGMD), facioscapular muscular dystrophy, peroneal muscular dystrophy, late adult onset distal myopathy, spinal muscular atrophy, scapuloperoneal muscular dystrophy, or amyotrophic lateral sclerosis (ALS) among others. Muscle biopsies showed rimmed vacuoles characteristic of an inclusion body myopathy in 7 of 18 patients (39%), however, inclusion body myopathy was correctly diagnosed among individuals in only families 5 and 15. Frontotemporal dementia (FTD) was diagnosed in 13 individuals (27%) at a mean age of 57 years (range 48.9-60.2 years); however, several individuals had been diagnosed with Alzheimer disease. Histopathological examination of brains of three affected individuals revealed a pattern of ubiquitin positive neuronal intranuclear inclusions and dystrophic neurites. These families expand the clinical phenotype in IBMPFD, a complex disorder caused by mutations in VCP. The presence of PDB in 28 (57%) individuals suggests that measuring serum alkaline phosphatase (ALP) activity may be a useful screen for IBMPFD in patients with myopathy.     

Linkage of scapuloperoneal spinal muscular atrophy to chromosome 12q24.1-q24.31

Scapuloperoneal (SP) syndromes are heterogeneous neuromuscular disorders which are characterized by weakness in the distribution of shoulder girdle and peroneal muscles. SP syndromes can resemble facioscapulohumeral muscular dystrophy (FSH) due to scapular weakness or Charcot-Marie-Tooth disease (CMT) due to atrophy of peroneal muscles. Both neurogenic and myopathic SP syndromes have been described. Locus for the myopathic form of SP syndrome (scapuloperoneal muscular dystrophy, SPMD) has recently been assigned to chromosome 12q. We previously described a large New England kindred exhibiting an autosomal dominant neurogenic SP syndrome (scapuloperoneal spinal muscular atrophy, SPSMA). Disease expression was more severe and progressive in successive generations, which suggested genetic anticipation. We performed genetic linkage analysis of this family with microsatellite markers and excluded the loci for FSH, CMT, SPMD and SMA (spinal muscular atrophy) in our family. Linkage in our SPSMA family (lod score > 3) was established to seven microsatellite markers that map to chromosome 12q24.1-q24.31. The highest lod score with two-point linkage analysis was 6.67 (theta = 0.00) with marker D12S353. Multipoint analysis gave maximum lod scores of 7.38 between D12S354 and D12S79, and also 7.38 between D12S369 and NOS1 (neuronal nitric oxide synthase). The gene for SPSMA lies within the 19 cM interval between D12S338 and D12S366. This report establishes a locus for the neurogenic form of SP syndrome approximately 20 cM telomeric to the one described for the myopathic form of SP syndrome.