Caveolin-1/3 double-knockout mice are viable,but lack both muscle and non-muscle caveolae,and develop a severe cardiomyopathic phenotype

The caveolin gene family consists of caveolins 1, 2, and 3. Caveolins 1 and 2 are co-expressed in many cell types, such as endothelial cells, fibroblasts, smooth muscle cells and adipocytes, where they form a heteroligomeric complex. In contrast, the expression of caveolin-3 is muscle-specific. Thus, the expression of caveolin-1 is required for caveolae formation in non-muscle cells, while the expression of caveolin-3 drives caveolae formation in striated muscle cell types (cardiac and skeletal). To create a truly caveolae-deficient mouse, we interbred Cav-1 null mice and Cav-3 null mice to generate Cav-1/Cav-3 double-knockout (Cav-1/3 dKO) mice. Here, we report that Cav-1/3 dKO mice are viable and fertile, despite the fact that they lack morphologically identifiable caveolae in endothelia, adipocytes, smooth muscle cells, skeletal muscle fibers, and cardiac myocytes. We also show that these mice are deficient in all three caveolin gene products, as caveolin-2 is unstable in the absence of caveolin-1. Interestingly, Cav-1/3 dKO mice develop a severe cardiomyopathy. At 2 months of age, analysis of Cav-1/3 dKO hearts via gated magnetic resonance imaging reveals a dramatic increase in left ventricular wall thickness, as compared with Cav-1-KO, Cav-3 KO, and wild-type mice. Further functional analysis of Cav-1/3 dKO hearts via transthoracic echocardiography demonstrates hypertrophy and dilation of the left ventricle, with a significant decrease in fractional shortening. As predicted, Northern analysis of RNA derived from the left ventricle of Cav-1/3 dKO mice shows a dramatic up-regulation of the atrial natriuretic factor message, a well-established biochemical marker of cardiac hypertrophy. Finally, histological analysis of Cav-1/3 dKO hearts reveals hypertrophy, disorganization, and degeneration of the cardiac myocytes, as well as chronic interstitial fibrosis and inflammation. Thus, dual ablation of both Cav-1 and Cav-3 genes in mice leads to a pleiotropic defect in caveolae formation and severe cardiomyopathy.     

Differential Effects of Myopathy-Associated Caveolin-3 Mutants on Growth Factor Signaling

Caveolin-3 is an important scaffold protein of cholesterol-rich caveolae. Mutations of caveolin-3 cause hereditary myopathies that comprise remarkably different pathologies. Growth factor signaling plays an important role in muscle physiology; it is influenced by caveolins and cholesterol-rich rafts and might thus be affected by caveolin-3 dysfunction. Prompted by the observation of a marked chronic peripheral neuropathy in a patient suffering from rippling muscle disease due to the R26Q caveolin-3 mutation and because TrkA is expressed by neuronal cells and skeletal muscle fibers, we performed a detailed comparative study on the effect of pathogenic caveolin-3 mutants on the signaling and trafficking of the TrkA nerve growth factor receptor and, for comparison, of the epidermal growth factor receptor. We found that the R26Q mutant slightly and the P28L strongly reduced nerve growth factor signaling in TrkA-transfected cells. Surface biotinylation experiments revealed that the R26Q caveolin-3 mutation markedly reduced the internalization of TrkA, whereas the P28L did not. Moreover, P28L expression led to increased, whereas R26Q expression decreased, epidermal growth factor signaling. Taken together, we found differential effects of the R26Q and P28L caveolin-3 mutants on growth factor signaling. Our findings are of clinical interest because they might help explain the remarkable differences in the degree of muscle lesions caused by caveolin-3 mutations and also the co-occurrence of peripheral neuropathy in the R26Q caveolinopathy case presented.Caveolae are flask-shaped invaginations of the plasma membrane implicated in membrane-trafficking and signal transduction. Caveolins are major scaffolding proteins of caveolae and lipid rafts. In these structures, cholesterol and lipid-modified signaling molecules such as Src-like kinases, H-Ras, and G-proteins accumulate.¹ Caveolin-3 (M-Caveolin) is one of three members of the caveolin protein family.² In contrast to caveolin-1 and caveolin-2, which are ubiquitously expressed, caveolin-3 is predominantly expressed in muscle fibers. The caveolin-3 proteins form homomeric complexes that are transported to the plasma membrane where they are associated with the dystrophin glycoprotein complex.³ Point mutations in caveolin-3 lead to a dissociation of the assembly process⁴ and a decrease of sarcolemmal caveolin-3 levels. The caveolin-3 mutant R26Q has been shown to accumulate in the Golgi complex,⁵ which decreases the half-life of both the mutated and the wild-type-caveolin-3 protein. Interestingly, the different caveolin-3 mutations lead to a wide spectrum of phenotypes ranging from clinically asymptomatic hyperCKemia to rippling muscle disease, distal myopathy, and limb girdle muscular dystrophy type 1C. For instance, the point mutation R26Q can cause heterogeneous disease patterns encompassing this whole spectrum of disorders.³ In contrast, the mutation P28L, which is located in close proximity to mutation R26Q, causes persistent elevated serum levels of creatine kinase only, without overt clinical symptoms.⁶ The caveolin-3 mutation A45T leads to the same heterogeneous phenotype as R26Q except for distal myopathy, whereas the G55S mutation is associated with the limb girdle muscular dystrophy type 1C phenotype.⁷Growth factors that signal through cell surface receptors are concentrated in caveolae and lipid rafts.⁸ Therefore, mutations in caveolin-3 might lead to alterations in signaling and trafficking of these factors and their receptors. In line with this hypothesis, Parton and co-workers⁹ showed in BHK fibroblasts and PC12 cells that the C71W caveolin-3 mutant strongly diminished signaling of H-Ras, an important downstream signal transducer of receptor tyrosine kinases. However, the consequences of caveolin-3 mutations on growth factor receptor activation at the cell surface are so far largely unexplored. Moreover, it is unclear how the different known caveolin-3 mutations trigger markedly different clinical phenotypes even when comparing cases with very similar mutations. This was recently exemplified by Minetti and co-workers,¹⁰ who showed that caveolin-3 T78M and T78K mutations lead to different phenotypes.In the present study we describe muscle and nerve biopsy findings of a patient who suffered from both rippling muscle disease due to the R26Q caveolin-3 mutation and chronic peripheral neuropathy. We found differential effects of the R26Q and the P28L caveolin-3 point mutants on nerve growth factor (NGF) versus epidermal growth factor (EGF) signaling in our in vitro experiments. Our results give rise to the intriguing hypothesis that differential effects of caveolin-3 mutants on various growth factor signaling pathways are causally related to the so far unexplained differences in clinical phenotypes.     

Phosphofructokinase muscle-specific isoform requires caveolin-3 expression for plasma membrane recruitment and caveolar targeting: implications for the pathogenesis of caveolin-related muscle diseases

Previous co-immunoprecipitation studies have shown that endogenous PFK-M (phosphofructokinase, muscle-specific isoform) associates with caveolin (Cav)-3 under certain metabolic conditions. However, it remains unknown whether Cav-3 expression is required for the plasma membrane recruitment and caveolar targeting of PFK-M. Here, we demonstrate that recombinant expression of Cav-3 dramatically affects the subcellular localization of PFK-M, by targeting PFK-M to the plasma membrane, and by trans-locating PFK-M to caveolae-enriched membrane domains. In addition, we show that the membrane recruitment and caveolar targeting of PFK-M appears to be strictly dependent on the concentration of extracellular glucose. Interestingly, recombinant expression of PFK-M with three Cav-3 mutants [DeltaTFT (63 to 65), P104L, and R26Q], which harbor the same mutations as seen in the human patients with Cav-3-related muscle diseases, causes a substantial reduction in PFK-M expression levels, and impedes the membrane recruitment of PFK-M. Analysis of skeletal muscle tissue samples from Cav-3(-/-) mice directly demonstrates that Cav-3 expression regulates the phenotypic behavior of PFK-M. More specifically, in Cav-3-null mice, PFK-M is no longer targeted to the plasma membrane, and is excluded from caveolar membrane domains. As such, our current results may be important in understanding the pathogenesis of Cav-3-related muscle diseases, such as limb-girdle muscular dystrophy-1C, distal myopathy, and rippling muscle disease, that are caused by mutations within the human Cav-3 gene.     

Impaired muscle growth and response to insulin-like growth factor 1 in dysferlin-mediated muscular dystrophy

Loss-of-function mutations in dysferlin cause muscular dystrophy, and dysferlin has been implicated in resealing membrane disruption in myofibers. Given the importance of membrane fusion in many aspects of muscle function, we studied the role of dysferlin in muscle growth. We found that dysferlin null myoblasts have a defect in myoblast-myotube fusion, resulting in smaller myotubes in culture. In vivo, dysferlin null muscle was found to have mislocalized nuclei and vacuolation. We found that myoblasts isolated from dysferlin null mice accumulate enlarged, lysosomal-associated membrane protein 2 (LAMP2)-positive lysosomes. Dysferlin null myoblasts accumulate transferrin-488, reflecting abnormal vesicular trafficking. Additionally, dysferlin null myoblasts display abnormal trafficking of the insulin-like growth factor (IGF) receptor, where the receptor is shuttled to LAMP2-positive lysosomes. We studied growth, in vivo, by infusing mice with the growth stimulant IGF1. Control IGF1-treated mice increased myofiber diameter by 30% as expected, whereas dysferlin null muscles had no response to IGF1, indicating a defect in myofiber growth. We also noted that dysferlin null fibroblasts also accumulate acidic vesicles, IGF receptor and transferrin, indicating that dysferlin is important for nonmuscle vesicular trafficking. These data implicate dysferlin in multiple membrane fusion events within the cell and suggest multiple pathways by which loss of dysferlin contributes to muscle disease.     

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.     

Simultaneous dystrophin and dysferlin deficiencies associated with high-level expression of the coxsackie and adenovirus receptor in transgenic mice

The Coxsackie and adenovirus receptor (CAR), a cell adhesion molecule of the immunoglobulin superfamily, is usually confined to the sarcolemma at the neuromuscular junction in mature skeletal muscle fibers. Previously, we reported that adenovirus-mediated gene transfer is greatly facilitated in hemizygous transgenic mice with extrasynaptic CAR expression driven by a muscle-specific promoter. However, in the present study, when these mice were bred to homozygosity, they developed a severe myopathic phenotype and died prematurely. Large numbers of necrotic and regenerating fibers were present in the skeletal muscle of the homozygous CAR transgenics. The myopathy was further characterized by increased levels of caveolin-3 and beta-dystroglycan and decreased levels of dystrophin, dysferlin, and neuronal nitric-oxide synthase. Even the hemizygotes manifested a subtle phenotype, displaying deficits in isometric force generation and perturbed mitogen-activated protein kinase (MAPK-erk1/2) activation during contraction. There are few naturally occurring or engineered mouse lines showing as severe a skeletal myopathy as observed with ectopic expression of CAR in the homozygotes. Taken together, these findings suggest that substantial overexpression of CAR may lead to physiological dysfunction by disturbing sarcolemmal integrity (through dystrophin deficiency), impairing sarcolemmal repair (through dysferlin deficiency), and interfering with normal signaling (through alterations in caveolin-3 and neuronal nitric-oxide synthase levels).     

Dysferlin and Myoferlin Regulate Transverse Tubule Formation and Glycerol Sensitivity

Dysferlin is a membrane-associated protein implicated in muscular dystrophy and vesicle movement and function in muscles. The precise role of dysferlin has been debated, partly because of the mild phenotype in dysferlin-null mice (Dysf). We bred Dysf mice to mice lacking myoferlin (MKO) to generate mice lacking both myoferlin and dysferlin (FER). FER animals displayed progressive muscle damage with myofiber necrosis, internalized nuclei, and, at older ages, chronic remodeling and increasing creatine kinase levels. These changes were most prominent in proximal limb and trunk muscles and were more severe than in Dysf mice. Consistently, FER animals had reduced ad libitum activity. Ultrastructural studies uncovered progressive dilation of the sarcoplasmic reticulum and ectopic and misaligned transverse tubules in FER skeletal muscle. FER muscle, and Dysf- and MKO-null muscle, exuded lipid, and serum glycerol levels were elevated in FER and Dysf mice. Glycerol injection into muscle is known to induce myopathy, and glycerol exposure promotes detachment of transverse tubules from the sarcoplasmic reticulum. Dysf, MKO, and FER muscles were highly susceptible to glycerol exposure in vitro, demonstrating a dysfunctional sarcotubule system, and in vivo glycerol exposure induced severe muscular dystrophy, especially in FER muscle. Together, these findings demonstrate the importance of dysferlin and myoferlin for transverse tubule function and in the genesis of muscular dystrophy.The muscular dystrophies are a heterogeneous group of genetic disorders characterized by progressive muscle loss and weakness. The mechanisms that underlie muscular dystrophy are diverse, including defective regeneration, plasma membrane instability, and defective membrane repair. Dysferlin (DYSF) has been implicated in all of these processes.^1,2 Autosomal recessive loss-of-function mutations in dysferlin cause three different forms of muscular dystrophy: limb-girdle muscular dystrophy type 2B, Miyoshi myopathy, and distal anterior compartment myopathy.^3–5 Mutations in dysferlin become clinically evident in the second to third decade or later, with muscle weakness. An early characteristic feature of dysferlin mutations is massively elevated serum creatine kinase levels. A spectrum of myopathic changes can be seen in muscle biopsy specimens from humans with dysferlin mutations, including dystrophic features, such as fibrofatty replacement and inflammatory infiltrates.Dysferlin is a 230-kDa membrane-inserted protein that contains at least six cytoplasmic C2 domains. C2 domains mediate protein-protein interactions and, in some cases, directly bind phospholipids and calcium. The C2 domains of dysferlin are highly related to those found in the membrane trafficking and fusion protein synaptotagmins.⁶ Dysferlin is highly expressed in adult skeletal muscle, whereas it is expressed at lower levels in muscle precursor cells, myoblasts.^1,7,8 On sarcolemma damage, dysferlin is found at the sites of membrane disruption and has been specifically implicated in resealing the sarcolemma.² Electron microscopy of skeletal muscle biopsy specimens from human dysferlin-mutant patients confirms discontinuity of the sarcolemma and reveals vesicles underneath the basal lamina, suggesting dysferlin plays an active role in vesicle fusion at the membrane lesion.⁹ Dysferlin also has been shown to interact with a variety of cytosolic and membrane-associated binding partners, including MG53, caveolin-3, AHNAK, and annexins A1 and A2.^10–13 Similar to dysferlin, MG53, caveolin-3, and the annexins have been implicated in membrane resealing, suggesting a large complex may act coordinately to seal the disrupted plasma membrane in a calcium-dependent manner.^13,14An increasing body of evidence suggests that dysferlin’s membrane-associated roles are not restricted to the sarcolemma. Dysferlin has been implicated in the development and maintenance of the transverse (T-) tubule, a muscle-specific membrane system essential for electromechanical coupling. The T-tubule is a membrane inversion of the sarcolemma that flanks the Z band of muscle, the anchor for sarcomeric proteins. Dysferlin associates with the T-tubule–like system in differentiated C2C12 myotubes,¹⁵ and dysferlin-null mouse muscle contains malformed T-tubules consistent with a role for dysferlin in the biogenesis and maintenance of the T-tubule system.¹⁶ In mature muscle damaged by stretch, dysferlin localizes to T-tubules, suggesting a reparative function for dysferlin at the T-tubule.¹⁷Dysferlin belongs to a family of proteins, the ferlins, that contains six family members. Myoferlin is a dysferlin homologue, which is 76% identical at the amino acid level.¹⁸ Such as dysferlin, myoferlin also contains at least six calcium-sensitive C2 domains, a carboxy-terminal transmembrane domain, an Fer domain, and a DysF domain.^17,19 Myoferlin is highly expressed in myoblasts and is markedly up-regulated in adult skeletal muscle on muscle damage.²⁰ Myoferlin, such as dysferlin, is required for normal myoblast fusion and muscle growth through regulating steps of vesicle trafficking and endocytic recycling.^1,20,21 Myoferlin, such as dysferlin, is required for the proper trafficking of and response to the insulin-like growth factor-1 receptor in muscle.²² Myoferlin interacts with endocytic recycling proteins EHD1 and EHD2, as well as AHNAK.^21,23,24 To date, no human forms of muscular dystrophy resulting from myoferlin mutations have been reported. However, mice lacking myoferlin show defects in muscle regeneration, establishing a role for myoferlin in muscle repair.²⁰We generated ferlin (FER) mice that carry both the dysferlin- and myoferlin-null loss of function mutations. We determined that FER mice have a more severe muscular dystrophy than dysferlin-null mice. In addition, FER muscle displays disorganization of the T-tubule system, dilated sarcoplasmic reticulum, and increased levels of serum glycerol. We revealed an enhanced sensitivity of Dysf, MKO, and especially FER myofibers to glycerol exposure, resulting in T-tubule vacuolation and disrupted membrane potential. Intramuscular glycerol injections into young FER muscle recapitulated the dystrophic phenotype characteristic of old FER muscle. Our data establish a role for both myoferlin and dysferlin in the biogenesis and remodeling of the sarcotubule system and suggest glycerol as a mediator of muscular dystrophy in dysferlin mutations.     

Disruption of muscle membrane and phenotype divergence in two novel mouse models of dysferlin deficiency

Limb girdle muscular dystrophy type 2B and Miyoshi myopathy are clinically distinct forms of muscular dystrophy that arise from defects in the dysferlin gene. Here, we report two novel lines of dysferlin-deficient mice obtained by (a) gene targeting and (b) identification of an inbred strain, A/J, bearing a retrotransposon insertion in the dysferlin gene. The mutations in these mice were located at the 3' and 5' ends of the dysferlin gene. Both lines of mice lacked dysferlin and developed a progressive muscular dystrophy with histopathological and ultrastructural features that closely resemble the human disease. Vital staining with Evans blue dye revealed loss of sarcolemmal integrity in both lines of mice, similar to that seen in mdx and caveolin-3 deficient mice. However, in contrast to the latter group of animals, the dysferlin-deficient mice have an intact dystrophin glycoprotein complex and normal levels of caveolin-3. Our findings indicate that muscle membrane disruption and myofiber degeneration in dysferlinopathy were directly mediated by the loss of dysferlin via a new pathogenic mechanism in muscular dystrophies. We also show that the mutation in the A/J mice arose between the late 1970s and the early 1980s, and had become fixed in the production breeding stocks. Therefore, all studies involving the A/J mice or mice derived from A/J, including recombinant inbred, recombinant congenic and chromosome substitution strains, should take into account the dysferlin defect in these strains. These new dysferlin-deficient mice should be useful for elucidating the pathogenic pathway in dysferlinopathy and for developing therapeutic strategies.     

Dysferlin is a plasma membrane protein and is expressed early in human development

Recently, a single gene, DYSF, has been identified which is mutated in patients with limb-girdle muscular dystrophy type 2B (LGMD2B) and with Miyoshi myopathy (MM). This is of interest because these diseases have been considered as two distinct clinical conditions since different muscle groups are the initial targets. Dysferlin, the protein product of the gene, is a novel molecule without homology to any known mammalian protein. We have now raised a monoclonal antibody to dysferlin and report on the expression of this new protein: immunolabelling with the antibody (designated NCL-hamlet) demonstrated a polypeptide of approximately 230 kDa on western blots of skeletal muscle, with localization to the muscle fibre membrane by microscopy at both the light and electron microscopic level. A specific loss of dysferlin labelling was observed in patients with mutations in the LGMD2B/MM gene. Furthermore, patients with two different frameshifting mutations demonstrated very low levels of immunoreactive protein in a manner reminiscent of the dystrophin expressed in many Duchenne patients. Analysis of human fetal tissue showed that dysferlin was expressed at the earliest stages of development examined, at Carnegie stage 15 or 16 (embryonic age 5-6 weeks). Dysferlin is present, therefore, at a time when the limbs start to show regional differentiation. Lack of dysferlin at this critical time may contribute to the pattern of muscle involvement that develops later, with the onset of a muscular dystrophy primarily affecting proximal or distal muscles.     

Zebrafish as a model for caveolin-associated muscle disease; caveolin-3 is required for myofibril organization and muscle cell patterning

Caveolae are an abundant feature of many animal cells. However, the exact function of caveolae remains unclear. We have used the zebrafish, Danio rerio, as a system to understand caveolae function focusing on the muscle-specific caveolar protein, caveolin-3 (Cav3). We have identified caveolin-1 (alpha and beta), caveolin-2 and Cav3 in the zebrafish. Zebrafish Cav3 has 72% identity to human CAV3, and the amino acids altered in human muscle diseases are conserved in the zebrafish protein. During embryonic development, cav3 expression is apparent by early segmentation stages in the first differentiating muscle precursors, the adaxial cells and slightly later in the notochord. cav3 expression appears in the somites during mid-segmentation stages and then later in the pectoral fins and facial muscles. Cav3 and caveolae are located along the entire sarcolemma of late stage embryonic muscle fibers, whereas beta-dystroglycan is restricted to the muscle fiber ends. Down-regulation of Cav3 expression causes gross muscle abnormalities and uncoordinated movement. Ultrastructural analysis of isolated muscle fibers reveals defects in myoblast fusion and disorganized myofibril and membrane systems. Expression of the zebrafish equivalent to a human muscular dystrophy mutant, CAV3P104L, causes severe disruption of muscle differentiation. In addition, knockdown of Cav3 resulted in a dramatic up-regulation of eng1a expression resulting in an increase in the number of muscle pioneer-like cells adjacent to the notochord. These studies provide new insights into the role of Cav3 in muscle development and demonstrate its requirement for correct intracellular organization and myoblast fusion.     

Muscular dystrophy with marked Dysferlin deficiency is consistently caused by primary dysferlin gene mutations

Dysferlin is a 237-kDa transmembrane protein involved in calcium-mediated sarcolemma resealing. Dysferlin gene mutations cause limb-girdle muscular dystrophy (LGMD) 2B, Miyoshi myopathy (MM) and distal myopathy of the anterior tibialis. Considering that a secondary Dysferlin reduction has also been described in other myopathies, our original goal was to identify cases with a Dysferlin deficiency without dysferlin gene mutations. The dysferlin gene is huge, composed of 55 exons that span 233 140 bp of genomic DNA. We performed a thorough mutation analysis in 65 LGMD/MM patients with ≤20% Dysferlin. The screening was exhaustive, as we sequenced both genomic DNA and cDNA. When required, we used other methods, including real-time PCR, long PCR and array CGH. In all patients, we were able to recognize the primary involvement of the dysferlin gene. We identified 38 novel mutation types. Some of these, such as a dysferlin gene duplication, could have been missed by conventional screening strategies. Nonsense-mediated mRNA decay was evident in six cases, in three of which both alleles were only detectable in the genomic DNA but not in the mRNA. Among a wide spectrum of novel gene defects, we found the first example of a ‘nonstop'' mutation causing a dysferlinopathy. This study presents the first direct and conclusive evidence that an amount of Dysferlin ≤20% is pathogenic and always caused by primary dysferlin gene mutations. This demonstrates the high specificity of a marked reduction of Dysferlin on western blot and the value of a comprehensive molecular approach for LGMD2B/MM diagnosis.     

Caveolin-1(-/-)- and caveolin-2(-/-)-deficient mice both display numerous skeletal muscle abnormalities, with tubular aggregate formation

Here, we examine the role of "non-muscle" caveolins (Cav-1 and Cav-2) in skeletal muscle biology. Our results indicate that skeletal muscle fibers from male Cav-1(-/-) and Cav-2(-/-) mice show striking abnormalities, such as tubular aggregates, mitochondrial proliferation/aggregation, and increased numbers of M-cadherin-positive satellite cells. Notably, these skeletal muscle defects were more pronounced with increasing age. Because Cav-2-deficient mice displayed normal expression levels of Cav-1, whereas Cav-1-null mice exhibited an almost complete deficiency in Cav-2, these skeletal muscle abnormalities seem to be due to loss of Cav-2. Thus, Cav-2(-/-) mice represent a novel animal model-and the first genetically well-defined mouse model-that can be used to study the pathogenesis of tubular aggregate formation, which remains a poorly understood age-related skeletal muscle abnormality. Finally, because Cav-1 and Cav-2 were not expressed within mature skeletal myofibers, our results indicate that development of these abnormalities probably originates in stem/precursor cells, such as satellite cells or myoblasts. Consistent with this hypothesis, skeletal muscle isolated from male Cav-3(-/-) mice did not show any of these abnormalities. As such, this is the first study linking stem cells with the genesis of these intriguing muscle defects.     

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.     

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.     

Clinical and functional significance of loss of caveolin-1 expression in breast cancer-associated fibroblasts

Loss of caveolin-1 (Cav-1) expression in breast cancer-associated fibroblasts (CAFs) is predictive of poor prognosis in breast cancer, but its function has not been established. Our study tested the hypotheses that loss of Cav-1 expression in breast fibroblasts was associated with poor prognosis in breast cancer, through promotion of breast cancer cell invasion. Cav-1 stromal expression was immunohistochemically assessed in 358 breast cancers. Cav-1 expression in primary breast fibroblasts was analysed by western blot. Modified Boyden chamber assays determined fibroblast ability to promote invasion of breast cancer cells. The impact of siRNA silencing of Cav-1 in fibroblasts was evaluated using invasion assays and 3D co-culture assays. Loss of Cav-1 expression in breast stroma was significantly associated with decreased breast cancer-specific and disease-free survival (p = 0.01). Mean survival was 72 months (Cav-1(+) group) versus 29.5 months (Cav-1(-) group). This was confirmed in multivariate analysis. Cav-1 expression was significantly decreased in CAFs compared to normal fibroblasts (p = 0.01) and was associated with increased invasion-promoting capacity. Cav-1 siRNA-treated fibroblasts promoted significantly increased invasion of MDA-MB-468 and T47D breast cancer cells from 27% (control) to 67% (p = 0.006) and from 37% to 56%, respectively (p = 0.01). 3D co-cultures of MDA-MB-468 cells with myoepithelial cells led to the formation of organized cohesive structures when cultured with conditioned media from fibroblasts but resulted in a disorganized appearance in the presence of conditioned media from Cav-1 siRNA-treated fibroblasts, accompanied by loss of E-cadherin expression in tumour cells. Our data confirm that loss of stromal Cav-1 in breast cancer predicts poor outcome. At a functional level, Cav-1-deficient CAFs are capable of significantly increasing the invasive capacity of breast cancer cells.     

Evidence for cyclooxygenase-2 association with caveolin-3 in primary cultured rat chondrocytes

The purpose of this study was to demonstrate the cellular localization of cyclooxygenase-2 (COX-2) and caveolin-3 (Cav-3) in primarily cultured rat chondrocytes. In normal rat chondrocytes, we observed relatively high levels of Cav-3 and a very low level of COX-2 mRNA and protein. Upon treating the chondrocytes with 5 microM of CdCl(2) (Cd) for 6 hr, the expressions of COX-2 mRNA and protein were increased with the decreased Cav-3 mRNA and protein expressions. The detergent insoluble caveolae-rich membranous fractions that were isolated from the rat chondrocytes and treated with Cd contained the both proteins of both COX-2 and Cav-3 in a same fraction. The immuno-precipitation experiments showed complex formation between the COX-2 and Cav-3 in the rat chondrocytes. Purified COX-2 with glutathione S-transferase-fused COX-2 also showed complex formation with Cav-3. Confocal and electron microscopy also demonstrated the co-localization of COX-2 and Cav-3 in the plasma membrane. The results from our current study show that COX-2 and Cav-3 are co-localized in the caveolae of the plasma membrane, and they form a protein-protein complex. The co-localization of COX-2 with Cav-3 in the caveolae suggests that the caveolins might play an important role for regulating the function of COX-2.     

非小细胞肺癌中窖蛋白1基因的蛋白表达和甲基化及其意义

目的 检测窖蛋白1(Car-1)在非小细胞肺癌(NSCLC)中的蛋白表达以及启动子的甲基化状况,探讨Cav-1基因在NSCLC中的作用及其临床意义.方法 应用免疫组织化学(sP法)和量子点Qd600染色检测123例NSCLC组织、17例良性病变肺组织中Cav一1蛋白表达和亚细胞定位.亚硫酸氢钠处理DNA,甲基化特异性PCR(MSP)检测Cav-1基因启动子区域的甲基化水平.结果 Cav-1蛋白在肺支气管黏膜上皮细胞、肺泡上皮细胞、毛细血管内皮细胞、成纤维细胞、平滑肌细胞的胞质和胞膜高表达.癌旁组织(对照)组和肺癌组中Cav-1蛋白的阳性率分别为17/17、43.1%(53/123),两组间差异有统计学意义(P=0.001);Cav-1蛋白在NSCLC不同的组织学类型(P=0.552)和分化程度(P=0.160)中差异均无统计学意义.Cav-1蛋白阳性率与NSCLC的TNM分期(P=0.001)以及淋巴结转移(P=0.001)均相关.在40例Cav-1蛋白表达为阴性的肺癌组织和12例癌旁肺组织,MSP法均未检测到Cav-1因启动子区域的甲基化.结论 Cav-1蛋白失表达的机制可能与启动子区是否甲基化无关.Cav-1蛋白高表达预示NSCLC恶化进展和高侵袭性.     

Novel sequence variants in dysferlin-deficient muscular dystrophy leading to mRNA decay and possible C2-domain misfolding

Mutations in the gene encoding dysferlin (DYSF) cause the allelic autosomal recessive disorders limb girdle muscular dystrophy 2B and Miyoshi myopathy. It encompasses 55 exons spanning 150 kb of genomic DNA. Dysferlin is involved in membrane repair in skeletal muscle. We identified three families with novel sequence variants in DYSF. All affected family members showed limb girdle weakness and had reduced or absent dysferlin protein on immunohistochemistry. All exons of DYSF were screened by genomic sequencing. Five novel variants in DYSF were found: two missense mutations (c.895G>A and c.4022T>C), one 5' donor splice-site variant (c.855+1delG), one nonsense mutation (c.1448C>A), and a variant in the 3'UTR of DYSF (c.*107T>A). All alterations were confirmed by restriction enzyme analysis and not found in 400 control alleles. Nonsense mediated RNA decay or changes in the three-dimensional protein structure resulting in intracellular dysferlin aggregates and finally the lack of dysferlin protein were identified as consequences of the novel DYSF variants.     

Myoferlin, a candidate gene and potential modifier of muscular dystrophy

Dysferlin, the gene product of the limb girdle muscular dystrophy (LGMD) 2B locus, encodes a membrane-associated protein with homology to Caenorhabditis elegans fer-1. Humans with mutations in dysferlin ( DYSF ) develop muscle weakness that affects both proximal and distal muscles. Strikingly, the phenotype in LGMD 2B patients is highly variable, but the type of mutation in DYSF cannot explain this phenotypic variability. Through electronic database searching, we identified a protein highly homologous to dysferlin that we have named myoferlin. Myoferlin mRNA was highly expressed in cardiac muscle and to a lesser degree in skeletal muscle. However, antibodies raised to myoferlin showed abundant expression of myoferlin in both cardiac and skeletal muscle. Within the cell, myoferlin was associated with the plasma membrane but, unlike dysferlin, myoferlin was also associated with the nuclear membrane. Ferlin family members contain C2 domains, and these domains play a role in calcium-mediated membrane fusion events. To investigate this, we studied the expression of myoferlin in the mdx mouse, which lacks dystrophin and whose muscles undergo repeated rounds of degeneration and regeneration. We found upregulation of myoferlin at the membrane in mdx skeletal muscle. Thus, myoferlin ( MYOF ) is a candidate gene for muscular dystrophy and cardiomyopathy, or possibly a modifier of the muscular dystrophy phenotype.