Human Molecular Genetics, 2001, Vol. 10, No. 3 173-178
© 2001 Oxford University Press
Transgenic mice expressing mutant caveolin-3 show severe myopathy associated with increased nNOS activity
1Division of Neurology, Department of Internal Medicine, Kawasaki Medical School, 577 Matsushima, Kurashiki-City, Okayama 701-0192, Japan, 2Department of Neurology and Neuroscience, Teikyo University School of Medicine, 2-11-1 Kaga, Itabashi-ku, Tokyo 173-8605, Japan and 3School of Pharmaceutical Sciences and 4Center for Biotechnology, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan
Received 29 August 2000; Revised and Accepted 30 November 2000.
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ABSTRACT |
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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.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Caveolae are 50–100 nm flask-shaped invaginations of the plasma membrane and have been implicated in a variety of cellular functions, including signal transduction, lipid metabolism, cell growth and apoptotic cell death (1). Caveolin family proteins, 21–24 kDa integral membrane proteins so far identified as caveolins-1, -2 and -3, are the principal components of caveolae (2–7). These caveolins serve as a scaffolding protein to concentrate caveolin-associated signaling molecules within caveolae and facilitate rapid and efficient coupling of activated receptors to cytoplasmic signaling molecules (8). Caveolins interact directly with a number of caveolin-associated signaling molecules such as H-Ras, heterotrimeric G proteins, epidermal growth factor receptor, protein kinase C, Src and nitric oxide synthase (NOS), and they also regulate the activities of these signaling molecules (9–12). For instance, direct binding of either caveolin-1 to endothelial (e) NOS or caveolin-3 to neuronal (n) NOS suppresses the catalytic activity of NOS.
Three distinct mammalian caveolin genes have been identified to date: caveolins-1, -2 and -3. The gene for caveolin-1 encodes two distinct isoforms via alternative translation initiation from methionines 1 and 32, resulting in caveolin-1
and -1ß (13). Caveolins-1 and -2 are expressed predominantly in endothelial cells and adipocytes, whereas the expression of caveolin-3 is confined to muscle tissues and found in skeletal, cardiac and smooth muscle cells. Caveolins form high molecular mass homo-oligomers consisting of 
14–16 caveolin monomers, and purified caveolin homo-oligomers have the capacity to self-associate into caveolae-like structures (14).
Caveolin-3 appears during the differentiation of skeletal myoblasts and is localized to the sarcolemma, where it interacts with dystrophin and its associated glycoproteins (15). This indicates the possibility that caveolin-3 may play a role in the pathogenesis of muscular dystrophy. In 1998 Minetti et al. (16) first reported two Italian families having heterozygous mutations in the caveolin-3 gene causing limb-girdle muscular dystrophy, now designated as LGMD1C. The clinical pictures of LGMD1C are characterized by calf hypertrophy, proximal muscle weakness, hyperCKemia and myopathic changes in muscle biopsies. They identified two distinct mutations in the caveolin-3 (CAV3) gene: (i) an in-frame 9 bp microdeletion involving Phe65 in the scaffolding domain; and (ii) a missense mutation, Pro104Leu, in the integral membrane domain. More recently a heterozygous missense mutation Arg26Gln was also identified in two unrelated children showing asymptomatic hyperCKemia (17).
Caveolin family proteins are evolutionarily conserved through worms to mammals; however, mammalian and Caenorhabditis elegans caveolin family proteins share only 12 identical amino acids (18), suggesting that these amino acids may be crucial for the protein structure or function of caveolins. Interestingly, of the 12 invariant amino acids, 3 (Arg26, Phe65 and Pro104) were altered in these patients (16,17), further supporting the aforementioned hypothesis. However, it remains unknown what is the molecular mechanism by which these heterozygous mutations cause the deficiency of caveolin-3 and eventually lead to muscle cell degeneration in LGMD1C. To address these questions, we generated transgenic mice expressing the Pro104Leu mutant caveolin-3 (TgCAV3M1) and demonstrated a dominant negative effect of the mutation causing the deficiency of caveolin-3 in skeletal muscle. In addition we showed a great increase of nNOS activity in transgenic mouse skeletal muscle, which may be involved in the pathogenesis of muscular degeneration in caveolin-3 deficiency.
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RESULTS |
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Analysis of caveolin-3 mRNAs in wild-type and TgCAV3M1 mice
We introduced Pro104Leu missense mutation into the mouse caveolin-3 cDNA by site-directed mutagenesis and made a mutant caveolin-3 expression vector pM1CV as shown in Fig. 1A. Tissue-specific expression of this mutant mouse caveolin-3 cDNA was controlled by regulatory elements of the mouse muscle creatine kinase (MCK) gene (19). Northern blot analysis of TgCAV3M1 mouse skeletal muscle showed the overexpression of the mutant caveolin-3 mRNA which was recognized as a signal slightly smaller than the endogenous caveolin-3 mRNA in wild-type littermates (Fig. 2A). The expression of the endogenous caveolin-3 mRNA in TgCAV3M1 mice was not clear from northern blot analysis. We analyzed differential expression of normal and mutant caveolin 3 mRNA by RT–PCR using two distinct reverse primers: the R1 primer (CV3R6) is specific for endogenous caveolin-3, whereas the R2 primer (SV40pAR) is specific for the mutant caveolin-3 as shown in Fig. 2C. RT–PCR using the R1 primer confirmed the expression of the endogenous caveolin-3 mRNA in TgCAV3M1 mice as well as in wild-type littermates (Fig. 2B).
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TgCAV3M1 mice show the myopathic phenotype
Transgenic mice showed poor growth and were significantly smaller than the normal littermates at 6 weeks of age. TgCAV3M1 mice developed kyphosis of spine and paralysis of hindlimbs by this time (Fig. 1B). Hematoxylin and eosin staining of skeletal muscle cryosections from TgCAV3M1 mice showed myopathic features: marked variation in fiber size, prominently atrophic fibers, internally placed nuclei and increased endomysium (Fig. 3).
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Protein analysis of caveolin-3 expression in wild-type and TgCAV3M1 mice
Immunohistochemical analysis revealed a continuous sarcolemmal localization of caveolin-3 in wild-type mouse skeletal muscle (Fig. 3). In TgCAV3M1 mice, on the other hand, the vast majority of muscle fibers lacked the sarcolemmal localization of caveolin-3, except a few small fibers showing a weak sarcolemmal immunosignal for caveolin-3 (Fig. 3). In some of the muscle fibers lacking the sarcolemmal localization of caveolin-3, in addition, small dot-like immunopositive deposits were scattered in the cytoplasm (Fig. 3). Drastic reduction of caveolin-3 in TgCAV3M1 mouse skeletal muscle was confirmed by immunoblot analysis (Fig. 4). The level of expression of dystrophin and ß-dystroglycan was indistinguishable between wild-type and TgCAV3M1 mice (Fig. 4). These findings indicate that the deficiency of caveolin-3 in transgenic mice was specific and not secondary to the muscle cell degeneration process.
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nNOS activity is elevated in TgCAV3M1 mouse skeletal muscle
Caveolins bind various types of signaling molecule, including NOS, and regulate signal transduction. Caveolin-3 interacts with nNOS in skeletal muscle and negatively regulates the catalytic activity of nNOS (20). We studied the status of nNOS in TgCAV3M1 mouse skeletal muscle to discover whether an alteration of nNOS activity is involved in the process of muscle degeneration in caveolin-3 deficiency. Immunohistochemical analysis showed a similar mosaic pattern of nNOS expression in the sarcolemma (Fig. 5B, left) and immunoblot analysis demonstrated that comparable amounts of nNOS were expressed in both wild-type and TgCAV3M1 mice (Fig. 5A). To establish the actual nNOS activity of individual muscle fibers, we performed the NADPH diaphorase (NDP) activity assay using the skeletal muscle sections (21). Both in wild-type and transgenic mice, muscle fibers without clear nNOS immunostaining were negative for NDP staining. Among nNOS-positive muscle fibers, NDP-positive muscle fibers were significantly more numerous in TgCAV3M1 mice than in control mice, indicating that nNOS activity was abnormally increased in the former (Fig. 5B, right, and C).
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DISCUSSION |
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In the present study we produced transgenic mice expressing the Pro104Leu mutant caveolin-3 which showed the myopathic phenotype mimicking human LGMD1C. Caveolin-3 protein was deficient in TgCAV3M1 mouse skeletal muscle although the mutant caveolin-3 mRNA was overexpressed in the presence of endogenous caveolin-3 mRNA. Deficiency of caveolin-3 resulting from co-expression of normal and mutant caveolin-3 proteins suggests a dominant negative effect of the mutation in the caveolin-3 gene. Interestingly, Galbiati et al. (22) reported that overexpression of the normal caveolin-3 induced a Duchenne-like muscular dystrophy phenotype in transgenic mice. Together with our data these results suggest that the presence of the mutant caveolin-3 plays a critical role in the pathogenesis of caveolin-3 deficiency in LGMD1C. How does the co-existence of normal and mutant caveolin-3 proteins result in the loss of caveolin-3 proteins in the sarcolemma? In this respect, it is noteworthy that Galbiati et al. (23) demonstrated recently that mutant caveolin-3 formed unstable homooligomers of a much larger size than those of wild-type caveolin-3. In addition, they showed that mutant homo-oligomers were retained within the Golgi complex and not targeted to the plasma membrane (23). We presume (i) that the presence of mutant caveolin-3 inhibits the formation of normal homo-oligomers and leads to accelerated degradation of caveolin-3 proteins; and (ii) that mutant homo-oligomers resulting from the overexpression of the mutant caveolin-3 in transgenic mice are unstable and not targeted to the plasma membrane correctly. Indeed, the cytoplasmic small dots which were immunopositive with anti-caveolin-3 antibody may represent the mutant homo-oligomers which failed to be targeted to the plasma membrane and accumulated in the cytoplasm.
The caveolin protein family comprises scaffolding proteins, which bind signaling molecules, including NOS, src-family kinases, receptor tyrosine kinases, G-protein and H-Ras (24). The caveolin protein family also modulates a signaling pathway (4,9,11). In skeletal muscle, caveolin-3 binds nNOS in the sarcolemma and inhibits its catalytic activity via the direct interaction of the scaffolding domain (10,20). In skeletal muscle, furthermore, nNOS binds 1-syntrophin which, in turn, is anchored to dystrophin and -dystrobrevin (15,25,26). Previously, it was reported that nNOS was deficient in Duchenne dystrophy skeletal muscle, suggesting that a decrease in nNOS activity might contribute to muscle fiber degeneration in this disease (27). More recently, nNOS was shown to be displaced from the sarcolemma in the -dystrobrevin knockout mice (26). They developed muscular dystrophy, suggesting that a disturbance in the nNOS signaling pathway might play a role in muscle fiber degeneration (26). On the other hand, however, the nNOS knockout mice did not show a myopathic phenotype (28). In addition, the disruption of the 1-syntrophin gene led to the displacement of nNOS from the sarcolemma but did not induce muscle fiber degeneration (21). Thus, the role of nNOS in the pathogenesis of muscle fiber degeneration still remains unclear.
In order to address this question, we compared the level of nNOS expression in transgenic and control mouse skeletal muscle. We found that the amount of nNOS expressed in the sarcolemma was comparable between transgenic and control mice. However, the actual nNOS activity was significantly elevated in TgCAV3M1 mouse skeletal muscle by the NDP activity assay using the skeletal muscle sections (21). Since caveolin-3 interacts with nNOS and inhibits its catalytic activity, it is most likely that the deficiency of caveolin-3 caused disinhibition of nNOS activity and led to abnormal increase of nNOS activity in transgenic mouse skeletal muscle. We propose that increased nNOS activity may play a role in the pathogenesis of muscle fiber degeneration in caveolin-3 deficiency.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Generation of transgenic mice
The full-length mouse caveolin-3 cDNA was cloned by RT–PCR from B10 mouse skeletal muscle. A missense mutation Pro104Leu was introduced into mouse caveolin-3 cDNA by site-directed mutagenesis. The mouse mutant caveolin-3 expression vector pM1CV was constructed as follows. The expression of the mutant caveolin-3 cDNA was regulated by 6.5 kb of MCK gene sequences, including 3300 bp upstream of exon 1, the complete first intron and a truncated exon 2 deleted just 5' of the initiator methionine (a kind gift from Dr Jeffrey S. Chamberlain, Michigan University). The SV40 polyadenylation site was inserted 3' of the mutant caveolin-3 cDNA. Transgenic mice TgCAV3M1 were generated by microinjection of the purified pM1CV insert into zygotes from scl:BDF1 x scl:BDF1 parents essentially as described (29). Positive transgenic mice were identified by genomic PCR using primers flanking MCK exon 2 and the mutant caveolin-3 construct. Several founder F0 mice were bred to generate offspring for analysis and breeding. Four transgenic mice from distinct lines were analyzed in this study.
Northern blot analysis
Total RNA (10 µg) was extracted from transgenic and control mouse quadriceps femoris muscle, separated on 1% agarose gels containing 7% formaldehyde and then blotted to Hybond-N+ membranes (Amersham). After prehybridization with SDS buffer (50% formamide, 0.1% N-laurylsarcosine, 7% SDS, 5x SSC, 1 mg/ml yeast RNA, 2% blocking reagent, 50 mM sodium phosphate buffer pH 7.0) for 3 h at 50°C, hybridized with DIG-labeled full-length mouse caveolin-3 cDNA probe overnight. To detect specific signals, we used the DIG Luminescent Detection Kit for Nucleic Acids (Roche Diagnostics). To detect ß-actin mRNA as an internal control, ß-actin cDNA probe was amplified from mouse skeletal muscle using the mouse ß-actin control amplifier set (Clontech) and labeled with DIG.
RT–PCR
cDNA templates were reverse transcribed from 2 µg of mouse muscle total RNA primed with an oligo(dT) primer and then subjected to PCR. For amplification of the endogenous caveolin-3 gene, we used CV3F2 (5'-CCCAGCCTCACAATGATGACCGAAG-3') and CV3R6 (5'-CATGTGAACGCAAAGCCTTGC-3'). For amplification of the transgene, we used CV3F2 and SV40pAR (5'-GCATTCTAGTTGTGGTTTGTCC-3').
Immunohistochemical analysis
Seven micrometer-thick cryosections from transgenic and control mouse quadriceps femoris muscle were fixed with 4% freshly depolymerized paraformaldehyde for 15 min at 4°C. After blocking with normal goat serum, sections were immunostained with a rabbit polyclonal antibody against caveolin-3 (Transduction Laboratories) for 1 h or a rabbit polyclonal antibody against nNOS (a kind gift from Dr Kevin P. Campbell, University of Iowa) for 18 h at room temperature. After extensive washing with phosphate-buffered saline (PBS), sections were incubated with FITC-labeled secondary antibody.
Western blot analysis
Transgenic or control mouse quadriceps femoris muscle was homogenized in 10 vol (v/v) of SDS extraction buffer (80 mM Tris–HCl pH 6.8, 0.115 M sucrose, 10% SDS, 1% ß-mercaptoethanol, 1 mM PMSF, 1 mM benzamidine, 1 mM EDTA) for 10 min at 50°C. After the homogenates were clarified by centrifugation at 15 000 g for 5 min, the supernatants were resolved by electrophoresis on 3–12% SDS–polyacrylamide gels and then electrophoretically transferred to PVDF membranes. The membranes were blocked with 5% milk in PBS and immunoreacted with a monoclonal antibody against ß-dystroglycan (Novo Castra), a rabbit polyclonal antibody against caveolin-3 (Transduction Laboratories) or a rabbit polyclonal antibody against nNOS (Santa Cruz Biotechnology) overnight at room temperature. After washing with PBS containing 0.1% Tween-20, the blots were incubated with horseradish peroxidase-labeled anti-mouse or rabbit IgG antibody. Immunoreactive bands were visualized with ECL (Amersham).
NDP activity assay
Fifteen micrometer-thick cryosections from transgenic and control mouse quadriceps femoris muscle were dried and fixed with 4% paraformaldehyde in PBS for 2 h at 4°C. After a brief rinse with PBS, sections were incubated in 0.2% Triton X-100 in PBS for 20 min at 37°C. The reaction was performed for 1 h in a dark humidified chamber at 37°C in 0.2% Triton X-100, 0.2 mM NADPH and 0.16 mg/ml nitro blue tertrazolium (NBT). The reaction was terminated by washing with water.
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ACKNOWLEDGEMENTS |
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We thank Drs Jeffrey Chamberlain and Kevin P. Campbell for their generous gifts of the MCK promoter and anti-nNOS antibody, respectively. We also thank Ms Sachiko Fujita and Miki Ikeda for their technical assistance. This study was supported by the Research Grant (10B-3 and 11B-1) for Nervous and Mental Disorders from the Ministry of Health and Welfare, research grants (nos 09470156, 09770460, 09877121, 10044319, 11470151, 11470152 and 11670644) from the Ministry of Education, Science, Sports and Culture, and the Health Science Research Grant ‘Research on Brain Science’ (H10-Brain-024).
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +81 86 462 1111; Fax: +81 86 462 1199; Email: ysunada@med.kawasaki-m.ac.jp
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REFERENCES |
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 R. Hnasko and M. P. Lisanti The Biology of Caveolae: Lessons from Caveolin Knockout Mice and Implications for Human Disease Mol. Interv., December 1, 2003; 3(8): 445 - 464. [Abstract] [Full Text] [PDF]
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 R. Gros, T. Afroze, X.-M. You, G. Kabir, R. Van Wert, W. Kalair, A. E. Hoque, I. N. Mungrue, and M. Husain Plasma Membrane Calcium ATPase Overexpression in Arterial Smooth Muscle Increases Vasomotor Responsiveness and Blood Pressure Circ. Res., October 3, 2003; 93(7): 614 - 621. [Abstract] [Full Text] [PDF]
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 B. M. Carlson, J. A. Carlson, E. I. Dedkov, and I. S. McLennan Concentration of Caveolin-3 at the Neuromuscular Junction in Young and Old Rat Skeletal Muscle Fibers J. Histochem. Cytochem., September 1, 2003; 51(9): 1113 - 1118. [Abstract] [Full Text] [PDF]
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 A. Piech, C. Dessy, X. Havaux, O. Feron, and J.-L. Balligand Differential regulation of nitric oxide synthases and their allosteric regulators in heart and vessels of hypertensive rats Cardiovasc Res, February 1, 2003; 57(2): 456 - 467. [Abstract] [Full Text] [PDF]
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P B Massion and J-L Balligand Modulation of cardiac contraction, relaxation and rate by the endothelial nitric oxide synthase (eNOS): lessons from genetically modified mice J. Physiol., January 1, 2003; 546(1): 63 - 75. [Abstract] [Full Text] [PDF]
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 R. H. CROSBIE, R. BARRESI, and K. P. CAMPBELL Loss of sarcolemma nNOS in sarcoglycan-deficient muscle FASEB J, November 1, 2002; 16(13): 1786 - 1791. [Abstract] [Full Text] [PDF]
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 B. Razani, S. E. Woodman, and M. P. Lisanti Caveolae: From Cell Biology to Animal Physiology Pharmacol. Rev., September 1, 2002; 54(3): 431 - 467. [Abstract] [Full Text] [PDF]
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 U. Schara, M. Vorgerd, N. Popovic, B. G.H. Schoser, K. Ricker, and W. Mortier Rippling Muscle Disease in Childhood J Child Neurol, July 1, 2002; 17(7): 483 - 490. [Abstract] [PDF]
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A. J. Carozzi, S. Roy, I. C. Morrow, A. Pol, B. Wyse, J. Clyde-Smith, I. A. Prior, S. J. Nixon, J. F. Hancock, and R. G. Parton Inhibition of Lipid Raft-dependent Signaling by a Dystrophy-associated Mutant of Caveolin-3 J. Biol. Chem., May 10, 2002; 277(20): 17944 - 17949. [Abstract] [Full Text] [PDF]
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 M. Vorgerd, K. Ricker, F. Ziemssen, W. Kress, H. H. Goebel, W. A. Nix, C. Kubisch, B. G.H. Schoser, and W. Mortier A sporadic case of rippling muscle disease caused by a de novo caveolin-3 mutation Neurology, December 26, 2001; 57(12): 2273 - 2277. [Abstract] [Full Text] [PDF]
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