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Caveolin-3 Null Mice Show a Loss of Caveolae, Changes in the Microdomain Distribution of the Dystrophin-Glycoprotein Complex, and T-tubule Abnormalities

2001; Elsevier BV; Volume: 276; Issue: 24 Linguagem: Inglês

10.1074/jbc.m100828200

ISSN

1083-351X

Autores

Ferruccio Galbiati, Jeffrey A. Engelman, Daniela Volonté, Xiao Lan Zhang, Carlo Minetti, Maomi Li, Harry Hou, Burkhard Kneitz, Winfried Edelmann, Michael P. Lisanti,

Tópico(s)

Metabolism, Diabetes, and Cancer

Resumo

Caveolin-3, a muscle-specific caveolin-related protein, is the principal structural protein of caveolae membrane domains in striated muscle cells. Recently, we identified a novel autosomal dominant form of limb-girdle muscular dystrophy (LGMD-1C) in humans that is due to mutations within the coding sequence of the human caveolin-3 gene (3p25). These LGMD-1C mutations lead to an ∼95% reduction in caveolin-3 protein expression, i.e. a caveolin-3 deficiency. Here, we created a caveolin-3 null (CAV3 −/−) mouse model, using standard homologous recombination techniques, to mimic a caveolin-3 deficiency. We show that these mice lack caveolin-3 protein expression and sarcolemmal caveolae membranes. In addition, analysis of skeletal muscle tissue from these caveolin-3 null mice reveals: (i) mild myopathic changes; (ii) an exclusion of the dystrophin-glycoprotein complex from lipid raft domains; and (iii) abnormalities in the organization of the T-tubule system, with dilated and longitudinally oriented T-tubules. These results have clear mechanistic implications for understanding the pathogenesis of LGMD-1C at a molecular level. Caveolin-3, a muscle-specific caveolin-related protein, is the principal structural protein of caveolae membrane domains in striated muscle cells. Recently, we identified a novel autosomal dominant form of limb-girdle muscular dystrophy (LGMD-1C) in humans that is due to mutations within the coding sequence of the human caveolin-3 gene (3p25). These LGMD-1C mutations lead to an ∼95% reduction in caveolin-3 protein expression, i.e. a caveolin-3 deficiency. Here, we created a caveolin-3 null (CAV3 −/−) mouse model, using standard homologous recombination techniques, to mimic a caveolin-3 deficiency. We show that these mice lack caveolin-3 protein expression and sarcolemmal caveolae membranes. In addition, analysis of skeletal muscle tissue from these caveolin-3 null mice reveals: (i) mild myopathic changes; (ii) an exclusion of the dystrophin-glycoprotein complex from lipid raft domains; and (iii) abnormalities in the organization of the T-tubule system, with dilated and longitudinally oriented T-tubules. These results have clear mechanistic implications for understanding the pathogenesis of LGMD-1C at a molecular level. wild-type limb-girdle muscular dystrophy knockout band knockout mice monoclonal antibody kilobase pair(s) Tris-buffered saline with Tween 20 4-morpholineethanesulfonic acid phosphate-buffered saline polyacrylamide gel electrophoresis ryanodine receptor dihydropyridine receptor Caveolae are 50–100-nm vesicular invaginations of the plasma membrane that participate in vesicular trafficking events and signal transduction processes (1Lisanti M.P. Scherer P. Tang Z.-L. Sargiacomo M. Trends Cell Biol. 1994; 4: 231-235Abstract Full Text PDF PubMed Scopus (587) Google Scholar, 2Couet J. Li S. Okamoto T. Scherer P.S. Lisanti M.P. Trends Cardiovasc. Med. 1997; 7: 103-110Crossref PubMed Scopus (111) Google Scholar, 3Okamoto T. Schlegel A. Scherer P.E. Lisanti M.P. J. Biol. Chem. 1998; 273: 5419-5422Abstract Full Text Full Text PDF PubMed Scopus (1338) Google Scholar, 4Engelman J.A. Zhang X.L. Galbiati F. Volonte D. Sotogia F. Pestell R.G. Minetti C. Scherer P.E. Okamoto T. Lisanti M.P. Am. J. Hum. Genet. 1998; 63: 1578-1587Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 5Smart E.J. Graf G.A. McNiven M.A. Sessa W.C. Engelman J.A. Scherer P.E. Okamoto T. Lisanti M.P. Mol. Cell. Biol. 1999; 19: 304-7289Crossref Scopus (918) Google Scholar). Caveolin, a 21–24-kDa integral membrane protein, is a principal component of caveolae membranes in vivo (6Glenney J.R. Soppet D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10517-10521Crossref PubMed Scopus (339) Google Scholar, 7Glenney J.R. FEBS Lett. 1992; 314: 45-48Crossref PubMed Scopus (189) Google Scholar, 8Glenney J.R. J. Biol. Chem. 1989; 264: 20163-20166Abstract Full Text PDF PubMed Google Scholar, 9Rothberg K.G. Heuser J.E. Donzell W.C. Ying Y. Glenney J.R. Anderson R.G.W. Cell. 1992; 68: 673-682Abstract Full Text PDF PubMed Scopus (1853) Google Scholar, 10Kurzchalia T. Dupree P. Parton R.G. Kellner R. Virta H. Lehnert M. Simons K. J. Cell Biol. 1992; 118: 1003-1014Crossref PubMed Scopus (462) Google Scholar). Caveolin is only the first member of a new gene family; as a consequence, caveolin has been re-termed caveolin-1 (11Scherer P.E. Okamoto T. Chun M. Nishimoto I. Lodish H.F. Lisanti M.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 131-135Crossref PubMed Scopus (490) Google Scholar).The mammalian caveolin gene family now consists of caveolin-1, -2, and -3 (3Okamoto T. Schlegel A. Scherer P.E. Lisanti M.P. J. Biol. Chem. 1998; 273: 5419-5422Abstract Full Text Full Text PDF PubMed Scopus (1338) Google Scholar, 11Scherer P.E. Okamoto T. Chun M. Nishimoto I. Lodish H.F. Lisanti M.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 131-135Crossref PubMed Scopus (490) Google Scholar, 12Parton R.G. Curr. Opin. Cell Biol. 1996; 8: 542-548Crossref PubMed Scopus (494) Google Scholar, 13Tang Z.-L. Scherer P.E. Okamoto T. Song K. Chu C. Kohtz D.S. Nishimoto I. Lodish H.F. Lisanti M.P. J. Biol. Chem. 1996; 271: 2255-2261Abstract Full Text Full Text PDF PubMed Scopus (605) Google Scholar). Caveolin-1 and -2 are co-expressed and form a hetero-oligomeric complex (14Scherer P.E. Lewis R.Y. Volonte D. Engelman J.A. Galbiati F. Couet J. Kohtz D.S. van Donselaar E. Peters P. Lisanti M.P. J. Biol. Chem. 1997; 272: 29337-29346Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar) in many cell types, with particularly high levels in adipocytes, whereas expression of caveolin-3 is muscle-specific and found in both cardiac and skeletal muscle, as well as smooth muscle cells (15Song K.S. Scherer P.E. Tang Z.-L. Okamoto T. Li S. Chafel M. Chu C. Kohtz D.S. Lisanti M.P. J. Biol. Chem. 1996; 271: 15160-15165Abstract Full Text Full Text PDF PubMed Scopus (606) Google Scholar). Expression of caveolin-3 is induced during the differentiation of skeletal myoblasts, and caveolin-3 is localized to the muscle cell plasma membrane (sarcolemma), where it forms a complex with dystrophin and its associated glycoproteins (15Song K.S. Scherer P.E. Tang Z.-L. Okamoto T. Li S. Chafel M. Chu C. Kohtz D.S. Lisanti M.P. J. Biol. Chem. 1996; 271: 15160-15165Abstract Full Text Full Text PDF PubMed Scopus (606) Google Scholar). It has been proposed that caveolin family members function as scaffolding proteins (16Sargiacomo M. Scherer P.E. Tang Z.-L. Kubler E. Song K.S. Sanders M.C. Lisanti M.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9407-9411Crossref PubMed Scopus (475) Google Scholar) to organize and concentrate specific lipids (cholesterol and glycosphingolipids; Refs. 17Li S. Song K.S. Lisanti M.P. J. Biol. Chem. 1996; 271: 568-573Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 18Murata M. Peranen J. Schreiner R. Weiland F. Kurzchalia T. Simons K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10339-10343Crossref PubMed Scopus (761) Google Scholar, 19Fra A.M. Masserini M. Palestini P. Sonnino S. Simons K. FEBS Lett. 1995; 375: 11-14Crossref PubMed Scopus (160) Google Scholar) and lipid modified signaling molecules (Src-like kinases, Ha-Ras, endothelial nitric-oxide synthase, and G-proteins; Refs. 17Li S. Song K.S. Lisanti M.P. J. Biol. Chem. 1996; 271: 568-573Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar and 20Li S. Okamoto T. Chun M. Sargiacomo M. Casanova J.E. Hansen S.H. Nishimoto I. Lisanti M.P. J. Biol. Chem. 1995; 270: 15693-15701Abstract Full Text Full Text PDF PubMed Scopus (556) Google Scholar, 21Song K.S. Li S. Okamoto T. Quilliam L. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1996; 271: 9690-9697Abstract Full Text Full Text PDF PubMed Scopus (916) Google Scholar, 22Li S. Couet J. Lisanti M.P. J. Biol. Chem. 1996; 271: 29182-29190Abstract Full Text Full Text PDF PubMed Scopus (670) Google Scholar, 23Shaul P.W. Smart E.J. Robinson L.J. German Z. Yuhanna I.S. Ying Y. Anderson R.G.W. Michel T. J. Biol. Chem. 1996; 271: 6518-6522Abstract Full Text Full Text PDF PubMed Scopus (624) Google Scholar, 24Garcia-Cardena G. Oh P. Liu J. Schnitzer J.E. Sessa W.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6448-6453Crossref PubMed Scopus (573) Google Scholar) within caveolae membranes.Caveolin-3 is most closely related to caveolin-1 based on protein sequence homology; caveolin-1 and caveolin-3 are ∼65% identical and ∼85% similar (13Tang Z.-L. Scherer P.E. Okamoto T. Song K. Chu C. Kohtz D.S. Nishimoto I. Lodish H.F. Lisanti M.P. J. Biol. Chem. 1996; 271: 2255-2261Abstract Full Text Full Text PDF PubMed Scopus (605) Google Scholar). However, caveolin-3 mRNA is expressed predominantly in muscle tissue types (skeletal muscle, diaphragm, and heart) (13Tang Z.-L. Scherer P.E. Okamoto T. Song K. Chu C. Kohtz D.S. Nishimoto I. Lodish H.F. Lisanti M.P. J. Biol. Chem. 1996; 271: 2255-2261Abstract Full Text Full Text PDF PubMed Scopus (605) Google Scholar). Identification of a muscle-specific member of the caveolin gene family has implications for understanding the role of caveolins in different muscle cell types, as previous morphological studies have demonstrated that caveolae are abundant in these cells. This indicates that muscle cell caveolae may play an important role in muscle membrane biology.Tight regulation of caveolin-3 expression appears essential for maintaining normal muscle homeostasis, as we have demonstrated that transgenic overexpression of wild-type (WT)1 caveolin-3 in mouse skeletal muscle fibers induces a Duchenne-like muscular dystrophy phenotype (25Galbiati F. Volonte D. Chu J.B. Li M. Fine S.W. Fu M. Bermudez J. Pedemonte M. Weidenheim K.M. Pestell R.G. Minetti C. Lisanti M.P. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 94-9689Crossref Scopus (130) Google Scholar). Analysis of skeletal muscle tissue from transgenic mice overexpressing caveolin-3 revealed: (i) a dramatic increase in sarcolemmal caveolae; (ii) hypertrophic, necrotic, and regenerating skeletal muscle fibers with central nuclei; and (iii) down-regulation of dystrophin and β-dystroglycan protein expression.One possibility is that overexpression of wild-type caveolin-3 disrupts the normal processing or stoichiometry of the dystrophin complex, leading to its degradation. In support of this hypothesis, we have recently demonstrated that a novel WW-like domain within caveolin-3 directly recognizes the extreme C terminus of β-dystroglycan that contains a PPXY motif (26Sotgia F. Lee J.K. Das K. Bedford M. Petrucci T.C. Macioce P. Sargiacomo M. Bricarelli F.D. Minetti C. Sudol M. Lisanti M.P. J. Biol. Chem. 2000; 275: 38048-38058Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). As the WW domain of dystrophin recognizes the same site within β-dystroglycan, caveolin-3 can effectively block the interaction of dystrophin with β-dystroglycanin vitro (26Sotgia F. Lee J.K. Das K. Bedford M. Petrucci T.C. Macioce P. Sargiacomo M. Bricarelli F.D. Minetti C. Sudol M. Lisanti M.P. J. Biol. Chem. 2000; 275: 38048-38058Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar), suggesting competitive regulation of the recruitment of dystrophin to the sarcolemma in vivo.In collaboration with Minetti and colleagues (27Minetti C. Sotgia F. Bruno C. Scartezzini P. Broda P. Bado M. Masetti E. Mazzocco P. Egeo A. Donati M.A. Volonte D. Galbiati F. Cordone G. Bricarelli F.D. Lisanti M.P. Zara F. Nat. Genet. 1998; 18: 365-368Crossref PubMed Scopus (487) Google Scholar), we have identified an autosomal dominant form of limb-girdle muscular dystrophy (LGMD-1C) in two Italian families that is due to a deficiency in caveolin-3 expression. Analysis of their genomic DNA reveals two distinct mutations in caveolin-3: (i) a 9-base pair microdeletion that removes the sequence TFT from the caveolin scaffolding domain, and (ii) a missense mutation that changes a proline to a leucine (Pro → Leu) in the transmembrane domain (27Minetti C. Sotgia F. Bruno C. Scartezzini P. Broda P. Bado M. Masetti E. Mazzocco P. Egeo A. Donati M.A. Volonte D. Galbiati F. Cordone G. Bricarelli F.D. Lisanti M.P. Zara F. Nat. Genet. 1998; 18: 365-368Crossref PubMed Scopus (487) Google Scholar). Both mutations lead to a loss of ∼90–95% of caveolin-3 protein expression.Using heterologous expression in cultured cells, we have recently demonstrated that LGMD-1C mutants of caveolin-3 behave in a dominant-negative fashion, causing the intracellular retention and degradation of wild-type caveolin-3 via the proteasome system (28Galbiati F. Volonte D. Minetti C. Chu J.B. Lisanti M.P. J. Biol. Chem. 1999; 274: 25632-25641Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Interestingly, treatment with proteasomal inhibitors blocks the dominant negative effect of LGMD-1C mutants and rescues wild-type caveolin-3 (29Galbiati F. Volonte D. Minetti C. Bregman D.B. Lisanti M.P. J. Biol. Chem. 2000; 275: 37702-37711Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar).Here, using a gene targeting approach, we generated mice lacking caveolin-3 protein expression (CAV3 −/−). Analysis of skeletal muscle fibers from these caveolin-3 null mice reveals mild myopathic changes, with a loss of sarcolemmal caveolae, that is consistent with what is observed in patients with LGMD-1C. In addition, skeletal muscle fibers from these caveolin-3 null mice are characterized by alterations in targeting of the dystrophin-glycoprotein complex to lipid raft microdomains and abnormalities in the organization of the T-tubule system. These data suggest that mislocalization of the dystrophin complex and abnormal T-tubule development may underlie the pathogenesis of LGMD-1C in humans.DISCUSSIONLGMD-1C is an autosomal dominant form of limb-girdle muscular dystrophy that is genetically caused by mutations within the coding regions of the caveolin-3 gene. In collaboration with Minetti and colleagues (27Minetti C. Sotgia F. Bruno C. Scartezzini P. Broda P. Bado M. Masetti E. Mazzocco P. Egeo A. Donati M.A. Volonte D. Galbiati F. Cordone G. Bricarelli F.D. Lisanti M.P. Zara F. Nat. Genet. 1998; 18: 365-368Crossref PubMed Scopus (487) Google Scholar), we recently identified two different Italian families with this autosomal dominant form of limb-girdle muscular dystrophy that is due to a deficiency in caveolin-3 expression. In these patients, by quantitative immunofluorescence and Western blot analysis, the levels of the caveolin-3 protein were reduced by ∼90–95%. Additionally, muscle biopsies from these patients showed muscle damage of mild-to-moderate severity (27Minetti C. Sotgia F. Bruno C. Scartezzini P. Broda P. Bado M. Masetti E. Mazzocco P. Egeo A. Donati M.A. Volonte D. Galbiati F. Cordone G. Bricarelli F.D. Lisanti M.P. Zara F. Nat. Genet. 1998; 18: 365-368Crossref PubMed Scopus (487) Google Scholar).Here, we created a caveolin-3-deficient (CAV3 −/−) mouse model, using standard homologous recombination techniques, to mimic the situation in patients with LGMD-1C. Importantly, loss of caveolin-3 protein expression resulted in loss of caveolae at the sarcolemma (Fig.2 C). This result clearly indicates that caveolin-3 is required for caveolae formation in skeletal muscle cells in vivo. Analysis of skeletal muscle tissue from caveolin-3 null mice revealed mild myopathic changes, with variability in the size of the muscle fibers and the presence of necrotic fibers (Fig. 3). Taken together, these results indicate that loss of caveolin-3 and sarcolemmal caveolae is sufficient to induce a mild myopathy that is similar in its severity to that seen in patients with LGMD-1C.In patients with LGMD-1C, the level of expression and sarcolemma localization of dystrophin and dystrophin-associated glycoproteins is not affected by loss of caveolin-3 expression (27Minetti C. Sotgia F. Bruno C. Scartezzini P. Broda P. Bado M. Masetti E. Mazzocco P. Egeo A. Donati M.A. Volonte D. Galbiati F. Cordone G. Bricarelli F.D. Lisanti M.P. Zara F. Nat. Genet. 1998; 18: 365-368Crossref PubMed Scopus (487) Google Scholar). Similarly, analysis of skeletal muscle fibers from caveolin-3 null mice showed that the expression levels and macroscopic localization of dystrophin, α-sarcoglycan, and β-dystroglycan was not affected (Fig. 4,A and B).However, the precise distribution of dystrophin and its associated glycoproteins within the sarcolemma in absence of caveolin-3 protein expression has not been addressed. Here, we demonstrate that dystrophin, α-sarcoglycan, and β-dystroglycan are all excluded from cholesterol-sphingolipid rafts, in the absence of caveolin-3 expression (Fig. 4 C). Thus, one function of caveolin-3 is to recruit the dystrophin-glycoprotein complex to cholesterol-sphingolipid rafts/caveolae in normal muscle fibers. As dystrophin and dystrophin-associated glycoproteins are important for normal skeletal muscle functioning, these findings suggest a possible mechanism for understanding the pathogenesis of LGMD-1C in humans.In fully differentiated skeletal muscle fibers, caveolin-3 is associated with sarcolemmal caveolae (53Parton R.G. Way M. Zorzi N. Stang E. J. Cell Biol. 1997; 136: 137-154Crossref PubMed Scopus (295) Google Scholar). However, early morphological studies suggested that T-tubules form from the repeated budding of caveolae (37Franzini-Armstrong C. Dev. Biol. 1991; 146: 63-353Google Scholar, 54Ishikawa H. J. Cell Biol. 1968; 38: 51-66Crossref PubMed Scopus (188) Google Scholar). In addition, caveolin-3 is transiently associated with T-tubules during the differentiation of primary cultured cells and the development of mouse skeletal muscle fibers (53Parton R.G. Way M. Zorzi N. Stang E. J. Cell Biol. 1997; 136: 137-154Crossref PubMed Scopus (295) Google Scholar). These results suggest that a functional relationship may exist between caveolin-3 expression, caveolae formation, and T-tubule biogenesis. It remains unknown whether caveolin-3 expression is required for proper T-tubule biogenesis.Thus, we next assessed the status of the T-tubule system in caveolin-3 null mice. Here, we show that two T-tubule marker proteins (dihydropyridine receptor-1α and ryanodine receptor) are mislocalized in skeletal muscle fibers from caveolin-3 null mice. The localization of these marker proteins also provides an indication that the T-tubule system is disorganized or immature in caveolin-3 null mice (Figs. 5(B–E) and 6).In accordance with this interpretation, electron micrographs of longitudinal sections from caveolin-3 null mice indicate that the T-tubules are dilated/swollen and run in irregular directions (Fig. 7,A and B). Interestingly, the T-tubule network has an exclusively longitudinal orientation at early stages of muscle differentiation and only becomes transversely oriented in fully differentiated skeletal muscle fibers (37Franzini-Armstrong C. Dev. Biol. 1991; 146: 63-353Google Scholar). As the T-tubule system in caveolin-3 null mice showed a clear tendency to run longitudinally, these results suggest that caveolin-3 expression and caveolae formation are required to generate a highly organized/fully mature T-tubule system in vivo. Thus, a disorganized immature T-tubule system may also contribute to the pathogenesis of LGMD-1C in humans. Caveolae are 50–100-nm vesicular invaginations of the plasma membrane that participate in vesicular trafficking events and signal transduction processes (1Lisanti M.P. Scherer P. Tang Z.-L. Sargiacomo M. Trends Cell Biol. 1994; 4: 231-235Abstract Full Text PDF PubMed Scopus (587) Google Scholar, 2Couet J. Li S. Okamoto T. Scherer P.S. Lisanti M.P. Trends Cardiovasc. Med. 1997; 7: 103-110Crossref PubMed Scopus (111) Google Scholar, 3Okamoto T. Schlegel A. Scherer P.E. Lisanti M.P. J. Biol. Chem. 1998; 273: 5419-5422Abstract Full Text Full Text PDF PubMed Scopus (1338) Google Scholar, 4Engelman J.A. Zhang X.L. Galbiati F. Volonte D. Sotogia F. Pestell R.G. Minetti C. Scherer P.E. Okamoto T. Lisanti M.P. Am. J. Hum. Genet. 1998; 63: 1578-1587Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 5Smart E.J. Graf G.A. McNiven M.A. Sessa W.C. Engelman J.A. Scherer P.E. Okamoto T. Lisanti M.P. Mol. Cell. Biol. 1999; 19: 304-7289Crossref Scopus (918) Google Scholar). Caveolin, a 21–24-kDa integral membrane protein, is a principal component of caveolae membranes in vivo (6Glenney J.R. Soppet D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10517-10521Crossref PubMed Scopus (339) Google Scholar, 7Glenney J.R. FEBS Lett. 1992; 314: 45-48Crossref PubMed Scopus (189) Google Scholar, 8Glenney J.R. J. Biol. Chem. 1989; 264: 20163-20166Abstract Full Text PDF PubMed Google Scholar, 9Rothberg K.G. Heuser J.E. Donzell W.C. Ying Y. Glenney J.R. Anderson R.G.W. Cell. 1992; 68: 673-682Abstract Full Text PDF PubMed Scopus (1853) Google Scholar, 10Kurzchalia T. Dupree P. Parton R.G. Kellner R. Virta H. Lehnert M. Simons K. J. Cell Biol. 1992; 118: 1003-1014Crossref PubMed Scopus (462) Google Scholar). Caveolin is only the first member of a new gene family; as a consequence, caveolin has been re-termed caveolin-1 (11Scherer P.E. Okamoto T. Chun M. Nishimoto I. Lodish H.F. Lisanti M.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 131-135Crossref PubMed Scopus (490) Google Scholar). The mammalian caveolin gene family now consists of caveolin-1, -2, and -3 (3Okamoto T. Schlegel A. Scherer P.E. Lisanti M.P. J. Biol. Chem. 1998; 273: 5419-5422Abstract Full Text Full Text PDF PubMed Scopus (1338) Google Scholar, 11Scherer P.E. Okamoto T. Chun M. Nishimoto I. Lodish H.F. Lisanti M.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 131-135Crossref PubMed Scopus (490) Google Scholar, 12Parton R.G. Curr. Opin. Cell Biol. 1996; 8: 542-548Crossref PubMed Scopus (494) Google Scholar, 13Tang Z.-L. Scherer P.E. Okamoto T. Song K. Chu C. Kohtz D.S. Nishimoto I. Lodish H.F. Lisanti M.P. J. Biol. Chem. 1996; 271: 2255-2261Abstract Full Text Full Text PDF PubMed Scopus (605) Google Scholar). Caveolin-1 and -2 are co-expressed and form a hetero-oligomeric complex (14Scherer P.E. Lewis R.Y. Volonte D. Engelman J.A. Galbiati F. Couet J. Kohtz D.S. van Donselaar E. Peters P. Lisanti M.P. J. Biol. Chem. 1997; 272: 29337-29346Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar) in many cell types, with particularly high levels in adipocytes, whereas expression of caveolin-3 is muscle-specific and found in both cardiac and skeletal muscle, as well as smooth muscle cells (15Song K.S. Scherer P.E. Tang Z.-L. Okamoto T. Li S. Chafel M. Chu C. Kohtz D.S. Lisanti M.P. J. Biol. Chem. 1996; 271: 15160-15165Abstract Full Text Full Text PDF PubMed Scopus (606) Google Scholar). Expression of caveolin-3 is induced during the differentiation of skeletal myoblasts, and caveolin-3 is localized to the muscle cell plasma membrane (sarcolemma), where it forms a complex with dystrophin and its associated glycoproteins (15Song K.S. Scherer P.E. Tang Z.-L. Okamoto T. Li S. Chafel M. Chu C. Kohtz D.S. Lisanti M.P. J. Biol. Chem. 1996; 271: 15160-15165Abstract Full Text Full Text PDF PubMed Scopus (606) Google Scholar). It has been proposed that caveolin family members function as scaffolding proteins (16Sargiacomo M. Scherer P.E. Tang Z.-L. Kubler E. Song K.S. Sanders M.C. Lisanti M.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9407-9411Crossref PubMed Scopus (475) Google Scholar) to organize and concentrate specific lipids (cholesterol and glycosphingolipids; Refs. 17Li S. Song K.S. Lisanti M.P. J. Biol. Chem. 1996; 271: 568-573Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 18Murata M. Peranen J. Schreiner R. Weiland F. Kurzchalia T. Simons K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10339-10343Crossref PubMed Scopus (761) Google Scholar, 19Fra A.M. Masserini M. Palestini P. Sonnino S. Simons K. FEBS Lett. 1995; 375: 11-14Crossref PubMed Scopus (160) Google Scholar) and lipid modified signaling molecules (Src-like kinases, Ha-Ras, endothelial nitric-oxide synthase, and G-proteins; Refs. 17Li S. Song K.S. Lisanti M.P. J. Biol. Chem. 1996; 271: 568-573Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar and 20Li S. Okamoto T. Chun M. Sargiacomo M. Casanova J.E. Hansen S.H. Nishimoto I. Lisanti M.P. J. Biol. Chem. 1995; 270: 15693-15701Abstract Full Text Full Text PDF PubMed Scopus (556) Google Scholar, 21Song K.S. Li S. Okamoto T. Quilliam L. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1996; 271: 9690-9697Abstract Full Text Full Text PDF PubMed Scopus (916) Google Scholar, 22Li S. Couet J. Lisanti M.P. J. Biol. Chem. 1996; 271: 29182-29190Abstract Full Text Full Text PDF PubMed Scopus (670) Google Scholar, 23Shaul P.W. Smart E.J. Robinson L.J. German Z. Yuhanna I.S. Ying Y. Anderson R.G.W. Michel T. J. Biol. Chem. 1996; 271: 6518-6522Abstract Full Text Full Text PDF PubMed Scopus (624) Google Scholar, 24Garcia-Cardena G. Oh P. Liu J. Schnitzer J.E. Sessa W.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6448-6453Crossref PubMed Scopus (573) Google Scholar) within caveolae membranes. Caveolin-3 is most closely related to caveolin-1 based on protein sequence homology; caveolin-1 and caveolin-3 are ∼65% identical and ∼85% similar (13Tang Z.-L. Scherer P.E. Okamoto T. Song K. Chu C. Kohtz D.S. Nishimoto I. Lodish H.F. Lisanti M.P. J. Biol. Chem. 1996; 271: 2255-2261Abstract Full Text Full Text PDF PubMed Scopus (605) Google Scholar). However, caveolin-3 mRNA is expressed predominantly in muscle tissue types (skeletal muscle, diaphragm, and heart) (13Tang Z.-L. Scherer P.E. Okamoto T. Song K. Chu C. Kohtz D.S. Nishimoto I. Lodish H.F. Lisanti M.P. J. Biol. Chem. 1996; 271: 2255-2261Abstract Full Text Full Text PDF PubMed Scopus (605) Google Scholar). Identification of a muscle-specific member of the caveolin gene family has implications for understanding the role of caveolins in different muscle cell types, as previous morphological studies have demonstrated that caveolae are abundant in these cells. This indicates that muscle cell caveolae may play an important role in muscle membrane biology. Tight regulation of caveolin-3 expression appears essential for maintaining normal muscle homeostasis, as we have demonstrated that transgenic overexpression of wild-type (WT)1 caveolin-3 in mouse skeletal muscle fibers induces a Duchenne-like muscular dystrophy phenotype (25Galbiati F. Volonte D. Chu J.B. Li M. Fine S.W. Fu M. Bermudez J. Pedemonte M. Weidenheim K.M. Pestell R.G. Minetti C. Lisanti M.P. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 94-9689Crossref Scopus (130) Google Scholar). Analysis of skeletal muscle tissue from transgenic mice overexpressing caveolin-3 revealed: (i) a dramatic increase in sarcolemmal caveolae; (ii) hypertrophic, necrotic, and regenerating skeletal muscle fibers with central nuclei; and (iii) down-regulation of dystrophin and β-dystroglycan protein expression. One possibility is that overexpression of wild-type caveolin-3 disrupts the normal processing or stoichiometry of the dystrophin complex, leading to its degradation. In support of this hypothesis, we have recently demonstrated that a novel WW-like domain within caveolin-3 directly recognizes the extreme C terminus of β-dystroglycan that contains a PPXY motif (26Sotgia F. Lee J.K. Das K. Bedford M. Petrucci T.C. Macioce P. Sargiacomo M. Bricarelli F.D. Minetti C. Sudol M. Lisanti M.P. J. Biol. Chem. 2000; 275: 38048-38058Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). As the WW domain of dystrophin recognizes the same site within β-dystroglycan, caveolin-3 can effectively block the interaction of dystrophin with β-dystroglycanin vitro (26Sotgia F. Lee J.K. Das K. Bedford M. Petrucci T.C. Macioce P. Sargiacomo M. Bricarelli F.D. Minetti C. Sudol M. Lisanti M.P. J. Biol. Chem. 2000; 275: 38048-38058Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar), suggesting competitive regulation of the recruitment of dystrophin to the sarcolemma in vivo. In collaboration with Minetti and colleagues (27Minetti C. Sotgia F. Bruno C. Scartezzini P. Broda P. Bado M. Masetti E. Mazzocco P. Egeo A. Donati M.A. Volonte D. Galbiati F. Cordone G. Bricarelli F.D. Lisanti M.P. Zara F. Nat. Genet. 1998; 18: 365-368Crossref PubMed Scopus (487) Google Scholar), we have identified an autosomal dominant form of limb-girdle muscular dystrophy (LGMD-1C) in two Italian families that is due to a deficiency in caveolin-3 expression. Analysis of their genomic DNA reveals two distinct mutations in caveolin-3: (i) a 9-base pair microdeletion that removes the sequence TFT from the caveolin scaffolding domain, and (ii) a missense mutation that changes a proline to a leucine (Pro → Leu) in the transmembrane domain (27Minetti C. Sotgia F. Bruno C. Scartezzini P. Broda P. Bado M. Masetti E. Mazzocco P. Egeo A. Donati M.A. Volonte D. Galbiati F. Cordone G. Bricarelli F.D. Lisanti M.P. Zara F. Nat. Genet. 1998; 18: 365-368Crossref PubMed Scopus (487) Google Scholar). Both mutations lead to a loss of ∼90–95% of caveolin-3 protein expression. Using heterologous expression in cultured cells, we have recently demonstrated that LGMD-1C mutants of caveolin-3 behave in a dominant-negative fashion, causing the intracellular retention and degradation of wild-type caveolin-3 via the proteasome system (28Galbiati F. Volonte D. Minetti C. Chu J.B. Lisanti M.P. J. Biol. Chem. 1999; 274: 25632-25641Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Interestingly, treatment with proteasomal inhibitors blocks the dominant negative effect of LGMD-1C mutants and rescues wild-type caveolin-3 (29Galbiati F. Volonte D. Minetti C. Bregman D.B. Lisanti M.P. J. Biol. Chem. 2000; 275: 37702-37711Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Here, using a gene targeting approach, we generated mice lacking caveolin-3 protein expression (CAV3 −/−). Analysis of skeletal muscle fibers from these caveolin-3 null mice reveals mild myopathic changes, with a loss of sarcolemmal caveolae, that is consistent with what is observed in patients with LGMD-1C. In addition, skeletal muscle fibers from these caveolin-3 null mice are characterized by alterations in targeting of the dystrophin-glycoprotein complex to lipid raft microdomains and abnormalities in the organization of the T-tubule system. These data suggest that mislocalization of the dystrophin complex and abnormal T-tubule development may underlie the pathogenesis of LGMD-1C in humans. DISCUSSIONLGMD-1C is an autosomal dominant form of limb-girdle muscular dystrophy that is genetically caused by mutations within the coding regions of the caveolin-3 gene. In collaboration with Minetti and colleagues (27Minetti C. Sotgia F. Bruno C. Scartezzini P. Broda P. Bado M. Masetti E. Mazzocco P. Egeo A. Donati M.A. Volonte D. Galbiati F. Cordone G. Bricarelli F.D. Lisanti M.P. Zara F. Nat. Genet. 1998; 18: 365-368Crossref PubMed Scopus (487) Google Scholar), we recently identified two different Italian families with this autosomal dominant form of limb-girdle muscular dystrophy that is due to a deficiency in caveolin-3 expression. In these patients, by quantitative immunofluorescence and Western blot analysis, the levels of the caveolin-3 protein were reduced by ∼90–95%. Additionally, muscle biopsies from these patients showed muscle damage of mild-to-moderate severity (27Minetti C. Sotgia F. Bruno C. Scartezzini P. Broda P. Bado M. Masetti E. Mazzocco P. Egeo A. Donati M.A. Volonte D. Galbiati F. Cordone G. Bricarelli F.D. Lisanti M.P. Zara F. Nat. Genet. 1998; 18: 365-368Crossref PubMed Scopus (487) Google Scholar).Here, we created a caveolin-3-deficient (CAV3 −/−) mouse model, using standard homologous recombination techniques, to mimic the situation in patients with LGMD-1C. Importantly, loss of caveolin-3 protein expression resulted in loss of caveolae at the sarcolemma (Fig.2 C). This result clearly indicates that caveolin-3 is required for caveolae formation in skeletal muscle cells in vivo. Analysis of skeletal muscle tissue from caveolin-3 null mice revealed mild myopathic changes, with variability in the size of the muscle fibers and the presence of necrotic fibers (Fig. 3). Taken together, these results indicate that loss of caveolin-3 and sarcolemmal caveolae is sufficient to induce a mild myopathy that is similar in its severity to that seen in patients with LGMD-1C.In patients with LGMD-1C, the level of expression and sarcolemma localization of dystrophin and dystrophin-associated glycoproteins is not affected by loss of caveolin-3 expression (27Minetti C. Sotgia F. Bruno C. Scartezzini P. Broda P. Bado M. Masetti E. Mazzocco P. Egeo A. Donati M.A. Volonte D. Galbiati F. Cordone G. Bricarelli F.D. Lisanti M.P. Zara F. Nat. Genet. 1998; 18: 365-368Crossref PubMed Scopus (487) Google Scholar). Similarly, analysis of skeletal muscle fibers from caveolin-3 null mice showed that the expression levels and macroscopic localization of dystrophin, α-sarcoglycan, and β-dystroglycan was not affected (Fig. 4,A and B).However, the precise distribution of dystrophin and its associated glycoproteins within the sarcolemma in absence of caveolin-3 protein expression has not been addressed. Here, we demonstrate that dystrophin, α-sarcoglycan, and β-dystroglycan are all excluded from cholesterol-sphingolipid rafts, in the absence of caveolin-3 expression (Fig. 4 C). Thus, one function of caveolin-3 is to recruit the dystrophin-glycoprotein complex to cholesterol-sphingolipid rafts/caveolae in normal muscle fibers. As dystrophin and dystrophin-associated glycoproteins are important for normal skeletal muscle functioning, these findings suggest a possible mechanism for understanding the pathogenesis of LGMD-1C in humans.In fully differentiated skeletal muscle fibers, caveolin-3 is associated with sarcolemmal caveolae (53Parton R.G. Way M. Zorzi N. Stang E. J. Cell Biol. 1997; 136: 137-154Crossref PubMed Scopus (295) Google Scholar). However, early morphological studies suggested that T-tubules form from the repeated budding of caveolae (37Franzini-Armstrong C. Dev. Biol. 1991; 146: 63-353Google Scholar, 54Ishikawa H. J. Cell Biol. 1968; 38: 51-66Crossref PubMed Scopus (188) Google Scholar). In addition, caveolin-3 is transiently associated with T-tubules during the differentiation of primary cultured cells and the development of mouse skeletal muscle fibers (53Parton R.G. Way M. Zorzi N. Stang E. J. Cell Biol. 1997; 136: 137-154Crossref PubMed Scopus (295) Google Scholar). These results suggest that a functional relationship may exist between caveolin-3 expression, caveolae formation, and T-tubule biogenesis. It remains unknown whether caveolin-3 expression is required for proper T-tubule biogenesis.Thus, we next assessed the status of the T-tubule system in caveolin-3 null mice. Here, we show that two T-tubule marker proteins (dihydropyridine receptor-1α and ryanodine receptor) are mislocalized in skeletal muscle fibers from caveolin-3 null mice. The localization of these marker proteins also provides an indication that the T-tubule system is disorganized or immature in caveolin-3 null mice (Figs. 5(B–E) and 6).In accordance with this interpretation, electron micrographs of longitudinal sections from caveolin-3 null mice indicate that the T-tubules are dilated/swollen and run in irregular directions (Fig. 7,A and B). Interestingly, the T-tubule network has an exclusively longitudinal orientation at early stages of muscle differentiation and only becomes transversely oriented in fully differentiated skeletal muscle fibers (37Franzini-Armstrong C. Dev. Biol. 1991; 146: 63-353Google Scholar). As the T-tubule system in caveolin-3 null mice showed a clear tendency to run longitudinally, these results suggest that caveolin-3 expression and caveolae formation are required to generate a highly organized/fully mature T-tubule system in vivo. Thus, a disorganized immature T-tubule system may also contribute to the pathogenesis of LGMD-1C in humans. LGMD-1C is an autosomal dominant form of limb-girdle muscular dystrophy that is genetically caused by mutations within the coding regions of the caveolin-3 gene. In collaboration with Minetti and colleagues (27Minetti C. Sotgia F. Bruno C. Scartezzini P. Broda P. Bado M. Masetti E. Mazzocco P. Egeo A. Donati M.A. Volonte D. Galbiati F. Cordone G. Bricarelli F.D. Lisanti M.P. Zara F. Nat. Genet. 1998; 18: 365-368Crossref PubMed Scopus (487) Google Scholar), we recently identified two different Italian families with this autosomal dominant form of limb-girdle muscular dystrophy that is due to a deficiency in caveolin-3 expression. In these patients, by quantitative immunofluorescence and Western blot analysis, the levels of the caveolin-3 protein were reduced by ∼90–95%. Additionally, muscle biopsies from these patients showed muscle damage of mild-to-moderate severity (27Minetti C. Sotgia F. Bruno C. Scartezzini P. Broda P. Bado M. Masetti E. Mazzocco P. Egeo A. Donati M.A. Volonte D. Galbiati F. Cordone G. Bricarelli F.D. Lisanti M.P. Zara F. Nat. Genet. 1998; 18: 365-368Crossref PubMed Scopus (487) Google Scholar). Here, we created a caveolin-3-deficient (CAV3 −/−) mouse model, using standard homologous recombination techniques, to mimic the situation in patients with LGMD-1C. Importantly, loss of caveolin-3 protein expression resulted in loss of caveolae at the sarcolemma (Fig.2 C). This result clearly indicates that caveolin-3 is required for caveolae formation in skeletal muscle cells in vivo. Analysis of skeletal muscle tissue from caveolin-3 null mice revealed mild myopathic changes, with variability in the size of the muscle fibers and the presence of necrotic fibers (Fig. 3). Taken together, these results indicate that loss of caveolin-3 and sarcolemmal caveolae is sufficient to induce a mild myopathy that is similar in its severity to that seen in patients with LGMD-1C. In patients with LGMD-1C, the level of expression and sarcolemma localization of dystrophin and dystrophin-associated glycoproteins is not affected by loss of caveolin-3 expression (27Minetti C. Sotgia F. Bruno C. Scartezzini P. Broda P. Bado M. Masetti E. Mazzocco P. Egeo A. Donati M.A. Volonte D. Galbiati F. Cordone G. Bricarelli F.D. Lisanti M.P. Zara F. Nat. Genet. 1998; 18: 365-368Crossref PubMed Scopus (487) Google Scholar). Similarly, analysis of skeletal muscle fibers from caveolin-3 null mice showed that the expression levels and macroscopic localization of dystrophin, α-sarcoglycan, and β-dystroglycan was not affected (Fig. 4,A and B). However, the precise distribution of dystrophin and its associated glycoproteins within the sarcolemma in absence of caveolin-3 protein expression has not been addressed. Here, we demonstrate that dystrophin, α-sarcoglycan, and β-dystroglycan are all excluded from cholesterol-sphingolipid rafts, in the absence of caveolin-3 expression (Fig. 4 C). Thus, one function of caveolin-3 is to recruit the dystrophin-glycoprotein complex to cholesterol-sphingolipid rafts/caveolae in normal muscle fibers. As dystrophin and dystrophin-associated glycoproteins are important for normal skeletal muscle functioning, these findings suggest a possible mechanism for understanding the pathogenesis of LGMD-1C in humans. In fully differentiated skeletal muscle fibers, caveolin-3 is associated with sarcolemmal caveolae (53Parton R.G. Way M. Zorzi N. Stang E. J. Cell Biol. 1997; 136: 137-154Crossref PubMed Scopus (295) Google Scholar). However, early morphological studies suggested that T-tubules form from the repeated budding of caveolae (37Franzini-Armstrong C. Dev. Biol. 1991; 146: 63-353Google Scholar, 54Ishikawa H. J. Cell Biol. 1968; 38: 51-66Crossref PubMed Scopus (188) Google Scholar). In addition, caveolin-3 is transiently associated with T-tubules during the differentiation of primary cultured cells and the development of mouse skeletal muscle fibers (53Parton R.G. Way M. Zorzi N. Stang E. J. Cell Biol. 1997; 136: 137-154Crossref PubMed Scopus (295) Google Scholar). These results suggest that a functional relationship may exist between caveolin-3 expression, caveolae formation, and T-tubule biogenesis. It remains unknown whether caveolin-3 expression is required for proper T-tubule biogenesis. Thus, we next assessed the status of the T-tubule system in caveolin-3 null mice. Here, we show that two T-tubule marker proteins (dihydropyridine receptor-1α and ryanodine receptor) are mislocalized in skeletal muscle fibers from caveolin-3 null mice. The localization of these marker proteins also provides an indication that the T-tubule system is disorganized or immature in caveolin-3 null mice (Figs. 5(B–E) and 6). In accordance with this interpretation, electron micrographs of longitudinal sections from caveolin-3 null mice indicate that the T-tubules are dilated/swollen and run in irregular directions (Fig. 7,A and B). Interestingly, the T-tubule network has an exclusively longitudinal orientation at early stages of muscle differentiation and only becomes transversely oriented in fully differentiated skeletal muscle fibers (37Franzini-Armstrong C. Dev. Biol. 1991; 146: 63-353Google Scholar). As the T-tubule system in caveolin-3 null mice showed a clear tendency to run longitudinally, these results suggest that caveolin-3 expression and caveolae formation are required to generate a highly organized/fully mature T-tubule system in vivo. Thus, a disorganized immature T-tubule system may also contribute to the pathogenesis of LGMD-1C in humans. We thank Dr. Frank Macaluso for expertise in T-tubule staining and Jorge Bermudez for help in collecting frozen sections of skeletal muscle tissue.

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