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Human Molecular Genetics Pages 2135-2140  

Collagen VI deficiency induces early onset myopathy in the mouse: an animal model for Bethlem myopathy
Introduction
Results
   Targeted inactivation of the col6a1 gene
   Triple-helical collagen VI molecules are absent in col6a1 -/- mutant mice
   Deficiency of the col6a1 gene induces myopathy
Discussion
Materials And Methods
   Construction of targeting vector
   Embryonic stem cells and animals
   Southern blot analysis
   Embryonic fibroblast cultures
   Northern blot analysis
   Purification of collagen VI from whole animals
   Immunofluorescence analysis
   Histological analysis
Abbreviations
Acknowledgements
References

Collagen VI deficiency induces early onset myopathy in the mouse: an animal model for Bethlem myopathy

Collagen VI deficiency induces early onset myopathy in the mouse: an animal model for Bethlem myopathy

Paolo Bonaldo, Paola Braghetta, Miriam Zanetti, Stefano Piccolo, Dino Volpin and Giorgio M. Bressan*

Institute of Histology and Embryology, University of Padova, Via G.Colombo 3, 35100 Padova, Italy

Received July 24, 1998; Revised and Accepted September 24, 1998

To gain insight into the function of type VI collagen, the col6a1 gene was inactivated by targeted gene disruption in the mouse. The homozygous mutants lacked collagen VI in the tissues and showed histological features of myopathy such as fiber necrosis and phagocytosis and a pronounced variation in the fiber diameter. Muscles also showed signs of stimulated regeneration of fibers. Necrotic fibers were particularly frequent in the diaphragm at all ages examined. Similar, although milder, alterations were detected in heterozygous mutant mice, indicating haploinsufficiency of the col6a1 gene function. The data led us to conclude that collagen VI is necessary for maintenance of the integrity of muscle fibers and that the col6a1-deficient mouse can be considered an animal model of Bethlem myopathy.

INTRODUCTION

Type VI collagen, a major protein of interstitial matrix, is composed of three genetically distinct [alpha] chains. The molecule consists of a central short collagenous region flanked by several von Willebrand factor type A modules at the N- and C-terminal ends (1). Both types of domain participate in specific molecular interactions: the collagenous domain binds cells (2), proteoglycans like decorin, biglycan and fibromodulin and collagen II (3); type A modules associate with collagens type I, IV and VI as well as with fibronectin, heparin and hyaluronic acid (4-6). Secreted collagen VI molecules form an extended microfilament network (7) particularly abundant close to the cells, which has been suggested to play a role in anchoring the basement membrane of non-epithelial cells to the underlying connective tissue (8,9). Linkage and mutation analyses in human have associated the genes encoding the three collagen VI polypeptide chains with Bethlem myopathy (10), an early onset benign myopathy with autosomal dominant inheritance (11,12). To further define the function of type VI collagen, we inactivated the col6a1 gene in the mouse by targeted gene disruption and found that the mutation induces an early onset myopathy.

RESULTS

Targeted inactivation of the col6a1 gene

A targeting vector containing a neomycin resistance cassette in the second exon (Fig. 1a) was used to inactivate the col6a1 gene in embryonic stem cells (ES cells) (Fig. 1b). Heterozygous animals for the null mutation were generated by crossing founder male chimeras with C57BL/6 females. Genotyping of weanlings from heterozygous intercrosses (Fig. 1c) showed the expected Mendelian ratio of allelic distribution. Homozygous mutant mice developed normally, were fertile and no obvious differences with their control littermates were noted during the period of observation, which is now >1 year for the oldest animals. [alpha]1(VI) mRNA was undetectable by northern blot analysis of total RNA extracted from embryo fibroblasts of homozygous mutant mice (Fig. 1d). The lack of this species did not affect the [alpha]2(VI) and [alpha]3(VI) mRNAs levels (Fig. 1d). Hybridization of the filters with a probe derived from the neomycin resistance cassette revealed two bands (Fig. 1d). The bands have the size expected for transcripts starting at the thymidine kinase (tk) promoter within the cassette and at the promoter of the col6a1 gene itself, suggesting that absence of the [alpha]1(VI) mRNA is mostly due to transcription termination at the neomycin resistance cassette.


Figure 1. Generation of col6a1 null mice. (a) (Top) Genomic organization of the 5[prime] portion of the murine col6a1 gene; (middle) targeting construct; (bottom) resulting targeted locus. The first three exons are represented by numbered filled boxes. Insertion of the neo cassette into the second exon interrupts the col6a1 coding sequence at amino acid 66 and increases the DraI fragment recognized by the probe from 5.6 to 6.8 kb. (b) Southern blot analysis of ES cells. wt, parental R1 cells containing the normal 5.6 kb DraI fragment; T, clone containing the targeted allele, as indicated by the presence of the additional band of 6.8 kb. (c) Southern blot analysis of tail biopsies of pups generated by crossing heterozygous F1 mice for the targeted allele. (d) Northern blot analysis of total RNA extracted from primary embryonic fibroblasts obtained from heterozygous F1 intercrosses. Cells which were -/- after genotyping did not express [alpha]1(VI) mRNA, whereas the [alpha]2(VI) and the [alpha]3(VI) mRNAs were not affected. Two bands were detected with the neo probe. The bands have the size expected for the mRNA species transcribed from the tk promoter of the neo cassette (lower band, 1100 bp) and from the promoter of the col6a1 gene (upper band, 1500 bp).

Triple-helical collagen VI molecules are absent in col6a1 -/- mutant mice

Although experimental data accumulated so far favor the [alpha]1(VI)·[alpha]2(VI)·[alpha]3(VI) heterotrimer as the only molecular form of type VI collagen (13), the possibility of triple helical molecules made exclusively of [alpha]2(VI) and [alpha]3(VI) chains cannot be excluded. Therefore, the presence of triple helical collagen VI molecules was carefully analyzed in the homozygous mutant animals. Pepsin-resistant collagen VI was purified from the eviscerated carcasses of one control and one col6a1 -/- mouse. The final resolution of the protein mixture by gel filtration chromatography showed immunoreactivity for collagen VI in a group of fractions from the control animal, whereas the reactivity was almost completely lacking in the extract from the col6a1 -/- mouse (Fig. 2, upper). The absence of peptides with the expected size for the collagenous domains of [alpha]1(VI), [alpha]2(VI) and [alpha]3(VI) chains in the -/- mouse was confirmed by gel electrophoresis and Coomassie blue staining of the fractions (Fig. 2, lower). Thus, as a consequence of the elimination of the [alpha]1(VI) chain, no triple helical collagen VI molecules are produced in the mutant mouse. In keeping with this result, when observed by immunofluorescence collagen VI was not detectable in tissues of col6a1 -/- embryos and the molecule was selectively missing from the extracellular matrix deposited by fibroblasts obtained from -/- embryos (Fig. 3).


Figure 2. Analysis of collagen VI polypeptide chains produced by wild-type and col6a1 null mice. (Top) Purification of pepsin-resistant fragments by gel filtration chromatography. Whole carcasses of mice were homogenized, digested with pepsin, subjected to various steps of salt precipitation and separated in a Sephacryl S-400 column (25). After measuring OD235 (control mouse, dashed line; col6a1 -/- mouse, continuous line), ELISA was performed on individual fractions with rabbit antiserum against collagen VI and analyzed at OD405 (control mouse, open squares; col6a1 -/- mouse, filled circles). (Bottom) Coomassie blue R250 staining of SDS-PAGE gels analyzing the indicated fractions from the Sephacryl S-400 column. Pepsin-resistant [alpha](VI) chains are found in the control (+/+), but not in the homozygous mutant (-/-) mouse. The absence of the polypeptides in the fractions from homozygous null mice was also confirmed by western blotting (data not shown).

Deficiency of the col6a1 gene induces myopathy


Figure 3. Immunodetection analysis of collagen VI. (a and b) Cryostat sections of thoracic wall from 16-day-old wild-type (+/+) and col6a1 null homozygous (-/-) embryos stained by immunofluorescence with rabbit IgG against mouse collagen VI. (c-f) Immunofluorescence analysis of collagen VI synthesized by embryonic fibroblasts. Cells isolated from col6a1 null homozygous (-/-) and wild-type (+/+) 14.5-day-old embryos were treated with rabbit anti-collagen VI IgG (anti-VI) and a monoclonal antibody against fibronectin (anti-FN), followed by the appropriate fluorescent secondary antibody. Collagen VI, but not fibronectin, staining was absent in cells from homozygous mutant mice. Bar, 50 µm.

Despite the absence of clear signs of phenotypic abnormalities by visual inspection of the mice, a detailed comparison of the histology of tissues from homozygous null and wild-type littermates was performed. Alterations in the histological structure were only observed in skeletal muscles of mutant animals, where general signs of myopathy such as muscle necrosis and phagocytosis and a more pronounced variation in fiber diameter were detected (Fig. 4a and b). Vital injection of Evans blue dye, which reveals the presence of hypercontracted or necrotic fibers (14), stained muscle fibers in col6a1 -/-, but not in control animals. The highest frequency of altered fibers was found in diaphragm (up to 20% of fibers affected) (Fig. 4c-f). Other muscles in which necrotic fibers were detected, although at lower frequencies, included intercostal muscles, external oblique and straight abdominal muscles and femoral medial large muscle. Altered fibers were less common in the remaining muscles. Necrotic fibers were present as both small groups or individual entities. Fiber necrosis was also detectable in mice heterozygous for the mutation, where it was less severe (Fig. 4d). An additional histological abnormality was the increase in central nuclei in col6a1 -/- compared with wild-type animals, a finding which indicates muscle fiber regeneration (15). Although not very high, this increase was significant and was observed in both homozygous and heterozygous mice (Table 1). At some sites the percentage of central nuclei was very high (>50%; Fig. 4b). In order to determine when necrosis first appeared and the pattern of its progression, the histological analysis was performed in mice of 3, 10, 20 and 40 days and 4 and 5 months after Evans blue dye injection. Necrosis was present both at all ages examined in homozygous and heterozygous individuals and the pattern of distribution was similar to that described above (data not shown). This indicates that alterations have an early onset and a limited or absent progression and that their pattern of distribution is independent of muscle type. The serum levels of creatine kinase in the mutant animals were normal.

To test if the myopathy impaired performance of the muscular system, 1-month-old col6a1 -/- and wild-type mice were housed in wheel cages and allowed to run for 2 months ad libitum. Although the average daily distance run each week was consistently lower for the mutant mice, the difference between the two groups of animals was not significant (data not shown). Comparable results were obtained when the same mice were similarly tested at 1 year of age (data not shown).

DISCUSSION

The data presented in this paper extend to the mouse previous observations in human on the association of collagen VI gene mutations with alterations of skeletal muscle and establish an animal model for Bethlem myopathy.

As indicated by northern blotting with the neomycin resistance gene probe, insertion of the gene targeting cassette into the second exon of the col6a1 gene in the sense orientation resulted in truncation of transcripts from the col6a1 gene promoter at the cassette termination signals and no [alpha]1(VI) mRNA was produced from the mutant allele. This condition did not affect the steady-state levels of the mRNA for the other two chains, suggesting that regulation of expression of the different collagen VI [alpha] chains is independent. Due to the lack of mRNA, the [alpha]1(VI) polypeptide was not synthesized by cells of col6a1 -/- mice. As a consequence, no triple-helical collagen VI molecules were deposited in the extracellular matrix. As observed for the [alpha]1(VI) and the [alpha]2(VI) chains in a cell line in which synthesis of the [alpha]3(VI) chain was defective (13), it is very likely that the [alpha]2(VI) and [alpha]3(VI) chains were degraded inside the cells in our col6a1 -/- mice.

Although the lack of collagen VI in the extracellular matrix of col6a1 -/- animals had no obvious phenotypic consequences, histological signs of myopathy were always detected in skeletal muscle of both homo- and heterozygous mutant animals. The myopathy of col6a1 knockout mice had several features in common with Bethlem myopathy, a human inherited syndrome which has been correlated with collagen VI genes (11,12). Both are mild, appear early in life and show no substantial worsening with time. A second correlation between the two myopathies is that the dominant character of Bethlem syndrome in some clinical cases may be due to haploinsufficiency. In fact, a family with Bethlem myopathy was recently reported in which the [alpha]1(VI) mRNA and protein were reduced to 50% of the normal amount as a consequence of a premature stop codon, which induced rapid degradation of the mRNA synthesized from the mutant allele (16). This condition is similar to that of the heterozygous col6a1 knockout mice, which, likewise, exhibited muscle necrosis. The functional deficit of the locomotor system was very limited in the mouse, where the myopathy is apparently asymptomatic. A major difference between the human and the mouse pathologies was the preferred location of lesions: extensor muscles of limbs in Bethlem myopathy and diaphragm and auxiliary respiratory muscles in mice. A similar difference in the distribution of damage was found in Duchenne muscular dystrophy patients and in adult mice with X-linked muscular dystrophy (mdx), two conditions determined by defects of the dystrophin gene (17). The distinct pattern of dystrophic changes in the two species has been attributed to the different obligatory respiratory work rate per unit mass (~5 times lower in human), a condition which would make the respiratory muscles of the mouse more prone to mechanical damage than the limb ones (18). Owing to these considerations, the col6a1-deficient mouse can be regarded as a model for Bethlem myopathy. Both conditions are probably the consequence of failure of the bridging function of collagen VI, which connects the collagen IV network (6) within the basement membrane with collagen I fibers, fibronectin and some proteoglycans (4-6) in the interstitium.

Table 1. Increased presence of central nuclei in muscle fibers of col6a1 mutant mice
Muscle col6a1 +/+ col6a1 +/- col6a1 -/-
Brachial triceps 1.8 ± 0.9 5.5 ± 0.5 (P < 0.0001) 6.6 ± 2.7 (P < 0.0001)
Femoral 1.3 ± 0.4 2.8 ± 0.3 (P < 0.0001) 6.3 ± 3.9 (P < 0.003)
External intercostal 2.5 ± 0.7 5.6 ± 0.7 (P <0.002) 7.6 ± 1.2 (P < 0.0001)
Diaphragm 2.6 ± 0.8 5.1 ± 1.2 (P < 0.05) 9.0 ± 2.0 (P < 0.001)
Indicated muscles were dissected from one col6a1 -/-, one col6a1 +/- and one col6a1 +/+ 5-month-old mouse, embedded in paraffin and histological sections stained with hematoxylin and eosin. The percentage of fibers with central nuclei was determined on at least four pictures taken from different microscopic fields. Values are expressed as means ± SD. The data obtained from mutant +/- and -/- animals were compared with those of control mice using Student's t-test and the significance is given in parentheses.

MATERIALS AND METHODS

Construction of targeting vector


Figure 4. Alterations of skeletal muscle in col6a1 knockout mice. (a) Longitudinal section of femoral quadriceps muscle from 40-day-old col6a1 -/- mouse showing necrosis and macrophage infiltration of a muscle fiber. (b) Femoral quadriceps muscle from 4-month-old col6a1 -/- mouse showing a focal site where most fibers have central nuclei. (c-e) Whole mount preparation of col6a1 -/- (c), +/- (d) and +/+ (e) diaphragms from 20-day-old littermates after vital injection of Evans blue dye. Necrotic fibers, identified by deep blue striations, are present only in the mutant mice. (f and g) Paraffin sections prepared from the two diaphragms shown in (c) and (e), respectively, and analyzed in the fluorescence microscope. Necrotic and hypercontracted fibers appear red (14), whereas normal intact fibers are light green, due to fixation of the sample with paraformaldehyde. Bar, 50 µm (a, f and g); 25 µm (b).

The isolation and characterization of mG3, a 15.5 kb genomic clone from a 129Sv [lambda]EMBL3 library containing the 5[prime] region of col6a1, was previously described (19). A 9.4 kb SphI-BamHI fragment including ~7.5 kb of 5[prime]-flanking region, the first two exons, the first intron and part of the second intron was cloned into pBluescript II KS(+) (Stratagene). The targeting vector contained the 9.4 kb SphI-BamHI fragment, the neomycin resistance gene (neo) under control of the Herpes simplex virus tk promoter (pMC1neo-pA; Stratagene) inserted in forward orientation into the Eco47III site in exon 2 and the Herpes simplex virus tk gene and promoter inserted into a SalI site at the 3[prime] end of the homology locus. The neo cassette interrupts the coding sequence of col6a1 at amino acid 66, leaving only the signal peptide and 47 amino acids of the N-terminal A[prime]3 domain (19). The resulting construct was purified by double sedimentation in a CsCl gradient and linearized with NotI before electroporation.

Embryonic stem cells and animals

Samples of 2 × 107 R1 ES cells (20) were electroporated with 50 µg linearized targeting vector at 250 V and 500 µF in phosphate-buffered saline (PBS). After electroporation, cells were seeded on a monolayer of neomycin-resistant primary mouse embryonic fibroblasts and selected for 7-10 days with 350 µg/ml G418 and 2 µM ganciclovir (Syntex). Resistant clones (390) were expanded for Southern blot analysis and three correctly targeted clones were identified. Chimeric animals were produced from ES cell clones by microinjection into C57BL/6 host blastocysts (21). Male chimeras were mated with C57BL/6 female mice to obtain heterozygous F1 mice which were intercrossed to produce homozygous mutants. Germline transmission of the mutation was obtained from one of the correctly targeted clones.

Southern blot analysis

DNA was extracted from ES cells or tail biopsies by digestion with proteinase K, phenol extraction and isopropanol precipitation (22). The DNA was digested with DraI, separated in 1% agarose gel, blotted into GeneScreen (Dupont) membranes and probed with a 32P-labeled fragment, obtained by PCR cloning, which lies outside the targeting construct and extends from the BamHI site in the second intron into the third exon (base 481 of the cDNA; 19). The membranes were rehybridized with a neo probe to verify that a single integration had occurred.

Embryonic fibroblast cultures

Fibroblasts were prepared from 13.5-day-old embryos from heterozygous F1 × F1 intercrosses as described (21) and grown in Dulbecco's minimal essential medium supplemented with 10% fetal calf serum. The genotype of the embryos was determined by Southern blot analysis of DNA purified from each individual liver. For immunofluorescence, the cells were plated into 8-well Chamber Slides (NUNC) (80 000 cells/well) and grown for 3-5 days. These cells and those used for immunoprecipitation were cultured in the presence of ascorbic acid (50 µg/ml culture medium).

Northern blot analysis

Total RNA was extracted from second passage confluent embryonic fibroblasts with RNAfast-II (Molecular Systems, San Diego, CA) as recommended by the manufacturer. Aliquots of 15 µg total RNA were separated in 1% agarose-formaldehyde gels, blotted into Hybond-N membranes (Amersham), and hybridized successively with the following 32P-labeled probes: (i) [alpha]1(VI) cDNA spanning nt 652-1992 (19); (ii) [alpha]2(VI) cDNA, a 0.7 kb fragment obtained by RT-PCR of total mouse lung RNA (P. Bonaldo, unpublished data); (iii) [alpha]3(VI) cDNA (clone mCol6a3-2, a gift from M.-L. Chu) (23); (iv) neo, excised from pMC1neo-pA by digestion with XhoI and SalI; and (v) a cDNA clone for human glyceraldehyde 3-phosphate dehydrogenase (24). Before application of one radioactive probe, the previous probe was stripped from the filters by incubation at 65°C for 90 min in 5 mM Tris-HCl, pH 7.6, 2 mM EDTA, 0.1% Denhardt's solution.

Purification of collagen VI from whole animals

Pepsin-resistant type VI collagen was purified from the skinned carcasses of one control and one null homozygous 1-month-old animal as described (25). The protein products were resolved by chromatography on a Sephacryl S-400 column (1.6 × 50 cm) and aliquots of the fractions tested by ELISA with a rabbit antiserum against mouse collagen VI (26). Aliquots of pooled fractions were analyzed by SDS-PAGE and Coomassie blue R-250 staining.

Immunofluorescence analysis

Mouse embryos (16 days old) were frozen in isopentane cooled in liquid nitrogen and stored at -80°C until processing. Thick frozen sections (6 µm) were collected on gelatin-subbed slides and air dried. Embryonic fibroblasts grown in Chamber Slides (Nunc) were washed twice with PBS and air dried. Antibodies were rabbit IgG against mouse collagen VI (a gift from A. Colombatti) (26) and a monoclonal antibody to fibronectin produced by a hybridoma supplied by the American Type Culture Collection (ATCC HB91). After treatment for 30 min with normal goat serum, the slides were incubated for 2 h at room temperature with the appropriate dilution of the primary antibody. After washing in PBS, TRITC-conjugated goat anti-mouse Ig or biotinylated goat anti-rabbit IgG followed by FITC-conjugated avidin was applied for 1 h at room temperature. The slides were washed, mounted in 50% glycerol in PBS and observed in a Zeiss Axioplan microscope equipped with epifluorescence optics.

Histological analysis

Tissue specimens were dissected from mice and fixed for 1 day in 4% paraformaldehyde in PBS at 4°C. To better investigate the presence of necrosis in the muscular system, some mice were injected i.p. with 0.05-0.2 ml Evans blue dye (10 mg/ml in PBS). After 6 h the animals were killed with ether, skinned and eviscerated. The carcasses were fixed with 4% paraformaldehyde in PBS for 1 day at 4°C and further fixed and decalcified for 30 days in 4% formic acid, 12% formaldehyde at room temperature. After fixation, the samples were dehydrated in a graded series of water/ethanol mixtures and embedded in paraffin. Thick sections (6 µm) were cut and stained with hematoxylin/eosin or observed in the fluorescence microscope to locate the Evans blue dye distribution after removal of paraffin and mounting in Permount.

ABBREVIATIONS

ES cells, embryonic stem cells; neo, neomycin resistance gene; PBS, phosphate-buffered saline; tk, thymidine kinase.

ACKNOWLEDGEMENTS

We would like to thank Alfonso Colombatti and Mon-Li Chu for reagents, Andras Nagy for the R1 ES cell line and Alessandra Zatti and Francesca Pampinella for help in performing Southern blot analyses. We also thank Mr Mauro Ghidotti for the maintenance of mouse colonies. The financial support of Telethon, Italy (grant nos E.22 and E.367) is gratefully acknowledged.

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*To whom correspondence should be addressed. Tel: +39 049 827 6086; Fax: +39 049 827 6079; Email: bressan@civ.bio.unipd.it

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