Emerin-Lacking Mice Show Minimal Motor and Cardiac Dysfunctions with Nuclear-Associated Vacuoles
2006; Elsevier BV; Volume: 168; Issue: 3 Linguagem: Inglês
10.2353/ajpath.2006.050564
ISSN1525-2191
AutoresRitsuko Ozawa, Yukiko Hayashi, Megumu Ogawa, Rumi Kurokawa, Hiroshi Matsumoto, S. Noguchi, Ikuya Nonaka, Ichizo Nishino,
Tópico(s)RNA regulation and disease
ResumoEmery-Dreifuss muscular dystrophy is an inherited muscular disorder clinically characterized by slowly progressive weakness affecting humero-peroneal muscles, early joint contractures, and cardiomyopathy with conduction block. The X-linked recessive form is caused by mutation in the EMD gene encoding an integral protein of the inner nuclear membrane, emerin. In this study, mutant mice lacking emerin were produced by insertion of a neomycin resistance gene into exon 6 of the coding gene. Tissues taken from mutant mice lacked emerin. The mutant mice displayed a normal growth rate indistinguishable from their littermates and were fertile. No marked muscle weakness or joint abnormalities were observed; however, rotarod test revealed altered motor coordination. Electrocardiography showed mild prolongation of atrioventricular conduction time in emerin-lacking male mice older than 40 weeks of age. Electron microscopic analysis of skeletal and cardiac muscles from emerin-lacking mice revealed small vacuoles, which mostly bordered the myonuclei. Our results suggest that emerin deficiency causes minimal motor and cardiac dysfunctions in mice with a structural fragility of myonuclei. Emery-Dreifuss muscular dystrophy is an inherited muscular disorder clinically characterized by slowly progressive weakness affecting humero-peroneal muscles, early joint contractures, and cardiomyopathy with conduction block. The X-linked recessive form is caused by mutation in the EMD gene encoding an integral protein of the inner nuclear membrane, emerin. In this study, mutant mice lacking emerin were produced by insertion of a neomycin resistance gene into exon 6 of the coding gene. Tissues taken from mutant mice lacked emerin. The mutant mice displayed a normal growth rate indistinguishable from their littermates and were fertile. No marked muscle weakness or joint abnormalities were observed; however, rotarod test revealed altered motor coordination. Electrocardiography showed mild prolongation of atrioventricular conduction time in emerin-lacking male mice older than 40 weeks of age. Electron microscopic analysis of skeletal and cardiac muscles from emerin-lacking mice revealed small vacuoles, which mostly bordered the myonuclei. Our results suggest that emerin deficiency causes minimal motor and cardiac dysfunctions in mice with a structural fragility of myonuclei. Emery-Dreifuss muscular dystrophy (EDMD) is an inherited disorder characterized clinically by the triad of 1) slowly progressive weakness and wasting that affects the humero-peroneal muscles in the early stages; 2) early contractures of the elbows, Achilles tendons, and postcervical muscles; and 3) cardiomyopathy with conduction defects that would result in high-risk sudden death.1Emery AE Emery-Dreifuss syndrome.J Med Genet. 1989; 26: 637-641Crossref PubMed Scopus (180) Google Scholar, 2Morris GE Manilal S Heart to heart: from nuclear proteins to Emery-Dreifuss muscular dystrophy.Hum Mol Genet. 1999; 8: 1847-1851Crossref PubMed Scopus (85) Google Scholar Three forms of inheritance are known, X-linked recessive (X-EDMD), autosomal dominant (AD-EDMD), and autosomal recessive (AR-EDMD) forms. 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On the other hand, there has been no vertebrate animal model for emerin deficiency. It was therefore the main aim of this study to produce mice lacking emerin. The mouse emerin gene (Emd) is composed of six exons and encodes a serine-rich protein comprising 259 amino acids and is 73% identical to human emerin.34Small K Wagener M Warren ST Isolation and characterization of the complete mouse emerin gene.Mamm Genome. 1997; 8: 337-341Crossref PubMed Scopus (19) Google Scholar Mice were engineered to lack most of exon 6 of the gene, including the part encoding the transmembrane domain at the C-terminal end of emerin. The resulting emerin-lacking mice showed minimal motor and cardiac dysfunctions. Electron microscopic analysis of skeletal and cardiac muscles showed vacuoles mostly associated with myonuclei. Genomic clones of Emd were isolated from a 129/SvJ library from Genome Systems (St. Louis, MO). Identities of the isolated clones were confirmed by DNA sequencing. The targeting vector was designed to carry a deletion of 84 amino acids, including the transmembrane domain of emerin, by replacing 702 bp (3167 to 3868) with a bovine growth hormone polyadenylation (bGHpA) sequence and neomycin resistance gene (Neo) obtained from Genome Systems (Figure 1A). The targeting vector was transfected into 129/SvJ embryonic stem cells, and clones carrying the specific targeted locus were confirmed by Southern blotting. A total of 20 μg of genomic DNA from ES cell clones was digested with XhoI (for 5′ and Neo probes) and EcoRV (for 3′ probe) and processed for Southern blot analysis (Figure 1B). Using the 5′ probe, hybridization bands of ∼11.5 kb, which corresponded in size to wild-type allele, and >17.7-kb band, which corresponded to homologous recombinant allele, were detected. The 3′ probe hybridized ∼13.5-kb wild-type and ∼9.8-kb recombinant alleles. Two recombinant embryonic stem cell lines were confirmed by 5′,3′ and Neo probes and injected into C57/BL6 blastocysts. Five chimeras were obtained and crossed with C57/BL6 wild-type mice to obtain heterozygous mice. The genotypes of the mice were verified by polymerase chain reaction using primer sets for Emd (forward: 5′-CCTAATTATTCTGCAGGTGCG-3′, reverse: 5′-AGGAAGAGTAACAGCTGGCC-3′) and Neo (forward: 5′-GCTTGGGTGGAGAGGCTATTC-3′, reverse: 5′-CAAGGTGAGATGACAGGA-GATC-3′). The mutant mice were backcrossed with C57/BL6 wild-type mice for 10 generations. Wild-type littermates were used as controls. Animals were housed and all experimental procedures were performed in accordance with the guidelines for the care and use of experimental animals at the National Institute of Neuroscience, National Center of Neurology and Psychiatry. Total RNA was extracted from skeletal muscles of emerin-lacking, heterozygous, and wild-type littermates at 5 and 46 weeks of age, by using a standard technique. Total RNA was also extracted from cardiac muscles from 5-week female littermates with each genotype. Quantitative RT-PCR was performed using iCycler (Bio-Rad Laboratories Inc., Richmond, CA) according to the manu-facturing protocols. Primer sets for emerin (forward: 5′-GTTATTTGACCACCAAGACATACGGG-3′, reverse: 5′-GGTGATGGAAGGTATCAGCATCTACA-3′) and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) (forward: 5′-CTGGAGAAACCTGCCAAGTATG-3′, reverse: 5′-TTGAAGTCGCAGGAGACAACCTG-3′) were used. The values of mRNA for emerin were normalized to that of G3PDH. Each mouse was weighed every 2 weeks from 3 weeks of age. Muscle strength was evaluated by using the hanging basket test. Mice were hung on a 20 cm × 20 cm upsetting wire basket for 10 minutes, and the time to drop was noted. Rotarod balance test was performed to evaluate motor coordination under load using Rotarod Treadmil (Ugo Basile Biological). After practice, each mouse was forced to walk on the rotating rod maintained at a constant speed of 50 rpm/minute for 10 minutes. Each mouse was given three trials and the longest latency to fall down from the rod was recorded. Statistical analyses were performed using an unpaired Student's t-test. To determine the involvement of both the skeletal and cardiac muscles, mice were placed into a 50 cm × 40 cm × 20 cm plastic tank filled with water for 5 minutes and monitored for their swimming and recovery process. The Softron ECG Processor SP2000 (Softron Co., Japan) was used for electrocardiogram data analysis. Under ether anesthesia, mice underwent digitalized electrocardiogram measurement of lead I and lead II using subcutaneous needle electrodes. To avoid the possible influence of anesthesia, recording was started just before recovery, wherein the heart rate had returned to near normal level (more than 500 bpm). Electrocardiogram recordings were performed three times with 2-second durations each. The P-wave duration, PR interval, QRS complex duration, QT interval, and RR interval were calculated and averaged. Long lead recording for 1 minute was also performed and repeated three times to detect arrhythmias. Muscles were taken from bilateral triceps, quadriceps femoris, hamstrings, anterior tibialis, gastrocnemius, paravertebral, diaphragm, and heart. Muscle samples were flash-frozen in isopentane and chilled with liquid nitrogen. Serial frozen sections (6 μm) of skeletal and cardiac muscles were stained with a battery of histochemical reagents and immunostained with the following antibodies: anti-emerin (Novocastra Laboratory, Newcastle-upon-Tyne, UK), anti-lamin A and anti-lamin C.8Sakaki M Koike H Takahashi N Sasagawa N Tomioka S Arahata K Ishiura S Interaction between emerin and nuclear lamins.J Biochem (Tokyo). 2001; 129: 321-327Crossref PubMed Scopus (121) Google Scholar Sections of cardiac muscle were also stained with anti-connexin 40 (Zymed Laboratories Inc., South San Francisco, CA) and anti-connexin 43 (Zymed Laboratories Inc.). Alexa 488- or 568-labeled goat anti-rabbit or anti-mouse IgG (Molecular Probes, Eugene, OR) was used as secondary antibody. Immunoblotting analysis was performed as previously described.35Hayashi YK Ogawa M Tagawa K Noguchi S Ishihara T Nonaka I Arahata K Selective deficiency of alpha-dystroglycan in Fukuyama-type congenital muscular dystrophy.Neurology. 2001; 57: 115-121Crossref PubMed Scopus (215) Google Scholar The density of the band corresponding to myosin heavy chain on postblotted sodium dodecyl sulfate-polyacrylamide gel was estimated, and the amount of skeletal muscle protein was normalized. Two emerin antibodies were used: a monoclonal anti-emerin (Novocastra) that was raised against N-terminal 222 amino acids for human emerin and polyclonal anti-emerin (FL-254; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) that was raised against human emerin corresponding to 3 to 254 amino acids. Rabbit antisera for lamin A and lamin C8Sakaki M Koike H Takahashi N Sasagawa N Tomioka S Arahata K Ishiura S Interaction between emerin and nuclear lamins.J Biochem (Tokyo). 2001; 129: 321-327Crossref PubMed Scopus (121) Google Scholar were also used. Histofine Simple Stain Max PO (Multi) (Nichirei Co., Japan) was used as secondary antibody. The immunoreactive bands on the membranes were visualized using the POD Immunostain Set (Wako Co., Japan). Similar amount of myosin heavy chain was detected in each lane. Muscle specimens were fixed in 2% glutaraldehyde in phosphate buffer and were kept in 0.1 mol/L calcium cacodylate at 4°C. After incubation in a mixture of 4% osmium tetroxide, 1.5% lanthanum nitrate, and 0.2 mol/L s-collidine buffer for 2 to 3 hours, samples were embedded in epoxy resin. Ultrathin sections (50 nm thick) were double-stained with uranyl acetate and lead citrate (Raynolds) and examined using a Hitachi H-7100 electron microscope. Cardiac muscles at 1, 21, 87, and 97 weeks of age and skeletal muscle at 78 weeks of age from emerin-lacking male mice were examined. Muscles from wild-type mice at 1 and 81 weeks of age were also examined. Skeletal and cardiac myocytes and skin fibroblasts from mice at 3 weeks of age were prepared by standard technique with minor modifications. Cells were incubated in Dulbecco's modified Eagle's medium-F12 supplemented with 20% fetal bovine serum and 1% penicillin/streptomycin at 37°C in a humidified atmosphere containing 5% CO2. Immunocytochemistry was performed using anti-emerin (Santa Cruz Biotechnology, Inc.), anti-lamin A, and anti-lamin C,8Sakaki M Koike H Takahashi N Sasagawa N Tomioka S Arahata K Ishiura S Interaction between emerin and nuclear lamins.J Biochem (Tokyo). 2001; 129: 321-327Crossref PubMed Scopus (121) Google Scholar anti-lamin B2 (Novocastra Laboratories Ltd.) and anti-LAP2 (BD Transduction Laboratories, Lexington, KY) antibodies. Emd mutant mice were produced with a deletion of emerin's C-terminal 84 amino acids, which include the transmembrane domain. Male (Emd−/y), and female (Emd+/− and Emd−/−) offspring with the mutant allele and wild-type littermates (Emd+/y and Emd+/+) were obtained and analyzed. Quantitative RT-PCR revealed normal expression levels of mRNA for emerin in both skeletal and cardiac muscles from mutant mice at 5 weeks of age (Figure 2A) and skeletal muscle at 46 weeks of age (data not shown). Immunohistochemical analysis was performed using skeletal and cardiac muscles and brain. Emerin was present at nuclear membrane in tissues from wild-type mice; however, the immunoreaction was completely negative in Emd−/y and Emd−/− mice (Figure 2B). Positive/negative mosaic expression in heterozygous female mice was seen (data not shown). The expression and distribution of lamin A and lamin C showed no alteration in both skeletal and cardiac muscles and brain of the mutant mice. On immunoblotting analysis, the two different anti-emerin antibodies detected a protein with an apparent molecular mass of 38 kd in muscles from wild-type mice, a protein that is larger than human emerin. This protein of 38 kd, which had been recognized by two different anti-emerin antibodies, was completely absent from muscles of Emd−/y and Emd−/− mice and noted to be reduced in muscles of Emd+/− mice (Figure 2C). The amounts of lamin A (data not shown) and lamin C (Figure 2C) were the same in the wild-type and mutant mice. Mice lacking emerin displayed similar growth rate and survival curve pattern when compared to their heterozygous and wild-type littermates (Figure 3). They were able to maintain sexual fertility and breed 10 generations of offsprings, as of the preparation of this article. Emerin-lacking mice were not observed to have waddling gait, scoliosis/kyphosis, or contractures as described in Lmna−/− mice.30Sullivan T Escalante-Alcalde D Bhatt H Anver M Bhat N Nagashima K Stewart CL Burke B Loss of A-type lamin expression compromises nuclear envelope integrity leading to muscular dystrophy.J Cell Biol. 1999; 147: 913-920Crossref PubMed Scopus (968) Google Scholar They showed no obvious muscle weakness, and except for a few overweight mice, could suspend on a wire basket for more than 10 minutes, as observed in wild-type mice. Mice lacking emerin could swim for more than 5 minutes in a water pool and recovered to an active state within 30 seconds. Although no apparent muscle weakness was seen, balance testing using rotarod revealed significant differences in motor function between emerin-lacking, heterozygous, and wild-type mice. There was no difference between male and female mice. Only 43% of emerin-lacking male mice, 30% of emerin-lacking female mice, and 58% of heterozygous female mice were able to remain on the rotating rod for 10 minutes, whereas all wild-type littermates younger than 20 weeks of age could remain on the rotating rod for more than 10 minutes (Figure 4A). A greater number of younger (4 to 10 weeks) mutant mice was able to remain on the rotating rod longer than older (11 to 20 weeks) mutant mice. The time that the mutant mice could keep on the rod was quite variable from less than 1 minute to more than 10 minutes. The mean latency to drop down in the wild-type male, wild-type female, heterozygous female, emerin-lacking male, and emerin-lacking female mice were 600, 600, 394, 301, and 243 seconds, respectively.Figure 4Result of rotarod test. A: The table shows number and ratio of mice that could walk on the rod for 10 minutes. All wild-type mice could complete the task but only 43% of emerin-lacking male mice, 30% of emerin-lacking female mice, and 58% of heterozygous female mice could complete the task. B: Average time that mice were able to remain on the rod was calculated from the same experiment as in A. Although some mutant mice could remain on the rod more than 10 minutes, others dropped within a minute. The mean time that mutant mice could remain on the rotating rod is significantly shorter than wild-type littermates (**P < 0.01, *P < 0.05). Older mutant mice were observed to have worse motor coordination, whereas no difference was seen among the wild-type mice regardless of age.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Cardiomyopathy with conduction block is one of the major clinical features in human patients with EDMD. To investigate cardiac involvement in mice lacking emerin, limb-lead electrocardiograms were recorded on 122 emerin-lacking male, 75 emerin-lacking female, 58 heterozygous female, 66 wild-type male, and 82 wild-type female littermates from 3 to 100 weeks of age. The PR intervals in emerin-lacking male mice tend to prolong with aging (Figure 5). The mean PR interval in emerin-lacking male mice from 41 to 100 weeks of age was 42.5 ± 3.1 (n = 44), whereas that in wild-type littermates was 41.0 ± 2.1 (n = 21) (P < 0.05). No significant differences in PR intervals were seen between mice with any genotype younger than 40 weeks of age, and the mean PR intervals were 39.1 ± 3.4 and 38.8 ± 4.0 in wild-type and mutant mice, respectively. Interestingly in female mice, no significant difference in the mean PR interval from 41 to 100 weeks of age was observed between wild-type (41.7 ± 3.0, n = 26), heterozygous (41.3 ± 0.4, n = 2), and homozygous emerin-lacking mice (40.0 ± 3.6, n = 26). The PR:RR ratio was evaluated to avoid the influence of heart rate. Again, male mice were observed to have prolonged PR:RR intervals with aging (data not shown). The mean PR:RR ratio of wild-type male, emerin-lacking male, wild-type female, heterozygous female, and emerin-lacking female mice from 41 to 100 weeks of age were 0.37 ± 0.07, 0.40 ± 0.05, 0.41 ± 0.07, 0.42 ± 0.05, and 0.39 ± 0.05, respectively. The P waves, QRS complexes, QT intervals, and RR intervals were comparable among the five groups. No arrhythmia was observed during the 3-minute recording. Histological analysis of skeletal and cardiac muscles from emerin-lacking mice and heterozygous female mice from 3 to 127 weeks of age did not reveal notable pathological changes compared to wild-type controls (Figure 6). The structural integrity of the myofibers was well preserved with no necrotic or regenerating fibers noted. Skeletal muscles from the emerin-lacking male mice had many tubular aggregates at 24 weeks of age or older, and the number and size of tubular aggregates increased with age (Figure 6B, arrow). However, wild-type male littermates were also noted to have tubular aggregates of similar number and size (Figure 6A, arrow). No tubular aggregates were seen in female mice even at 64 weeks of age (Figure 6, E and F). Tubular aggregates in muscles from both mutant and wild-type mice did not immunostain for anti-emerin antibodies (data not shown). Intramuscular peripheral nerves were well myelinated and showed no notable abnormality. To investigate the cardiac conduction system, the expression and localization of connexin 40 and connexin 43 were examined by immunohistochemistry. A strong immunostaining reaction to connexin 40 had been observed in cells associated with the conduction system from the atrioventricular node to the Purkinje cells in hearts of both wild-type and mutant mice. Connexin 43 was located at the intercalated disks of the myocardium. The staining patterns and intensity for both connexin 40 and connexin 43 were indistinguishable in mutant and wild-type mice. Amounts of connexin 40 and connexin 43 detected on immunoblots were identical in cardiac muscles from both wild-type and mutant mice (data not shown). Toluidin
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