Preservation of Muscle Force in Mdx3cv Mice Correlates with Low-Level Expression of a Near Full-Length Dystrophin Protein
2008; Elsevier BV; Volume: 172; Issue: 5 Linguagem: Inglês
10.2353/ajpath.2008.071042
ISSN1525-2191
AutoresDejia Li, Yongping Yue, Dongsheng Duan,
Tópico(s)Advanced Sensor and Energy Harvesting Materials
ResumoThe complete absence of dystrophin causes Duchenne muscular dystrophy. Its restoration by greater than 20% is needed to reduce muscle pathology and improve muscle force. Dystrophin levels lower than 20% are considered therapeutically irrelevant but are associated with a less severe phenotype in certain Becker muscular dystrophy patients. To understand the role of low-level dystrophin expression, we compared muscle force and pathology in mdx3cv and mdx4cv mice. Dystrophin was eliminated in mdx4cv mouse muscle but was expressed in mdx3cv mice as a near full-length protein at ∼5% of normal levels. Consistent with previous reports, we found dystrophic muscle pathology in both mouse strains. Surprisingly, mdx3cv extensor digitorium longus muscle showed significantly higher tetanic force and was also more resistant to eccentric contraction-induced injury than mdx4cv extensor digitorium longus muscle. Furthermore, mdx3cv mice had stronger forelimb grip strength than mdx4cv mice. Immunostaining revealed utrophin up-regulation in both mouse strains. The dystrophin-associated glycoprotein complex was also restored in the sarcolemma in both strains although at levels lower than those in normal mice. Our results suggest that subtherapeutic expression levels of near full-length, membrane-bound dystrophin, possibly in conjunction with up-regulated utrophin levels, may help maintain minimal muscle force but not arrest muscle degeneration or necrosis. Our findings provide valuable insight toward understanding delayed clinical onset and/or slow disease progression in certain Becker muscular dystrophy patients. The complete absence of dystrophin causes Duchenne muscular dystrophy. Its restoration by greater than 20% is needed to reduce muscle pathology and improve muscle force. Dystrophin levels lower than 20% are considered therapeutically irrelevant but are associated with a less severe phenotype in certain Becker muscular dystrophy patients. To understand the role of low-level dystrophin expression, we compared muscle force and pathology in mdx3cv and mdx4cv mice. Dystrophin was eliminated in mdx4cv mouse muscle but was expressed in mdx3cv mice as a near full-length protein at ∼5% of normal levels. Consistent with previous reports, we found dystrophic muscle pathology in both mouse strains. Surprisingly, mdx3cv extensor digitorium longus muscle showed significantly higher tetanic force and was also more resistant to eccentric contraction-induced injury than mdx4cv extensor digitorium longus muscle. Furthermore, mdx3cv mice had stronger forelimb grip strength than mdx4cv mice. Immunostaining revealed utrophin up-regulation in both mouse strains. The dystrophin-associated glycoprotein complex was also restored in the sarcolemma in both strains although at levels lower than those in normal mice. Our results suggest that subtherapeutic expression levels of near full-length, membrane-bound dystrophin, possibly in conjunction with up-regulated utrophin levels, may help maintain minimal muscle force but not arrest muscle degeneration or necrosis. Our findings provide valuable insight toward understanding delayed clinical onset and/or slow disease progression in certain Becker muscular dystrophy patients. Duchenne muscular dystrophy (DMD) results from mutations in the dystrophin gene.1Kunkel LM 2004 William Allan Award Address. Cloning of the DMD gene.Am J Hum Genet. 2005; 76: 205-214Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar In the striated muscle of DMD patients, dystrophin is essentially eliminated. Dystrophin is an important cytoskeleton protein. It protects the sarcolemma from the shearing stress generated during muscle contraction. In the absence of dystrophin, sarcolemma integrity is compromised. This sensitizes myofibers to contraction-induced injury. As a result, muscle cells undergo degeneration and necrosis. Eventually, muscle is replaced by adipose and fibrous tissues and loses contractility. DMD patients experience difficulties in moving and/or climbing at 3 to 5 years of age. Thereafter, clinical progression follows a catastrophic downhill track. Patients are confined to a wheelchair at ∼11 years of age and die prematurely before age 30.2Emery AEH Muntoni F Emery AEH Muntoni F Duchenne Muscular Dystrophy. Oxford University Press, New York2003: 26-44Google ScholarDystrophin gene mutation also causes Becker muscular dystrophy (BMD), a milder allelic form. In BMD patients, symptoms usually start in the teenage years and progress slowly. In contrast to DMD, dystrophin expression is not lost in BMD patients.3Hoffman EP Fischbeck KH Brown RH Johnson M Medori R Loike JD Harris JB Waterston R Brooke M Specht L Kupsky W Chamberlain J Caskey Y Shapiro F Kunkel LM Characterization of dystrophin in muscle-biopsy specimens from patients with Duchenne's or Becker's muscular dystrophy.N Engl J Med. 1988; 318: 1363-1368Crossref PubMed Scopus (753) Google Scholar In general, it falls into two categories. Some BMD patients express an internally truncated but partially functional dystrophin. Some of these abbreviated dystrophin isoforms can be quite effective in halting disease progression.4England SB Nicholson LV Johnson MA Forrest SM Love DR Zubrzycka-Gaarn EE Bulman DE Harris JB Davies KE Very mild muscular dystrophy associated with the deletion of 46% of dystrophin.Nature. 1990; 343: 180-182Crossref PubMed Scopus (480) Google Scholar, 5Harper SQ Hauser MA DelloRusso C Duan D Crawford RW Phelps SF Harper HA Robinson AS Engelhardt JF Brooks SV Chamberlain JS Modular flexibility of dystrophin: implications for gene therapy of Duchenne muscular dystrophy.Nat Med. 2002; 8: 253-261Crossref PubMed Scopus (452) Google Scholar In fact, several gene therapy strategies are based on expressing internally deleted dystrophin. Among these, microdystrophin, minidystrophin, and exon-skipping approaches have shown great promise in ameliorating disease in animal models and are currently in the early phase of clinical trials.In other BMD patients, dystrophin expression is reduced. In these patients, there seems a clear correlation between the amount of dystrophin protein and the clinical phenotype. Patients who have ≥20% of the normal dystrophin levels usually display mild disease.6Hoffman EP Kunkel LM Angelini C Clarke A Johnson M Harris JB Improved diagnosis of Becker muscular dystrophy by dystrophin testing.Neurology. 1989; 39: 1011-1017Crossref PubMed Google Scholar, 7Bulman DE Murphy EG Zubrzycka-Gaarn EE Worton RG Ray PN Differentiation of Duchenne and Becker muscular dystrophy phenotypes with amino- and carboxy-terminal antisera specific for dystrophin.Am J Hum Genet. 1991; 48: 295-304PubMed Google Scholar, 8Byers TJ Neumann PE Beggs AH Kunkel LM ELISA quantitation of dystrophin for the diagnosis of Duchenne and Becker muscular dystrophies.Neurology. 1992; 42: 570-576Crossref PubMed Google Scholar Most of them are ambulant beyond age 20.6Hoffman EP Kunkel LM Angelini C Clarke A Johnson M Harris JB Improved diagnosis of Becker muscular dystrophy by dystrophin testing.Neurology. 1989; 39: 1011-1017Crossref PubMed Google Scholar, 7Bulman DE Murphy EG Zubrzycka-Gaarn EE Worton RG Ray PN Differentiation of Duchenne and Becker muscular dystrophy phenotypes with amino- and carboxy-terminal antisera specific for dystrophin.Am J Hum Genet. 1991; 48: 295-304PubMed Google Scholar, 8Byers TJ Neumann PE Beggs AH Kunkel LM ELISA quantitation of dystrophin for the diagnosis of Duchenne and Becker muscular dystrophies.Neurology. 1992; 42: 570-576Crossref PubMed Google Scholar Patients who have less than 20% of the levels show an intermediate phenotype between typical DMD and BMD.6Hoffman EP Kunkel LM Angelini C Clarke A Johnson M Harris JB Improved diagnosis of Becker muscular dystrophy by dystrophin testing.Neurology. 1989; 39: 1011-1017Crossref PubMed Google Scholar, 9Beggs AH Hoffman EP Snyder JR Arahata K Specht L Shapiro F Angelini C Sugita H Kunkel LM Exploring the molecular basis for variability among patients with Becker muscular dystrophy: dystrophin gene and protein studies.Am J Hum Genet. 1991; 49: 54-67PubMed Google Scholar, 10Hoffman EP Genotype/phenotype correlations in Duchenne/Becker dystrophy.Mol Cell Biol Hum Dis Ser. 1993; 3: 12-36PubMed Google Scholar They become wheelchair-bound between 14 and 20 years of age, several years later than DMD patients.6Hoffman EP Kunkel LM Angelini C Clarke A Johnson M Harris JB Improved diagnosis of Becker muscular dystrophy by dystrophin testing.Neurology. 1989; 39: 1011-1017Crossref PubMed Google ScholarInvestigators have also studied the effect of varying dystrophin levels in mdx mice, a mouse model for DMD.11Wells DJ Wells KE Walsh FS Davies KE Goldspink G Love DR Chan-Thomas P Dunckley MG Piper T Dickson G Human dystrophin expression corrects the myopathic phenotype in transgenic mdx mice.Hum Mol Genet. 1992; 1: 35-40Crossref PubMed Scopus (61) Google Scholar, 12Wells DJ Wells KE Asante EA Turner G Sunada Y Campbell KP Walsh FS Dickson G Expression of human full-length and minidystrophin in transgenic mdx mice: implications for gene therapy of Duchenne muscular dystrophy.Hum Mol Genet. 1995; 4: 1245-1250Crossref PubMed Scopus (147) Google Scholar, 13Phelps SF Hauser MA Cole NM Rafael JA Hinkle RT Faulkner JA Chamberlain JS Expression of full-length and truncated dystrophin mini-genes in transgenic mdx mice.Hum Mol Genet. 1995; 4: 1251-1258Crossref PubMed Scopus (264) Google Scholar Uniform expression of full-length dystrophin or minidystrophin at 20% or higher levels results in remarkable improvement in muscle pathology and strength in transgenic mdx mice.12Wells DJ Wells KE Asante EA Turner G Sunada Y Campbell KP Walsh FS Dickson G Expression of human full-length and minidystrophin in transgenic mdx mice: implications for gene therapy of Duchenne muscular dystrophy.Hum Mol Genet. 1995; 4: 1245-1250Crossref PubMed Scopus (147) Google Scholar, 13Phelps SF Hauser MA Cole NM Rafael JA Hinkle RT Faulkner JA Chamberlain JS Expression of full-length and truncated dystrophin mini-genes in transgenic mdx mice.Hum Mol Genet. 1995; 4: 1251-1258Crossref PubMed Scopus (264) Google Scholar Muscle degeneration/regeneration, sarcolemma leakage, and muscle-specific force are all normalized. Few studies have evaluated the effect of low level (<20%) dystrophin expression in animal models of DMD. Wells and colleagues11Wells DJ Wells KE Walsh FS Davies KE Goldspink G Love DR Chan-Thomas P Dunckley MG Piper T Dickson G Human dystrophin expression corrects the myopathic phenotype in transgenic mdx mice.Hum Mol Genet. 1992; 1: 35-40Crossref PubMed Scopus (61) Google Scholar reported partial amelioration of muscle disease in transgenic mdx mice that express a minidystrophin gene at ∼17% of the normal level. They observed a significant reduction in muscle degeneration but the serum creatine kinase (CK) level remained high.11Wells DJ Wells KE Walsh FS Davies KE Goldspink G Love DR Chan-Thomas P Dunckley MG Piper T Dickson G Human dystrophin expression corrects the myopathic phenotype in transgenic mdx mice.Hum Mol Genet. 1992; 1: 35-40Crossref PubMed Scopus (61) Google ScholarTo better understand the effect of subtherapeutic level dystrophin expression in mice, we compared muscle pathology and force in the limb muscles of BL6, mdx3cv, and mdx4cv mice. Mdx3cv and mdx4cv mice are N-ethylnitrosourea (ENU)-induced mouse models for DMD.14Chapman VM Miller DR Armstrong D Caskey CT Recovery of induced mutations for X chromosome-linked muscular dystrophy in mice.Proc Natl Acad Sci USA. 1989; 86: 1292-1296Crossref PubMed Scopus (178) Google Scholar They are on the BL6 background. Different point mutations in the dystrophin gene disrupt normal dystrophin expression in these mice.15Cox GA Phelps SF Chapman VM Chamberlain JS New mdx mutation disrupts expression of muscle and nonmuscle isoforms of dystrophin.Nat Genet. 1993; 4: 87-93Crossref PubMed Scopus (165) Google Scholar, 16Im WB Phelps SF Copen EH Adams EG Slightom JL Chamberlain JS Differential expression of dystrophin isoforms in strains of mdx mice with different mutations.Hum Mol Genet. 1996; 5: 1149-1153Crossref PubMed Scopus (167) Google Scholar Mdx4cv muscle represents a true dystrophin-null model. However, mdx3cv muscle expresses a low-level near full-length dystrophin protein.15Cox GA Phelps SF Chapman VM Chamberlain JS New mdx mutation disrupts expression of muscle and nonmuscle isoforms of dystrophin.Nat Genet. 1993; 4: 87-93Crossref PubMed Scopus (165) Google Scholar, 16Im WB Phelps SF Copen EH Adams EG Slightom JL Chamberlain JS Differential expression of dystrophin isoforms in strains of mdx mice with different mutations.Hum Mol Genet. 1996; 5: 1149-1153Crossref PubMed Scopus (167) Google Scholar, 17Danko I Chapman V Wolff JA The frequency of revertants in mdx mouse genetic models for Duchenne muscular dystrophy.Pediatr Res. 1992; 32: 128-131Crossref PubMed Scopus (121) Google Scholar, 18Li S Kimura E Ng R Fall BM Meuse L Reyes M Faulkner JA Chamberlain JS A highly functional mini-dystrophin/GFP fusion gene for cell and gene therapy studies of Duchenne muscular dystrophy.Hum Mol Genet. 2006; 15: 1610-1622Crossref PubMed Scopus (47) Google Scholar, 19Judge LM Haraguchiln M Chamberlain JS Dissecting the signaling and mechanical functions of the dystrophin-glycoprotein complex.J Cell Sci. 2006; 119: 1537-1546Crossref PubMed Scopus (86) Google Scholar Consistent with previous reports, we found characteristic muscle pathology in both mdx3cv and mdx4cv mice.15Cox GA Phelps SF Chapman VM Chamberlain JS New mdx mutation disrupts expression of muscle and nonmuscle isoforms of dystrophin.Nat Genet. 1993; 4: 87-93Crossref PubMed Scopus (165) Google Scholar, 17Danko I Chapman V Wolff JA The frequency of revertants in mdx mouse genetic models for Duchenne muscular dystrophy.Pediatr Res. 1992; 32: 128-131Crossref PubMed Scopus (121) Google Scholar Surprisingly, the extensor digitorium longus (EDL) muscle strength was significantly preserved in mdx3cv mice albeit at a level lower than that of normal mice. The mdx3cv EDL muscle was also partially protected from eccentric contraction-induced injury. Furthermore, mdx3cv mice performed better than mdx4cv mice on a forelimb grip strength assay. Additional studies showed utrophin up-regulation in both mdx3cv and mdx4cv muscles. The dystrophin-associated glycoprotein complex (DGC) was also weakly restored in both strains. Using antibodies against different regions of dystrophin, we found uniform low-level expression of a membrane-bound dystrophin protein in mdx3cv, but not mdx4cv, muscles. Taken together, our results provide the first evidence that subtherapeutic levels of a membrane-bound dystrophin expression may help preserve minimal muscle force. However, this low-level dystrophin expression is insufficient to prevent sarcolemma injury neither can it stop myofiber degeneration.Materials and MethodsAnimalsAll animal experiments were approved by the Animal Care and Use Committee of the University of Missouri and were in accordance with National Institutes of Health guidelines. BL6 (C57BL/6J), mdx3cv (B6Ros.Cg-Dmdmdx-3Cv/J), and mdx4cv (B6Ros.Cg-Dmdmdx-4Cv/J) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Male mice (3, 6, and 12 months of age) were used in this study. All experimental mice were housed in a specific pathogen-free facility and were kept under a 12-hour light (25 lux)/12-hour dark cycle with free access to food and water.Histopathology StudiesHematoxylin and eosin (H&E) staining was used to reveal general histology and centrally nucleated myofibers. Macrophage infiltration in muscle was evaluated by nonspecific esterase (α-naphthyl butyrate esterase) staining according to a published protocol.20Lai Y Yue Y Liu M Ghosh A Engelhardt JF Chamberlain JS Duan D Efficient in vivo gene expression by trans-splicing adeno-associated viral vectors.Nat Biotechnol. 2005; 23: 1435-1439Crossref PubMed Scopus (173) Google Scholar, 21Yue Y Liu M Duan D C-terminal truncated microdystrophin recruits dystrobrevin and syntrophin to the dystrophin-associated glycoprotein complex and reduces muscular dystrophy in symptomatic utrophin/dystrophin double knock-out mice.Mol Ther. 2006; 14: 79-87Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar Macrophages are dark brown cells scatted between myofibers. Sarcolemma integrity was determined with an Evans blue dye (EBD) uptake assay described previously.20Lai Y Yue Y Liu M Ghosh A Engelhardt JF Chamberlain JS Duan D Efficient in vivo gene expression by trans-splicing adeno-associated viral vectors.Nat Biotechnol. 2005; 23: 1435-1439Crossref PubMed Scopus (173) Google Scholar Briefly, EBD (10 mg/ml in phosphate-buffered saline, 20 μl/g body weight) was injected into tail vein at 24 hours before muscle harvesting. At 2 hours before tissue collection, mice were run on a 15° downhill treadmill at the speed of 15 m/minute for 30 minutes. Freshly dissected muscle was snap-frozen in liquid nitrogen-cooled isopentane in optimal cutting temperature compound (Sakura Finetek Inc., Torrance, CA). Ten-μm cryosections were visualized under the Texas Red channel with an E800 fluorescence microscope (Nikon, Melville, NY). Nuclei were visualized with 4,6-diamidino-2-phenylindole staining (SlowFade light antifade kit with 4,6-diamidino-2-phenylindole, catalog no. S-24636; Molecular Probes, Eugene, OR).ImmunostainingDystrophin was examined with seven epitope-specific antibodies including a mouse monoclonal antibody against the N-terminal domain (Manex1A, 1:300, clone 4C7, IgG2b; a gift from Dr. Glenn Morris, The Robert Jones and Agnes Hunt Orthopaedic Hospital, Shropshire, UK),22Thanh LT Nguyen TM Helliwell TR Morris GE Characterization of revertant muscle fibers in Duchenne muscular dystrophy, using exon-specific monoclonal antibodies against dystrophin.Am J Hum Genet. 1995; 56: 725-731PubMed Google Scholar a rabbit polyclonal antibody against spectrin-like repeats 4 to 6 (1:400; Santa Cruz Biotechnology, Santa Cruz, CA), a mouse monoclonal antibody against spectrin-like repeat 11 (Mandys8, 1:200, IgG2b; Sigma, St. Louis, MO), a mouse monoclonal antibody against spectrin-like repeats 14 to 18 (Mandys105, 1:500, clone 8A4, IgG1; a gift from Dr. Glenn Morris),22Thanh LT Nguyen TM Helliwell TR Morris GE Characterization of revertant muscle fibers in Duchenne muscular dystrophy, using exon-specific monoclonal antibodies against dystrophin.Am J Hum Genet. 1995; 56: 725-731PubMed Google Scholar a mouse monoclonal antibody against spectrin-like repeat 19/hinge 3 (Manex50, 1:2000, clone 6A9, IgG1; a gift from Dr. Glenn Morris),22Thanh LT Nguyen TM Helliwell TR Morris GE Characterization of revertant muscle fibers in Duchenne muscular dystrophy, using exon-specific monoclonal antibodies against dystrophin.Am J Hum Genet. 1995; 56: 725-731PubMed Google Scholar a mouse monoclonal antibody against the C-terminal domain (Mandra1, 1:500, clone 7A10, IgG1; a gift from Dr. Glenn Morris),22Thanh LT Nguyen TM Helliwell TR Morris GE Characterization of revertant muscle fibers in Duchenne muscular dystrophy, using exon-specific monoclonal antibodies against dystrophin.Am J Hum Genet. 1995; 56: 725-731PubMed Google Scholar and another mouse monoclonal antibody against the C-terminal domain (Dys-2, 1:30, clone Dy8/6C5, IgG1; Novocastra, Newcastle, UK). Utrophin was examined with a mouse monoclonal antibody against the utrophin N-terminal domain (VP-U579, 1:20, clone DRP3/20C5, IgG1; Vector Laboratories, Burlingame, CA). β-Dystroglycan was revealed with a mouse monoclonal antibody against the C terminus (NCL-b-DG, 1:50, clone 43DAG1/8D5, IgG2a; Novocastra). β-Sarcoglycan was revealed with a mouse monoclonal antibody (NCL-b-SARC, 1:50, clone 5B1, IgG1; Novocastra). Dystrobrevin was revealed with a mouse monoclonal antibody (no. 610766, 1:200, clone 23, IgG1; BD Biosciences, San Diego, CA). Syntrophin was revealed with a pan-syntrophin mouse monoclonal antibody that recognized the PDZ domain (ab11425, 1:200, clone 1351, IgG1; Abcam, Cambridge, MA). Immunostaining was performed essentially as we described before.20Lai Y Yue Y Liu M Ghosh A Engelhardt JF Chamberlain JS Duan D Efficient in vivo gene expression by trans-splicing adeno-associated viral vectors.Nat Biotechnol. 2005; 23: 1435-1439Crossref PubMed Scopus (173) Google Scholar, 23Yue Y Li Z Harper SQ Davisson RL Chamberlain JS Duan D Microdystrophin gene therapy of cardiomyopathy restores dystrophin-glycoprotein complex and improves sarcolemma integrity in the mdx mouse heart.Circulation. 2003; 108: 1626-1632Crossref PubMed Scopus (131) Google Scholar To determine the relative immunofluorescence intensity in samples stained with anti-dystrophin or anti-utrophin antibodies, digitized images were quantified using the Image J software (version 1.36b; National Institutes of Health, Bethesda, MD). Three to five random fields were analyzed for each muscle section. At least three different sections were studied for each muscle sample.Western BlotWhole muscle lysate was prepared from freshly isolated tibialis anterior (TA) muscle. Briefly, muscle tissue was homogenized in a liquid nitrogen-cooled mortar in a buffer containing 10% sodium dodecyl sulfate, 5 mmol/L ethylenediaminetetraacetic acid, 62.5 mmol/L Tris, pH 6.8, and 1% protease inhibitor (Roche, Indianapolis, IN). After a 2-minute boiling, the homogenate was spun at 14,000 rpm for 2 minutes (Eppendorf centrifuge, model 5417C; Eppendorf-Netheler-Hinz GmbH, Hamburg, Germany). Supernatant was used for Western blot. Protein concentration was determined using a DC protein assay kit (Bio-Rad, Hercules, CA). Muscle lysate (180 μg) was loaded on a 6% sodium dodecyl sulfate-polyacrylamide gel. After electrophoresis, protein was transferred to a polyvinylidene difluoride membrane. Dystrophin was detected with the Mandra1 antibody (1:100, clone 7A10, IgG1; a gift from Dr. Glenn Morris).22Thanh LT Nguyen TM Helliwell TR Morris GE Characterization of revertant muscle fibers in Duchenne muscular dystrophy, using exon-specific monoclonal antibodies against dystrophin.Am J Hum Genet. 1995; 56: 725-731PubMed Google Scholar Utrophin was detected with a mouse monoclonal antibody against utrophin amino acid residues 768 to 874 (no. 610896, 1:250, clone 55, IgG1; BD Biosciences). As a loading control, membrane was also probed with an anti-α-tubulin antibody (1:3000, clone B-5-1-2; Sigma).EDL Muscle Force MeasurementsEDL contractile properties were examined using a previously described protocol.20Lai Y Yue Y Liu M Ghosh A Engelhardt JF Chamberlain JS Duan D Efficient in vivo gene expression by trans-splicing adeno-associated viral vectors.Nat Biotechnol. 2005; 23: 1435-1439Crossref PubMed Scopus (173) Google Scholar, 21Yue Y Liu M Duan D C-terminal truncated microdystrophin recruits dystrobrevin and syntrophin to the dystrophin-associated glycoprotein complex and reduces muscular dystrophy in symptomatic utrophin/dystrophin double knock-out mice.Mol Ther. 2006; 14: 79-87Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 24Liu M Yue Y Harper SQ Grange RW Chamberlain JS Duan D Adeno-associated virus-mediated micro-dystrophin expression protects young Mdx muscle from contraction-induced injury.Mol Ther. 2005; 11: 245-256Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar The freshly isolated EDL muscle was vertically mounted in a 30°C jacket organ bath containing oxygenated Ringer's solution (95% O2, 5% CO2). Muscle tendons were attached to a 300B dual-mode servomotor transducer (Aurora Scientific, Inc., Aurora, Canada) with sutures. Optimal muscle length (Lo) was determined as the muscle length at which the maximal twitch force was elicited. At first, all of the myofibers were activated with three 500-ms tetanic stimulations at 150 Hz. After 2 minutes of resting, the absolute twitch force was measured. Muscle force-frequency relationship was then determined with tetanic stimulation at 50, 80, 120, and 150 Hz, respectively. Muscle cross-sectional area was calculated according to the following equation, cross-sectional area = (muscle mass)/(0.44 × Lo × muscle density), where 0.44 represents the ratio of fiber length to optimal muscle length (Lf/Lo) for the EDL muscle. Muscle density is 1.06 g/cm3. The specific force (kN/m2) was calculated by normalizing the absolute muscle force with the cross-sectional area. After tetanic force measurement, the EDL muscle was subjected to an eccentric injury protocol. Briefly, the EDL muscle was stimulated at 150 Hz for 700 ms and an eccentric injury was applied during the last 200 ms by lengthening the muscle by 10% Lo at the speed of 0.5 Lo/second. A total of 10 cycles of eccentric contraction was applied. The maximal isometric tetanic force developed during the first 500 ms of stimulation of the first cycle was designated as 100% force (baseline force). The percentage of tetanic force drop after each eccentric injury was graphed to reflect the level of resistance against contraction-induced injury. Raw data were acquired and analyzed with DMC/DMA software (Version 3.12, Aurora Scientific).Forelimb Grip Strength MeasurementForelimb grip strength was measured with a computerized grip strength meter (Columbus Instruments, Columbus, OH). Five sets of measurements were performed in each mouse throughout a period of 60 minutes with a 10-minute rest between sets. In each set of measurements, the mouse was allowed three attempts. The highest force from the three attempts was recorded as the force score for the set. The average value from three highest sets was defined as the forelimb grip strength for the mouse.Serum CK AssayFresh serum was collected by retro-orbital bleeding. The CK level was determined with a CK liqui-UV test kit from Stanbio Laboratory (Boerne, TX).Statistical AnalysisData are presented as mean ± SE of mean. Statistical analysis was performed with the SPSS software (SPSS, Chicago, IL). Statistical significance for multiple group comparison was determined by one-way analysis of variance followed by Bonferroni post hoc analysis. Statistical significance for two-group comparison was determined by t-test. Difference was considered significant when P < 0.05.ResultsThe Mdx3cv EDL Muscle Showed Significantly Higher Tetanic Force and Was More Resistant to Eccentric Contraction-Induced Injury than the Mdx4cv EDL MuscleContractile property of the mdx3cv EDL muscle has not been studied before. We first compared the twitch and tetanic forces in 6-month-old mice. We used age- and sex-matched BL6 mice as the normal control. As expected, the BL6 EDL muscle displayed the highest specific forces under all stimulation conditions. They were significantly higher than those from mdx3cv and mdx4cv muscles (Figure 1). When single twitch and low frequency (50 Hz) tetanic stimulations were applied, mdx3cv and mdx4cv muscles showed similar responses. Surprisingly, when muscles were stimulated at higher stimulation frequencies (80 Hz, 120 Hz, and 150 Hz), the mdx3cv EDL muscle generated significantly stronger force than that of the mdx4cv EDL muscle (Figure 1B).A pivotal feature in DMD is contraction-induced muscle injury. We next examined whether mdx3cv muscle could be protected from eccentric contraction damage. In eccentric contraction, a muscle is stretched beyond its optimal length while the muscle is at its peak tetanic force. This is considered one of the most sensitive physiology assays in evaluating disease progression and therapeutic intervention in muscular dystrophy research. A better force preservation suggests good muscle protection. We applied 10 repeated cycles of eccentric contraction. The BL6 EDL muscle showed moderate force decline through the entire experimental protocol (Figure 1C). Muscle force rapidly dropped to less than 20% of the starting level in the mdx4cv EDL muscle. The mdx3cv EDL muscle also showed a quick drop but to a significantly less extent than that of the mdx4cv EDL muscle (Figure 1C).To further extend our observation, we examined muscle contraction in 12-month-old mice (Figure 2). A similar but weaker profile was obtained for the older mdx3cv EDL muscles. Their specific forces were significantly better than those of the mdx4cv EDL muscles at 120-Hz and 150-Hz stimulation frequencies (Figure 2B). In the eccentric contraction assay, they also showed a trend of better force preservation although it did not reach statistical significance (Figure 2C). Despite the difference in the tetanic force and eccentric contraction profiles between mdx3cv and mdx4cv mice, the anatomical features of the EDL muscles were identical between the two groups (Table 1). Both showed significant hypertrophy as demonstrated by the increased EDL muscle weight and the enlarged cross-sectional area (Table 1).Figure 2The EDL muscles of 12-month-old mdx3cv mice display better specific tetanic force than that of age-matched mdx4cv mice at high stimulation frequencies. A: Specific twitch force. B: The force-frequency relationship. C: Relative force decline after 10 rounds of eccentric contraction-induced injury. Sample size, n = 4 for BL6, n = 8 for mdx3cv, n = 6 for mdx4cv. Asterisk, values in BL6 are significantly different from those in mdx3cv and mdx4cv mice; dagger, values in mdx3cv are significantly different from those in BL6 and mdx4cv mice.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table 1Characterization of the EDL MuscleStrainAge (months)nWeight (mg)Lo (mm)CSA (mm2)BL66811.20 ± 0.44*Significantly different from those of age-matched mdx3cv and mdx4cv.13.20 ± 0.051.82 ± 0.07*Significantly different from those of age-matched mdx3cv and mdx
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