DUCHENNE MUSCULAR DYSTROPHY (DMD) is the most

Transcription

1 1572 Eccentric Contraction Injury in Dystrophic Canine Muscle Martin K. Childers, DO, Carol S. Okamura, PhD, Daniel J. Bogan, BA, Janet R. Bogan, BS, Gregory F. Petroski, MS, Kerry McDonald, PhD, Joe N. Kornegay, DVM, PhD ABSTRACT. Childers MK, Okamura CS, Bogan DJ, Bogan JR, Petroski GF, McDonald K, Kornegay JN. Eccentric contraction injury in dystrophic canine muscle. Arch Phys Med Rehabil 2002;83: Objective: To test the hypothesis that eccentric contractions induce greater injury in dystrophic compared with normal canine muscle. Design: Blinded cohort study. Setting: Animal laboratory. Animals: Ten dogs with a homologue to Duchenne muscular dystrophy (Golden retriever muscular dystrophy [GRMD]) and 10 normal littermates. Interventions: Contractions induced in tibiotarsal flexors and extensors by sciatic nerve stimulation. Because more powerful extensors overrode flexors, eccentric contractions occurred in flexors. Concentric contractions were induced in contralateral flexors by peroneal nerve stimulation. Main Outcome Measure: Tibiotarsal flexion force 3 days after contractions. Muscle was examined for injury (esterase activity, Evans blue dye penetration) and regeneration (embryonic myosin isoform expression). Results: Mean force deficit after eccentric flexor contractions was 43.3% 25.7% in GRMD dogs compared with 25.0% 18.4% in controls (P.04, Wilcoxon rank-sum test). Concentric contractions induced force deficits in GRMD but not normal dogs; however, the difference between the 2 groups was not significant (P.08, Wilcoxon rank-sum test). After concentric contractions in controls, force decrements correlated with esterase activity measured by area (r.794, P.006) and intensity (r.697, P.025, Spearman rank correlation). No other significant correlation was detected between force and biopsy data. Conclusions: Force data support the hypothesis that eccentric contractions induce greater injury in dystrophic compared with normal canine muscle. Phenotypic features of the dystrophic canine model used here are similar to those of humans with Duchenne s. Key Words: Dogs; Exercise; Muscles; Muscular dystrophy, animal; Muscular dystrophy, Duchenne; Rehabilitation by the American Congress of Rehabilitation Medi- From the Departments of Physical Medicine and Rehabilitation (Childers) and of Physiology (Childers, McDonald), School of Medicine; Department of Medicine and Surgery, College of Veterinary Medicine (Okamura, Bogan, Bogan, Kornegay); The Dalton Cardiovascular Research Center (Okamura, Bogan, Bogan, Kornegay); and the Biostatistics Unit, Integrated Technology Services (Petroski), University of Missouri-Columbia, Columbia, MO. Supported by the National Center for Medical Rehabilitation Research, National Institute of Child Health and Human Development, National Institutes of Health (grant no. KO8 HD ), The Parent Project for Muscular Dystrophy Research Inc, and the American Academy of Physical Medicine and Rehabilitation New Investigator Award. No commercial party having a direct financial interest in the results of the research supporting this article has or will confer a benefit upon the author(s) or upon any organization with which the author(s) is/are associated. Reprint requests to Martin K. Childers, DO, DCO 46.00, One Hospital Dr, University of Missouri-Columbia, Columbia, MO 65212, childersmk@ health.missouri.edu /02/ $35.00/0 doi: /apmr cine and the American Academy of Physical Medicine and Rehabilitation DUCHENNE MUSCULAR DYSTROPHY (DMD) is the most common inherited lethal childhood muscle disorder worldwide. 1 Affected children become progressively weaker because of muscle wasting and contractures, often leading to wheelchair use by the age of The primary biochemical cause of DMD is a deficiency of dystrophin, a protein normally found on the internal surface of the myofiber membrane. 3-5 However, factors that lead to disability in DMD remain obscure. Investigators 6 have speculated that dystrophin stabilizes the myofiber membrane during contraction. Thus, dystrophin-deficient muscles might be more susceptible than normal to contractioninduced injury. 5 This idea is supported by the observation in human myopathies that proximal muscles are involved in lengthening (eccentric) contractions to a greater extent than distal muscles. 7 Lengthening contractions occur when active muscles are extended, such as when lowering the body during stepping. Subsequent animal research 8-10 has shown that normal muscle is injured more after lengthening contractions compared with shortening contractions. A vicious cycle of weakness, stretch, damage, and further weakness might explain observations of selective involvement of eccentric-contracting dystrophic muscles. 6 Ethical concerns limit collection of experimental data from children with DMD. Animal models, however, provide an opportunity to thoroughly pursue scientific hypotheses in DMD. 11 Several dystrophin-deficient animal models have been characterized Mutation of the dystrophin gene causes a canine DMD homologue, termed golden retriever muscular dystrophy (GRMD). 17 Unlike the dystrophin-deficient mdx mouse, which remains relatively normal clinically, GRMD dogs suffer a rapidly progressive, fatal disease similar to DMD. 18 Incomplete muscle repair in both DMD and GRMD results in fibrosis, contracture, and weakness Moreover, GRMD dogs display selective muscle involvement analogous to that of DMD patients Because of these clinical similarities, studies in GRMD are more likely than those in the mdx mouse to predict pathogenesis and outcome of treatment in DMD. Furthermore, functional tests such as force measurements and joint range of motion can be used as outcome parameters in affected dogs Muscle injury increases directly with peak force 10 and is more pronounced after lengthening (eccentric) contractions versus shortening (concentric) contractions. 27 In our study, we hypothesized that eccentric contractions would induce greater muscle injury in GRMD compared with normal canine muscle. In the absence of fatigue, the force deficit is a reliable physiologic measure of muscle damage. 28,29 The force deficit, defined as the difference between maximum isometric force (P 0 ) before and after exercise, is expressed as a percent change of the control value for P 0 : force deficit ([P 0 P 0 after exercise ]/ P 0 ) 100. Because skeletal muscle damage caused by exercise peaks after 3 days in normal humans, 9,30 we compared the force deficit between GRMD and normal dogs 3 days after experimental contractions. As an additional measure, biopsy speci-

2 ECCENTRIC INJURY IN DYSTROPHY, Childers 1573 mens were examined for markers of myofiber injury and regeneration. METHODS Protocols were approved by the University of Missouri Animal Care and Use Committee and adhered to principles outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. A colony of GRMD dogs was used for all experiments. 18,31,32 Affected dogs were identified at 1 day of age based on dramatic elevation of serum creatine kinase (CK) and confirmed with polymerase chain reaction based genotyping. Characteristic clinical signs developed subsequently. Body weights were recorded several times daily for the first 3 weeks of life to ensure that GRMD pups were gaining weight, with subsequent measures taken at weekly intervals. Ten GRMD dogs aged 10 to months, and 10 age-matched, normal littermates were used. Contraction Force Measurements The force deficit was used to measure muscle injury. 33 Accordingly, maximal isometric force generated by flexor muscles of the cranial tibial compartment was measured by a blinded investigator (JNK) in anesthetized dogs immediately before and 3 days after eccentric (lengthening) or concentric (shortening) contractions. Serial measures of isometric contraction forces in the flexor compartment of GRMD dogs performed by using these methods was previously reported, with good retest reliability between right and left pelvic limbs: mean standard deviation (SD) of the difference between GRMD flexion forces was (P.36), with a maximum absolute difference of 2.45N. 26 Thus, repeated measures between right and left limbs did not differ statistically. In our investigation, the lever used during isometric contractions 26 was immovable during baseline and follow-up isometric force measures but moved freely during eccentric (fig 1) and concentric (fig 2) protocols. Dogs were premedicated with acepromazine maleate (.02mg/kg), butorphanol (.40mg/kg), and atropine sulfate (.04mg/kg); masked; intubated; and subsequently maintained with isoflurane. In a preliminary study, mean alveolar concentration values for isoflurane did not significantly affect force measurements. 26 Anesthetized dogs were positioned in dorsal recumbency. The pelvic limbs were alternately immobilized in a custom-made stereotactic frame that aligns the tibia parallel to the table at a 90 angle to the femur. In an earlier study, 26 the length-tension relationship was not shifted for GRMD (n 7) versus normal (n 4) dogs in which measurements were made at 10 increments between tibiotarsal joint angles 60 and 120. Baseline forces were measured by stimulating the common peroneal nerve by using paired stimulating and reference 27- gauge monopolar needle electrodes a placed just distal to the fibular head. As a result, the distal pelvic limb pulled a lever interfaced with a custom-made transducer and ergometer, a providing a measure of isometric force. Supramaximal 150V, 100 s pulses were applied b in a tetanic run of 250 pulses (50/s). Passive force was subtracted from total force produced; only active force generated by flexor muscles was measured. Because dogs varied in weight, the absolute tetanic force (in newtons) was divided by the body weight (in kilograms) to obtain weight-corrected force. Dogs were repositioned after each force measurement, and the mean of 2 weight-corrected forces was recorded. Experimental Contractions After randomization and coding, both eccentric and concentric contractions were induced in GRMD (n 10) and normal Fig 1. The eccentric contraction method. Supramaximal percutaneous stimulation of the sciatic nerve at mid-femur caused contraction of both tibiotarsal joint flexors and extensors. However, because the more powerful extensor muscles of the caudal tibial compartment predominated, flexor muscles of the cranial tibial compartment underwent eccentric (lengthening) contractions. (n 10) dogs. One limb in each dog was subjected to eccentric contractions while the contralateral pelvic limb underwent concentric contractions. Eccentric contractions in tibiotarsal flexor muscles of the cranial tibial compartment of the pelvic limb occurred by stimulating the sciatic nerve at mid-femur. The sciatic nerve innervates both the cranial (anterior) and caudal (posterior) tibial compartment muscles. Stimulation of the muscles of the caudal compartment, larger and stronger than muscles of the cranial compartment, resulted in extension of the tibiotarsal joint with eccentric contractions of the flexor muscles (fig 1). Concentric contractions of the cranial compartment in the contralateral pelvic limb occurred by percutaneous stimulation of the common peroneal nerve (fig 2). Stimulation Protocols Before beginning the experimental contractions, isometric forces from single muscle twitches were measured. The sciatic and common peroneal nerves were alternately stimulated by using square wave pulses of 100 s in duration. The percutaneous stimulating electrode was optimally positioned and voltage adjusted until twitch force (P t ) reached a maximum. To maximally activate the muscles of interest, stimulation voltage was 50% greater than necessary to achieve P t. Electromyographic activity was continuously monitored in the cranial

3 1574 ECCENTRIC INJURY IN DYSTROPHY, Childers without treatment. Sections for antibody staining, including 1 antibody-negative control, were incubated in precooled acetone at 20 C for 10 minutes and washed 3 10 minutes with phosphate-buffered saline before adding the antibody. Histochemistry for nonspecific esterase activity 35,36 was performed with an -napthyl acetate and pararosaniline substrate. Sections were stored at 80 C and thawed to room temperature just before imaging. Microscopy Three histochemical staining methods were used: Evans blue dye 34 to measure muscle injury, nonspecific esterase 35,36 for muscle necrosis, and embryonic myosin heavy chain (emhc) for muscle regeneration. 37,38 Degenerating muscle fibers emit bright red fluorescence under green light (wavelength, 590nm) activation after intravenous administration of Evans blue dye before biopsy. 34 Accordingly, cryosections were prepared and observed by using an Olympus IMT-2 d inverted epifluorescence microscope equipped with a green activation filter e permitting visualization of injured myofibers permeated by Evans blue dye (fig 3). Necrotic myofibers staining positive for a nonspecific esterase 35,36 were also evaluated in transverse cryosections by using light microscopy. After experimental muscle damage in humans and in mdx mice, regenerating myofibers can be identified based on their expression of emhc. 37,38 Therefore, we used emhc as a marker of myofiber regeneration. Muscle cryosections (10 m) were treated with monoclonal antibodies f to emhc. Primary antibodies were localized with a Cy2-conjugated goat antimouse IgG antibody g and visualized by using epifluorescence microscopy methods described previously. 34 Fig 2. Isometric force measures and the concentric contraction method. Anesthetized dogs were positioned in dorsal recumbency, with the tibia parallel to the table and perpendicular to the femur. The common peroneal nerve was stimulated distal to the stifle (knee) to measure maximal tetanic force. As a consequence of stimulation, the distal pelvic limb pulled (flexion) a lever interfaced with a force transducer and ergometer. tibialis muscle to ensure that maximal stimulation was maintained. If a decrease in motor unit action potential amplitude was observed, the stimulating needle was repositioned. Muscle contractions were induced using square wave pulses of 100 s in a tetanic run for a duration of 500ms and at a frequency of 50Hz. Pulses were applied to the pelvic limb over 20 minutes, at a rate of 1 train of pulses every 4 seconds. Thus, a rest time of 4 seconds occurred between stimulations. This resulted in 240 stimuli applied to the sciatic nerve to achieve eccentric contraction of muscles in the cranial tibial compartment. The common peroneal nerve was similarly stimulated in the contralateral pelvic limb to achieve concentric contractions in the same muscle group. Muscle Biopsies Samples were taken from the cranial tibialis muscle in anesthetized dogs according to established protocols. 34 Longitudinal sections of muscle (approximately 0.5cm 3 ) were removed sharply from 3 areas: the midsection and both proximal and distal ends, near the musculotendinous junctions. Samples were frozen in a freon substitute, c cooled with liquid nitrogen, and sectioned at 10 m. Cryosections to be examined for Evans blue dye penetration were analyzed Quantification of Muscle Injury, Necrosis, and Regeneration After randomization and coding, serial cryosections were analyzed by an investigator (MKC) who did not know the sources of the sections. Slides were previewed under low magnification to identify areas devoid of staining artifacts. For each of the 3 staining methods described earlier, a computer software program h was used to digitally measure the number of positively stained myofibers, their respective cross-sectional area, and relative pixel intensity. This was accomplished by digitally photographing a total of 1000 myofibers from each cryosection serially stained by the 3 histochemical methods. Slides were previewed under low magnification to identify areas devoid of staining artifacts. One hundred myofibers in 10 adjacent fields of view (to obtain 1000 total myofibers) were photographed under 200 magnification, measured, and recorded in a spreadsheet. These data provided a basis to estimate the amount of muscle injury, necrosis, and regeneration based on histochemical staining methods. Statistical Analysis All statistical analyses were performed by using SAS i statistical software. The Wilcoxon signed-rank test was used to test the hypothesis that muscle damage, as measured by the force deficit, resulted as a consequence of experimental contractions. The Wilcoxon rank-sum test was used to test for differences in normal and GRMD dogs with respect to percent change in force. Statistical significance was set at the 5% level (P.05). The Spearman rank correlation coefficient was used to examine associations between force measures and muscle biopsy data. Percentage of change in relative force was correlated with quantitative data (number of positively

4 ECCENTRIC INJURY IN DYSTROPHY, Childers 1575 Fig 3. Micrographs (200 ) of10 m cryosections from the cranial tibialis muscle of age-matched 12-month-old normal (top panels) and GRMD dog (bottom panels). Images were taken 3 days after eccentric contractions. Left panels: Evans blue dye (fluorescent at 510nm) is normally excluded from myofibers but permeates damaged (lightly colored) myofibers. This finding indicates myofiber damage in GRMD dogs. Middle panels: Nonspecific esterase activity in necrotic (darkly stained) myofibers. This finding denotes myofibers necrosis in GRMD dogs. Right panels: emhc expression in regenerating (darkly stained, bottom right panel) myofibers. Note the clusters of centrally located nuclei characteristic of myofiber regeneration in GRMD dogs.

5 1576 ECCENTRIC INJURY IN DYSTROPHY, Childers Table 1: Summary Statistics of Force Changes Relative to Baseline Values 3 Days After Experimental Contractions Group n Mean SD Median Minimum Maximum P * Concentric contractions GRMD Control Eccentric contractions GRMD Control NOTE. Values are percent changes from baseline in body-weight corrected isometric tetanic forces (N/kg) generated by tarsal joint flexion. Negative values indicate a reduction in force. * Wilcoxon signed-rank test, testing the hypothesis that no differences occurred in force relative to baseline. stained myofibers, cross-sectional area, and pixel intensity) derived from each of the 3 staining methods of the cranial tibialis muscle. Force data for individual dogs were matched with mean area and maximum pixel intensity of positively stained myofibers. Correlation analysis was performed for each intervention (eccentric and concentric contractions) and for each biopsy stain (esterase stain, Evans blue dye penetration, emhc expression). RESULTS Force Measures Force deficits 3 days after experimental interventions for normal and GRMD dogs are shown in table 1. Mean isometric force significantly (P.05) decreased under all conditions except after concentric paradigms in normal dogs (P 1.0). Thus, muscle damage as measured by changes in maximal isometric force, resulted as a consequence of some, but not all, experimental interventions. Eccentric paradigms induced significantly (P.04) greater force deficits in muscles of the cranial tibial compartment in GRMD dogs compared with controls. The mean force deficit after eccentric paradigms was 43.3% 25.7% in GRMD dogs compared with 25.0% 18.4% in controls (table 1, fig 4). As stated previously, concentric paradigms induced force deficits in GRMD dogs but not normal dogs (fig 5). However, the difference in force deficits between the 2 groups was not statistically significant (P.08). Although both types of experimental interventions caused a greater decrease in force in GRMD compared with normal dogs, the eccentric effect was more pronounced compared with the concentric effect. Taken together, these data support the hypothesis that eccentric contractions are more damaging than concentric contractions and induce greater injury in dystrophic compared with normal canine muscles. Histopathologic Measures Following concentric paradigms in normal dogs, force measures correlated with the esterase stain area average (r.794, P.006) and with the esterase stain area maximum pixel intensity (r.697, P.025). No other significant correlation was detected between force and biopsy stain data (Evans blue dye penetration, emhc expression) under any other experimental condition (data not shown). Fig 4. Boxplot of weight-corrected isometric flexion forces measured 3 days after eccentric contractions in GRMD (n 10) and normal (n 10) dogs. Bars extending below the 0 on the abscissa indicate force deficits. Legend:, mean; upper and lower ends of the boxes, 25th and 75th percentiles, respectively; line through the box, median; vertical lines, range of the data. Fig 5. Boxplot of weight-corrected isometric flexion forces measured 3 days after concentric contractions in GRMD (n 10) and normal (n 10) dogs. See figure 4 for explanations.

6 ECCENTRIC INJURY IN DYSTROPHY, Childers 1577 DISCUSSION Absence of dystrophin in DMD may lead to membrane instability and mechanical damage during eccentric muscle contractions and subsequent myofiber necrosis Controversy regarding the mechanical damage hypothesis comes from conflicting findings in the mdx mouse, the murine homologue to DMD. 35,43 In 1 study, 44 eccentric contractions did not result in increased CK or greater than normal force deficit. In contrast, after eccentric contractions, mdx muscles have been shown to take up greater-than-normal membrane-impermeable dye 42 and to have greater than normal necrosis. 45,46 Similarly, mdx muscles injected with a minidystrophin gene were protected from stretch-induced injury. 47 Findings from our canine study agree with the latter investigations and further support a role for dystrophin in protecting against muscle injury after eccentric contractions. In our study, normal dogs showed force deficits 3 days after eccentric but not concentric contractions, in keeping with previous reports GRMD dogs experienced greater than normal force decrements after concentric but even more so after eccentric contractions. Cycles of repeated injury might explain observations of selective involvement of eccentric-contracting dystrophic muscles. 6,34,48 Nonspecific esterase activity was the only histologic marker that correlated with force data in our study. This lack of consistent histopathologic correlation suggests 2 possibilities. First, muscle biopsy data support the null hypothesis, that is, eccentric contractions did not induce greater than normal injury in dystrophic myofibers. Second, histopathologic measures did not reflect differences between groups when differences existed. Although the first scenario cannot be excluded, the second possibility appears more likely. Phenotypic variability 17,26,32 in GRMD dogs, as reflected in our biopsy data, lessens the likelihood that significant differences will be seen with small sample sizes. This was complicated by the fact that only a small cross-section of muscle can be readily examined in dogs versus mice. In an attempt to account for this discrepancy of size, samples were examined from the proximal, middle, and distal ends of the cranial tibialis muscle. Nonetheless, it appears likely that the sample size was too small to offset phenotypic variability in the GRMD dogs. Clinical Implications The effects of exercise stress in DMD patients remain controversial. 2,49,50 Some patients have shown improved strength after 1 year of resistance training. 51 Although improvement in walking was found in 1 group of DMD patients receiving intensive physiotherapy, the treated group died significantly younger than the controls. 52 In contrast, others have reported myalgias and rhabdomyolysis after muscle contractions in patients with dystrophinopathies Our data suggest that experimental contractions induce injury in dystrophic muscle to a greater extent than normal, thereby supporting the idea that dystrophic muscles are preferentially injured by mechanical stress. CONCLUSION Data reported here support the hypothesis that dystrophic canine flexor muscles are more vulnerable than normal to eccentric contraction-induced injury as measured by force deficits 3 days following experimental interventions. Ethical concerns limit collection of experimental data from children with DMD, thus the present investigation was conducted in dogs with GRMD. Phenotypic features of the dystrophic canine model are similar to those of humans with DMD. Acknowledgments: We acknowledge the assistance of Shawn Bailes and Jessica Wagner in the Molecular Cytology Core Facility, Dept of Biological Sciences, College of Arts and Sciences, University of Missouri-Columbia. References 1. Monaco AP. Molecular human genetics and the Duchenne/Becker muscular dystrophy gene. In: Partridge T, editor. Molecular and cell biology of muscular dystrophy. London: Chapman & Hall; p Siegel IM. The management of muscular dystrophy: a clinical review. Muscle Nerve 1978;1: Watkins SC, Hoffman EP, Slayter HS, Kunkel LM. Immunoelectron microscopic localization of dystrophin in myofibres. Nature 1988;333: Cullen MJ, Walsh J, Nicholson LV, Harris JB. Ultrastructural localization of dystrophin in human muscle by using gold immunolabelling. Proc R Soc Lond B Biol Sci 1990;240: Karpati G, Carpenter SC. The deficiency of a sarcolemmal cytoskeletal protein (dystrophin) leads to necrosis of skeletal muscle fibres in Duchenne-Becker dystrophy. In: Sellin LS, Libellius R, Thesloff S, editors. Neuromuscular junction. Amsterdam: Elsevier, p Best TM, Hasselman CT, Garrett WE. Muscle strain injuries: biomechanical and structural studies. In: Salmons S, editor. Muscle damage. Oxford: Oxford Univ Pr; p Edwards RH, Newham DJ, Jones DA, Chapman SJ. Role of mechanical damage in pathogenesis of proximal myopathy in man. Lancet 1984;1: Brooks SV, Zerba E, Faulkner JA. Injury to muscle fibres after single stretches of passive and maximally stimulated muscles in mice. J Physiol 1995;488:(Pt 2): Faulkner JA, Brooks SV, Opiteck JA. Injury to skeletal muscle fibers during contractions: conditions of occurrence and prevention. Phys Ther 1993;73: McCully KK, Faulkner JA. Characteristics of lengthening contractions associated with injury to skeletal muscle fibers. J Appl Physiol 1986;61: Hoffman EP, Brown RH Jr, Kunkel LM. Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell 1987;51: Jackson MJ. Molecular mechanisms of muscle damage. In: Partridge T, editor. Molecular and cell biology of muscular dystrophy. London: Chapman & Hall; p Straub V, Rafael JA, Chamberlain JS, Campbell KP. Animal models for muscular dystrophy show different patterns of sarcolemmal disruption. J Cell Biol 1997;139: Bartlett RJ, Winand NJ, Secore SL, et al. Mutation segregation and rapid carrier detection of X-linked muscular dystrophy in dogs. Am J Vet Res 1996;57: Ervasti JM, Roberds SL, Anderson RD, Sharp NJ, Kornegay JN, Campbell KP. Alpha-dystroglycan deficiency correlates with elevated serum creatine kinase and decreased muscle contraction tension in golden retriever muscular dystrophy. FEBS Lett 1994; 350: Prattis SM, Horton SB, van Camp SD, Kornegay JN. Immunohistochemical detection of neural cell adhesion molecule and laminin in X-linked dystrophic dogs and mdx mice. J Comp Pathol 1994;110: Cooper BJ, Winand NJ, Stedman H, et al. The homologue of the Duchenne locus is defective in X-linked muscular dystrophy of dogs. Nature 1988;334: Kornegay JN, Tuler SM, Miller DM, Levesque DC. Muscular dystrophy in a litter of golden retriever dogs. Muscle Nerve 1988;11: Vignos PJ, Wagner MB, Karlinchak B, Katirji B. Evaluation of a program for long-term treatment of Duchenne muscular dystrophy. Experience at the University Hospitals of Cleveland. J Bone Joint Surg Am 1996;78: Oliveira AS, Gabbai AA, Kiyomoto BH, Ferreira Neto A, Schmidt B, Lima JG. [Congenital muscular dystrophy: clinical study of 17 patients] [Portuguese]. Arq Neuropsiquiatr 1991;49:

7 1578 ECCENTRIC INJURY IN DYSTROPHY, Childers 21. Seeger BR, Caudrey DJ, Little JD. Progression of equinus deformity in Duchenne muscular dystrophy. Arch Phys Med Rehabil 1985;66: Kornegay JN, Sharp NJ, Schueler RO, Betts CW. Tarsal joint contracture in dogs with golden retriever muscular dystrophy. Lab Anim Sci 1994;44: Johnson ER, Fowler WM Jr, Lieberman JS. Contractures in neuromuscular disease. Arch Phys Med Rehabil 1992;73: Sharp NJ, Kornegay JN, Bartlett RJ, Hung WY, Dykstra MJ. Notexin-induced muscle injury in the dog. J Neurol Sci 1993;116: Kornegay JN, Sharp NJ. A method to measure contraction tension sequentially from the peroneous longus muscle of dogs. Acta Cardiomiol 1995;7: Kornegay JN, Bogan DJ, Bogan JR, et al. Contraction force generated by tarsal joint flexion and extension in dogs with golden retriever muscular dystrophy. J Neurol Sci 1999;166: Armstrong RB, Warren GL, Warren JA. Mechanisms of exerciseinduced muscle fibre injury. Sports Med 1991;12: Faulkner JA, Jones DA, Round JM. Injury to skeletal muscles of mice by forced lengthening during contractions. Q J Exp Physiol 1989;74: Metzger JM, Fitts RH. Fatigue from high- and low-frequency muscle stimulation: contractile and biochemical alterations. J Appl Physiol 1987;62: Friden J, Lien IN. Muscle damage induced by cyclic eccentric contraction: biomechanical and structural studies. In: Salmons S, editor. Muscle damage. Oxford: Oxford Univ Pr; p Kornegay JN. Golden retriever muscular dystrophy: the model and relevance to developmental therapeutics. In: Kakulas BA, Howell JM, Roses AD, editors. Duchenne muscular dystrophy: animal models and genetic manipulation. New York: Raven Pr; p Valentine BA, Winand NJ, Pradhan D, et al. Canine X-linked muscular dystrophy as an animal model of Duchenne muscular dystrophy: a review. Am J Med Genet 1992;42: Brooks SV, Faulkner JA. The magnitude of the initial injury induced by stretches of maximally activated muscle fibres of mice and rats increases in old age. J Physiol 1996;497: Childers MK, Okamura CS, Bogan DJ, Bogan JR, Sullivan MJ, Kornegay JN. Myofiber injury and regeneration in a canine homologue of Duchenne muscular dystrophy. Am J Phys Med Rehabil 2001;80: Calore EE, Sesso A, Puga FR, Cavaliere MJ, Calore NM, Weg R. Sarcoplasmic lipase and non-specific esterase inhibition in myofibers of rats intoxicated with the organophosphate isofenphos. Exp Toxicol Pathol 1999;51: Abdel-Ghaffar AE, Essa TM, Nasr ME. Histopathological and immunohistochemical studies of skeletal muscle in mice experimentally infected with Schistosoma mansoni. J Egypt Soc Parasitol 1997;27: Wilson LA, Dux L, Cooper BJ, Dubowitz V, Sewry CA. Experimental regeneration in canine muscular dystrophy 2. Expression of myosin heavy chain isoforms. Neuromuscul Disord 1994; 4: Grounds MD, Yablonka-Reuveni Z. Molecular and cell biology of skeletal muscle regeneration. Mol Cell Biol Hum Dis Ser 1993; 3: Hutter OF. The membrane hypothesis of Duchenne muscular dystrophy: quest for functional evidence. J Inherit Metab Dis 1992;15: McArdle A, Edwards RH, Jackson MJ. Time course of changes in plasma membrane permeability in the dystrophin-deficient mdx mouse. Muscle Nerve 1994;17: McArdle A, Edwards RH, Jackson MJ. How does dystrophin deficiency lead to muscle degeneration? evidence from the mdx mouse. Neuromuscul Disord 1995;5: Petrof BJ, Shrager JB, Stedman HH, Kelly AM, Sweeney HL. Dystrophin protects the sarcolemma from stresses developed during muscle contraction. Proc Natl Acad Sci U S A 1993;90: Hutter OF, Burton FL, Bovell DL. Mechanical properties of normal and mdx mouse sarcolemma: bearing on function of dystrophin. J Muscle Res Cell Motil 1991;12: Sacco P, Jones DA, Dick JR, Vrbova G. Contractile properties and susceptibility to exercise-induced damage of normal and mdx mouse tibialis anterior muscle. Clin Sci (Lond) 1992;82: Weller B, Karpati G, Carpenter S. Dystrophin-deficient mdx muscle fibers are preferentially vulnerable to necrosis induced by experimental lengthening contractions. J Neurol Sci 1990;100: Vilquin JT, Brussee V, Asselin I, Kinoshita I, Gingras M, Tremblay JP. Evidence of mdx mouse skeletal muscle fragility in vivo by eccentric running exercise. Muscle Nerve 1998;21: Deconinck N, Ragot T, Marechal G, Perricaudet M, Gillis JM. Functional protection of dystrophic mouse (mdx) muscles after adenovirus-mediated transfer of a dystrophin minigene. Proc Natl Acad SciUSA1996;93: Petrof BJ. The molecular basis of activity-induced muscle injury in Duchenne muscular dystrophy. Mol Cell Biochem 1998;179: Lansman JB, Franco AJ. What does dystrophin do in normal muscle? J Muscle Res Cell Motil 1991;12: Iannaccone ST, Nanjiani Z. Duchenne muscular dystrophy. Curr Treat Options Neurol 2001;3: Vignos PJ Jr, Watkins MP. The effect of exercise in muscular dystrophy. JAMA 1966;197: Rideau Y. [Treatment of Duchenne s myopathy with early physiotherapy. Critical analysis] [French]. Arch Fr Pediatr 1985;42: Barbiroli B, McCully KK, Iotti S, Lodi R, Zaniol P, Chance B. Further impairment of muscle phosphate kinetics by lengthening exercise in DMD/BMD carriers. An in vivo 31P-NMR spectroscopy study. J Neurol Sci 1993;119: Figarella-Branger D, Baeta Machado AM, Putzu GA, Malzac P, Voelckel MA, Pellissier JF. Exertional rhabdomyolysis and exercise intolerance revealing dystrophinopathies. Acta Neuropathol (Berl) 1997;94: Malapert D, Recan D, Leturcq F, Degos JD, Gherardi RK. Sporadic lower limb hypertrophy and exercise induced myalgia in a woman with dystrophin gene deletion. J Neurol Neurosurg Psychiatry 1995;59: Suppliers a. Medical Research Electronics, CH-6314 Unterägeri Seestr 31a, Bremen, Germany. b. Model S48 Solid State Square Wave Stimulator; Astro-Med Inc, Astro-Med Industrial Pk, 600 E Greenwich Ave, West Warwick, RI c. Suva ; Dupont Corp, DuPont Bldg, 1007 Market St, Wilmington, DE d. Olympus Optical Co Ltd, Shinjuku Monolith, 3-1 Nishi-Shinjuku 2-chome, Shinjuku-ku, Tokyo , Japan. e. Omega Optical Inc, 210 Main St, Brattleboro, VT f. Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA g. Jackson ImmunoResearch Laboratories Inc, PO Box 9, 872 W Baltimore Pike, West Grove, PA h. Metamorph Imaging System; Universal Imaging Corp, 402 Boot Rd, Downingtown, PA i. Version 6; SAS Institute Inc, SAS Campus Dr, Cary, NC