SHORT REPORT

ABSTRACT: It has recently been suggested, based on studies of tissue pathology, that the limb muscles of old mdx mice may be a good model for the muscular changes seen in human Duchenne muscular dystrophy. To test this hypothesis, we measured force and stiffness of soleus and extensor digitorum longus (EDL) muscles of old (2021 months) mdx mice and agematched controls. The mdx and control muscles generated similar twitch, tetanic, and eccentric forces. They were also equally stiff. The results show that the mechanics of aged mdx limb muscles differ greatly from Duchenne muscular dystrophy in humans, and disagree with the hypothesis. 1998
John Wiley & Sons, Inc. Muscle Nerve 21: 536539, 1998

Key words: mdx mouse; force; stiffness; aging; Duchenne muscular dystrophy

FORCE AND STIFFNESS OF OLD DYSTROPHIC (MDX) MOUSE SKELETAL MUSCLES

JACQUES BOBET, PhD,1* ROBERT F. MOONEY, MSc,2 and TESSA GORDON, PhD3
1

Department of Physical Therapy, University of Alberta, Edmonton, Alberta, T6G 2E1, Canada 2 Faculty of Medicine, University of Alberta, Edmonton, Alberta T6G 2E1, Canada 3 Department of Pharmacology, University of Alberta, Edmonton, Alberta T6G 2E1, Canada Accepted 22 July 1997

Duchenne muscular dystrophy is an X-linked, recessive condition in which the gene for a cytoskeletal protein, dystrophin, is mutated. Although dystrophin is thought to help maintain the integrity of muscle cells, the mechanism by which its absence leads to the symptoms of the disease is not yet known.7 The lack of a good animal model for Duchenne muscular dystrophy has been a major impediment to understanding the disease process. Because its gene deletion and high serum creatine kinase levels mimic the inherited human disease, the mdx mouse promised to be a good animal model.3,9,18 However, the mechanical properties and morphology of skeletal muscles were found to be essentially normal in young and adult mdx mice, with the exception of the diaphragm which showed dramatic wasting and decline in force.5,19 Despite some cardiomyopathy and diaphragmatic weakness,
*Correspondence to: Dr. Jacques Bobet Contract grant sponsors: The Muscular Dystrophy Association of Canada; the Faculty of Medicine of the University of Alberta; and the Department of Physical Therapy of the University of Alberta. CCC 0148-639X/98/040536-04 1998 John Wiley & Sons, Inc.

the mouses lifespan was normal. Although muscle degeneration occurred, the high rate of replacement of muscle fibers suggested that the mdx mouse was a better model of muscle regeneration than of Duchenne muscular dystrophy.1 More recent data indicate that the muscle pathology of the mdx mouse is age dependent, with the muscles of old (2024 months) mdx mice exhibiting many of the pathological changes seen in Duchenne muscular dystrophy, as well as progressive loss of motor function.11,14,15 The question remains as to whether the skeletal muscles of old mdx mice also exhibit the muscle wasting and weakness which are characteristic of the human disease. This question was addressed in this study by examining muscle force and stiffness in limb muscles of old mdx mice.
METHODS

Soleus and extensor digitorum longus (EDL) muscles were dissected from 17 female mdx and 17 control (C57BL/ScSn) mice (Jackson Laboratories, Bar Harbor, ME) at 2021 months of age under sodium pentobarbital anesthesia (35 mg/kg IP). They were placed in a 20C bath of KrebsHenseleit solution bubbled with 95% O2/5% CO2. The distal ten-

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FIGURE 1. (a) Experimental protocol. Stimulation and imposed length for twitches, isometric tetanus, eccentric contraction, small passive stretch, and large passive stretch. (b) Absolute force of old mdx and old control EDL and soleus during twitches, isometric tetani, and eccentric contractions. The mdx muscle was not significantly weaker than control muscle under any condition. (c) Contractility (force per unit muscle weight) of old mdx and old control EDL and soleus during twitches, isometric tetani, and eccentric contractions. The mdx EDL was significantly weaker (P < .05; asterisks) than control EDL under all conditions. The mdx soleus was weaker than control only for isometric tetanus. (d) Stiffness of unstimulated old mdx and old control EDL and soleus during short stretches near rest length and large stretches to full extension. Old mdx mouse muscles were not significantly stiffer than controls for any condition.

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don was secured to the silicone lining of the bath. The proximal tendon was attached by 5-0 silk thread to a servomotor (Model 300B, Cambridge Technology, Watertown, MA) for length control and force measurements. Twitch and tetanic forces were elicited by supramaximal, bipolar stimulus pulses delivered to the muscle nerve by silver electrodes led from a Grass stimulator. The experimental protocol was: (1) stimulation with a single pulse at optimum length (twitch); (2) stimulation with a train of pulses (120 Hz for EDL, 80 Hz for soleus), at optimum length (tetanus); (3) a small (1 mm) muscle stretch, without stimulation (small passive stretch); (4) a second stretch. This second stretch was either: a 1-mm stretch imposed upon the plateau of a tetanic contraction (eccentric contraction) (10 muscles); or a 2.5-mm stretch imposed on an unstimulated muscle (large passive stretch) (7 muscles). These are illustrated schematically in Figure 1a. All stretches started at optimum length, and were done at 10 cm/s. All trials were done in the order indicated. For each trial, the maximum force in the trial was the measured variable. Differences between muscles in mdx and control mice were analyzed using an unpaired (one-tailed) t-test (P < 0.05), to test whether mdx muscles were stiffer or weaker than controls. Stiffness was calculated as the change in force during a stretch divided by the change in length. In several muscles, the silk tie was pulled off the tendon by the second stretch; if this happened, that measurement was ignored and the experiment was terminated.
RESULTS Muscle Force. The mdx mouse muscles were as strong as controls, with no significant differences between control and mdx muscles on any of the absolute force measures (Fig. 1b). The muscle forces in these older mice were lower than those reported in younger mice,1,4,6,10,13,14,16 due to their age2 and the low bath temperature.17 Some differences between muscles from mdx and control mice became evident when force was divided by muscle weight to give a measure of contractility (Fig. 1c). In the EDL, all these normalized forces were significantly less in mdx mice. In the soleus, only normalized tetanic force was significantly less. Muscle Stiffness. The mdx muscle was not stiffer than control, for either EDL or soleus muscle (Fig. 1d). Neither passive short-stretch nor passive largestretch stiffness differed from controls. Clearly, the dystrophic muscles were not stiffer in the normal

operating range, nor at the extremes of the range which produce the greatest passive stiffness of which the muscle is capable.
DISCUSSION

This study of contractile forces of skeletal muscles in old mice shows that the lack of dystrophin does not lead to increased muscle weakness in old age. Age brings weakness, but this weakness is similar in mdx and controls. Unlike strength, contractility of old mdx muscle was less. The mdx muscles produce the same force as controls, but require more bulk to do so. This is consistent with results in younger mice.1,6,10,13 This may mean that individual fibers are weaker, or that mdx muscle contains more noncontractile tissue (connective tissue or nectrotic fibers). Old mdx muscles were no stiffer than controls, despite reported increases in connective tissue in mdx mice.7,12,14 This agrees with reports that increased connective tissue does not produce more stiffness in old control mice.2 It is also possible that our mice had less connective tissue than mice in these other studies, being from a different breeding source. Our findings, therefore, indicate that the mechanical properties of limb muscles of old mdx mice are not a good model for Duchenne muscular dystrophy. Apparently, muscle regeneration in young animals is remarkably good at maintaining normal function in mdx mice for the duration of their lives.
We thank Dr. B.H. Bressler for providing animals for pilot studies.

REFERENCES 1. Anderson JE, Bressler BH, Ovalle WK: Functional regeneration in the hindlimb skeletal muscle of the mdx mouse. J Musc Res Cell Motil 1988;9:499515. 2. Brooks SV, Faulkner JA: Contractile properties of skeletal muscles from young, adult, and aged mice. J Physiol 1988;404: 7182. 3. Bulfield G, Siller WG, Wight PAL, Moore KJ: X chromosomelinked muscular dystrophy (mdx) in the mouse. Proc Natl Acad Sci USA 1984;81:11891192. 4. Coulton GR, Curtin NA, Morgan JE, Partridge TA: The mdx mouse skeletal muscle myopathy: II. Contractile properties. Neuropathol Appl Neurobiol 1988;14:299314. 5. Dangain J, Vrbova G: Muscle development in mdx mutant mice. Muscle Nerve 1984;7:700704. 6. Dupont-Versteegden EE, McCarter RJ: Differential expression of muscular dystrophy in diaphragm versus hindlimb muscles of mdx mice. Muscle Nerve 1992;15:11051110. 7. Emery AEH: Some unanswered questions in Duchenne muscular dystrophy. Neuromusc Disord 1994;4:301303. 8. Goldspink GK, Fernandes PE, Williams PE, Wells DJ: Agerelated changes in collagen gene expression in the muscles of mdx dystrophic and normal mice. Neuromusc Disord 1994;4: 183191. 9. Hoffman EP, Brown RH, Kunkel LM: Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell 1987; 51:919928.

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10. Kometani K, Tsugeno H, Yamada K: Mechanical and energetic properties of dystrophic (mdx) mouse muscle. Jpn J Physiol 1990;40:541549. 11. Lefaucheur JP, Pastoret C, Sebille A: Phenotype of dystrophinopathy in old MDX mice. Anat Record 1995;242:7076. 12. Marshall PA, Williams PE, Goldspink G: Accumulation of collagen and altered fiber-type ratios as indicators of abnormal muscle gene expression in the mdx dystrophic mouse. Muscle Nerve 1989;12:528537. 13. McArdle A, Edwards RHT, Jackson MJ: Effects of contractile activity on muscle damage in the dystrophin-deficient mdx mouse. Clin Sci 1991;80:367371. 14. Pastoret C, Sebille A: Further aspects of muscular dystrophy in mdx mice. Neuromusc Disord 1993;3:471475.

15. Pastoret C, Sebille A: mdx mice show progressive weakness and muscle deterioration with age. J Neurol Sci 1995;129: 97105. 16. Quinlan JG, Johnson SR, McKee MK, Lyden SP: Twitch and tetanus in mdx mouse muscle. Muscle Nerve 1992;15:837842. 17. Ranatunga KW: Influence of temperature on isometric tension development in mouse fast- and slow-twitch skeletal muscles. Exp Neurol 1980;70:211218. 18. Sicinski P, Geng Y, Ryder-Cook AS, Barnard EA, Darlison MG, Barnard PJ: The molecular basis of muscular dystrophy in the mdx mouse: a point mutation. Science 1989;244:15781580. 19. Stedman HH, Sweeney HL, Shrager JB, et al.: The mdx mouse diaphragm reproduces the degenerative changes of Duchenne muscular dystrophy. Nature 1991;352:536539.

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