Clinical Science (1992) 82,227-236 (Printed in Great Britain)
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Contractile properties and susceptibility to exerciseinduced damage of normal and rndx mouse tibialis anterior muscle P. SACCO; D. A. JONES,J. R. T. DICK* and G. VRBOVA* Department of Medicine, University College London, The Rayne Institute, London, U.K., and "Deportment of Anatomy and Developmental Biology, University College London, London, U.K. (Received 25 Junell2 September 199 I; accepted 20 September 199 I)
1. The functional properties of tibialis anterior muscles of normal adult (C57BL/10) and age-matched dystrophin-deficient (C57BL/10 mdx) mice have been investigated in situ. Comparisons were made between tibialis anterior muscle strength, rates of force development and relaxation, force-frequency responses and fatiguability. Subjecting rndx and C57 muscles to a regimen of eccentric exercise allowed the hypothesis to be tested that dystrophin-deficient muscles are more susceptible to exercise-induced muscle damage. 2. mdx muscles were, on average, 30% stronger than C57 muscles and almost 80% heavier, but both had similar muscle lengths. Thus, although mdx muscles were stronger in absolute terms, their estimated force per unit cross-sectional area was significantly less than that of C57 muscles. 3. The force-frequency relationships of C57 and rndr muscles differed in that whilst, at 40 Hz, the former developed 70% of the force developed at 1 0 0 Hz, the latter developed only 55%of the maximal force. Twitch force was normal in mdx muscles, but contraction time was shortened, and the consequent reduction in fusion frequency probably explains the force-frequency differences observed between the two groups. 4. mdx muscles were less fatiguable than normal muscles when stimulated repeatedly at a frequency of 40 Hz. It is possible that the lower relative force at 40 Hz in rndr muscles entailed a lower energy demand and thus a slower rate of fatigue than seen in normal muscles. 5. Eccentrically exercised C57 muscles showed a large loss of maximal force for up to 12 days after exercise. Maximal force loss occurred 3 days after exercise (55%of non-exercised tibialis anterior muscle), which also corresponded with the period of greatest fibre necrosis. C57 muscles showed a significantly reduced 40 Hz/100 Hz force-frequency ratio at 1and 3 days after exercise. This was primarily due to a reduced twitch amplitude rather than to a change in the time course of the twitch. It is unlikely, therefore, that the altered contractile charac-
teristics of mdx muscle were a result of the presence of damaged but otherwise normal fibres. 6. C57 and mdx tibialis anterior muscles displayed similar degrees of force loss after exercise. Furthermore, the rate of recovery after the nadir of force loss was very similar for the two groups. By 12 days after exercise, force recovered to 76% and 80%of control in C57 and mdu muscles, respectively. Our findings do not support the hypothesis that dystrophin-deficient muscle is more susceptible to exercise-induced muscle damage.
INTRODUCTION Identification of the protein dystrophin as the missing or defective gene product in Duchenne and Becker muscular dystrophies has been a major advance in understanding the pathogenesis of these disorders [ 11.Although dystrophin is now known to be associated with the sarcolemma1 membrane, its precise role in the muscle fibre and the reason why its absence leads to dystrophic changes remain to be elucidated. The discovery that dystrophin is absent in the mdx strain of mice and that the gene locus affected is homologous with that responsible for the human Duchenne and Becker muscular dystrophies [2] makes the mdx strain a valuable model for investigating the functional consequences of dystrophin deficiency. The ma'x mutant was originally described by Bulfield et af. [3] in a colony of C57BL/10 mice. These animals have chronically raised serum levels of muscle-specific creatine kinase and exhibit histological changes characteristic of a mild form of muscular dystrophy. Dangain & Vrbov5 [4] reported that muscles from mdx mice undergo an acute and massive fibre necrosis at approximately 3 weeks of age with a subsequent regeneration and recovery by 5 weeks, although degeneration and regeneration, on a more limited scale, continue throughout adult life [4-61. It is possible that the lack of dystrophin may be reflected in altered contractile properties [7] and fatiguability of
Key words: contractile properties, dystrophin, eccentric exercise, rndx mice, muscle damage. Abbreviations: MRR, maximum rate of relaxation: MRTD, maximum rate of tension development; TA, tibialis anterior; TP, time to peak tension. Correspondence: D r P. Sacco, Department of Medicine, The Rayne Institute, University Street, London WCI E 611, U.K.
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dystrophic muscle or in the susceptibility to damaging exercise and the ability to recover. We have compared the contractile properties and fatigue characteristics of mdx tibialis anterior (TA) muscle with those of normal mice. However, a difficulty in interpreting these results is the fact that dystrophic muscles contain populations of both necrotic and regenerating fibres, and it is possible that any abnormalities may be a reflection of the presence of these fibres. In the second part of the work presented here we have investigated th'e contractile properties of damaged and regenerating normal mouse muscle and compared these values with those of mdx muscle. Exercise in which active muscles are forcibly stretched (negative work or eccentric contractions) can lead to considerable loss of force, fibre degeneration and subsequent regeneration [8-101 even in normal muscles, and it has been suggested that dystrophic muscles may be more susceptible to the damaging effects of eccentric exercise [ 111. The recent speculation that dystrophin is a structural protein, which may serve to stabilize the fibre membrane against the damaging effects of mechanical stresses associated with muscle activity [ 121, is consistent with this hypothesis. In the third part of the work we have tested the hypothesis that the absence of dystrophin causes mdx muscle to be more vulnerable to stretch-induced muscle damage.
MATERIALS AND METHODS Experimental animals Myopathic mice ( m d x )were maintained as a breeding colony from stock donated by Dr G. Bulfield, Department of Genetics, University of Edinburgh. Normal animals were obtained from a colony of C57BL/10 mice, this being the strain in which the mdx mutant arose. Experiments were performed on female animals aged 16-26 weeks (mdx, mean=21.4 weeks) and 16-24 weeks (normal C57, mean= 21.0 weeks).
Force recordings All investigations were carried out using the TA muscle. Animals were anaesthetized by an intraperitoneal injection of chloral hydrate [4.5% (w/v) solution, 0.01 ml/g body weight]. The distal tendon of the TA was cut and the proximal end was tied to a Statham force transducer with fine silk suture. The sciatic nerve was isolated and cut, and the leg was secured by metal pins through the knee and ankle joints. A bipolar platinum stimulating electrode was positioned in contact with the peripheral stump of the sciatic nerve. Muscles were stimulated via the nerve with square wave pulses of 100 ,us duration and the voltage was adjusted so as to give supramaximal stimulation (5-10 V). An initial tetanic stimulation at 100 Hz for 0.5 s was used to remove any slack which might be present in the tendon attachments. The muscle length was then adjusted to give maximum twitch tension without a significant increase in
resting tension. Data were recorded (a) on a storage oscilloscope screen, (b) on a Devices chart recorder and (c) after digitization on a computer database for subsequent analysis. The latter was used to measure the time to peak tension (TP), maximum rate of tension development (MRTD) and maximum rate of relaxation (MRR). Since the MRTD and MRR are related to the absolute force of the contraction, they were normalized for peak force to obtain a value of the percentage change in peak force/ms.
Stimulation protocol A force-frequency relationship was determined for each muscle by stimulating for half a second at 1,40, 60, 80 and 100 Hz. At least 30 s recovery time was allowed between tetani. After 5 min recovery, muscles were subjected to a fatigue protocol consisting of supramaximal stimulation at 40 Hz for 250 ms every second for 3 min. The tetanic force after 3 min of fatigue was expressed as a percentage of the maximum tetanic force; this was usually the initial contraction, but occasionally there was some potentiation during the first 2-5 s, in which case the greatest value was used.
Exercise protocol Eccentric contractions were elicited in the dorsiflexor muscles of the right foot [13], the contralateral muscles serving as non-exercised controls. Mice were anaesthetized with an intraperitoneal injection of 4.5% (w/v) chloral hydrate (0.01 ml/g body weight) and the right knee was clamped firmly to restrict movement to the lower limb. The peroneal nerve was exposed just above the knee and was placed on a small hook electrode. The foot was taped to a perspex holder, which rotated around a pivot at the ankle. The foot holder was attached to the rim of a motor-driven wheel, the rotation of which caused the foot to travel from full flexion to full extension (approximately 100")and back in 600 ms. The TA was stimulated for 300 ms at 100 Hz using 100 ,us square wave pulses of supramaximal voltage beginning just before and continuing during the lengthening phase. The shortening phase was entirely passive. Lengthening contractions were repeated every 5 s for 240 repetitions, after which the incision was closed and the animals were allowed to recover. One, three, six or twelve days after exercise the animals were again anaesthetized, and the strength, contractile properties and fatiguability of exercised and contralateral muscles were determined as described above.
Post-mortem examination After the fatigue run, the mice were killed by a lethal dose of chloral hydrate. TA length was measured in situ in a number of mice. All muscles were carefully excised, weighed and a portion from the belly of the muscle was orientated to give transverse sections before being frozen in isopentane cooled with liquid nitrogen.
Contractile properties and susceptibility to damage of mdx mouse muscle
Histology and histochemistry Cryostat sections were cut at 8 p m and were stained with haematoxylin and eosin and for NADH-tetrazolium reductase.
Morphometry Fibre areas were measured by using semi-quantitative image analysis [14] on transverse sections stained with haematoxylin and eosin. One-hundred fibres were measured from each TA muscle in 10 adjacent fields covering the lateral portion of the muscle. To provide a quantitative estimate of damage the number of 'damage foci'/field were counted under x 400 magnification for every second field across the section (15 fields counted/section). Damage foci were defined as fibres which were infiltrated by mononuclear cells.
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where the TA muscle represented a significantly smaller portion of body weight (1.5%). Muscles from C57 mice were normal in appearance, fibres with internal nuclei being very rare (Fig. la). Examination of rndx muscles showed a wide variation in muscle fibre size, foci of fibre necrosis and infiltration with mononuclear cells (Fig. l b ) . A notable feature of mdx muscles was that the majority of fibres had one or more internally placed nuclei1 (Fig. l b ) . Staining for NADH-tetrazolium reductase showed similar proportions of oxidative and non-oxidative fibres in normal and rndx muscles. Muscle fibre areas of normal and rndx TA muscles are shown in Fig. 2 and Table 1. Mean fibre areas were very similar for normal and mdx muscles, but the latter showed a greater range of fibre areas, illustrated by the larger SD (Table 1).Thus 17% of the mdx fibres had an area less than 2000 pm2 compared with 10% of normal muscles, whereas 9% of the mdx fibres had an area greater than 6000 pm2 compared with 1% of the normal fibres.
Statistics Values for grouped mean data were compared by using Student's t-test; where not specifically stated, significance was set at the 5% level. Results are expressed as means f SEM.
RESULTS Muscle size and histology Details of animal and muscle weights are given in Table 1. rndx mice were, on average, 20% heavier than normal C57 mice, whereas the individual TA muscles from rndx mice were 80% heavier than those from normal animals. Both types of muscles were the same length, so that the calculated cross-sectional areas of mdx muscles were greater than those of the normal C57 muscles. The mdx TA muscle weights were approximately 2.3% of total body weight, in contrast to the normal group,
Table I. Physical properties of mdx and normal TA muscles. Values are given as meanstsm except for fibre areas, which are given as means tso. Abbreviation: CSA, cross-sectional area. Statistical significance: * P
227
Contractile properties and susceptibility to exerciseinduced damage of normal and rndx mouse tibialis anterior muscle P. SACCO; D. A. JONES,J. R. T. DICK* and G. VRBOVA* Department of Medicine, University College London, The Rayne Institute, London, U.K., and "Deportment of Anatomy and Developmental Biology, University College London, London, U.K. (Received 25 Junell2 September 199 I; accepted 20 September 199 I)
1. The functional properties of tibialis anterior muscles of normal adult (C57BL/10) and age-matched dystrophin-deficient (C57BL/10 mdx) mice have been investigated in situ. Comparisons were made between tibialis anterior muscle strength, rates of force development and relaxation, force-frequency responses and fatiguability. Subjecting rndx and C57 muscles to a regimen of eccentric exercise allowed the hypothesis to be tested that dystrophin-deficient muscles are more susceptible to exercise-induced muscle damage. 2. mdx muscles were, on average, 30% stronger than C57 muscles and almost 80% heavier, but both had similar muscle lengths. Thus, although mdx muscles were stronger in absolute terms, their estimated force per unit cross-sectional area was significantly less than that of C57 muscles. 3. The force-frequency relationships of C57 and rndr muscles differed in that whilst, at 40 Hz, the former developed 70% of the force developed at 1 0 0 Hz, the latter developed only 55%of the maximal force. Twitch force was normal in mdx muscles, but contraction time was shortened, and the consequent reduction in fusion frequency probably explains the force-frequency differences observed between the two groups. 4. mdx muscles were less fatiguable than normal muscles when stimulated repeatedly at a frequency of 40 Hz. It is possible that the lower relative force at 40 Hz in rndr muscles entailed a lower energy demand and thus a slower rate of fatigue than seen in normal muscles. 5. Eccentrically exercised C57 muscles showed a large loss of maximal force for up to 12 days after exercise. Maximal force loss occurred 3 days after exercise (55%of non-exercised tibialis anterior muscle), which also corresponded with the period of greatest fibre necrosis. C57 muscles showed a significantly reduced 40 Hz/100 Hz force-frequency ratio at 1and 3 days after exercise. This was primarily due to a reduced twitch amplitude rather than to a change in the time course of the twitch. It is unlikely, therefore, that the altered contractile charac-
teristics of mdx muscle were a result of the presence of damaged but otherwise normal fibres. 6. C57 and mdx tibialis anterior muscles displayed similar degrees of force loss after exercise. Furthermore, the rate of recovery after the nadir of force loss was very similar for the two groups. By 12 days after exercise, force recovered to 76% and 80%of control in C57 and mdu muscles, respectively. Our findings do not support the hypothesis that dystrophin-deficient muscle is more susceptible to exercise-induced muscle damage.
INTRODUCTION Identification of the protein dystrophin as the missing or defective gene product in Duchenne and Becker muscular dystrophies has been a major advance in understanding the pathogenesis of these disorders [ 11.Although dystrophin is now known to be associated with the sarcolemma1 membrane, its precise role in the muscle fibre and the reason why its absence leads to dystrophic changes remain to be elucidated. The discovery that dystrophin is absent in the mdx strain of mice and that the gene locus affected is homologous with that responsible for the human Duchenne and Becker muscular dystrophies [2] makes the mdx strain a valuable model for investigating the functional consequences of dystrophin deficiency. The ma'x mutant was originally described by Bulfield et af. [3] in a colony of C57BL/10 mice. These animals have chronically raised serum levels of muscle-specific creatine kinase and exhibit histological changes characteristic of a mild form of muscular dystrophy. Dangain & Vrbov5 [4] reported that muscles from mdx mice undergo an acute and massive fibre necrosis at approximately 3 weeks of age with a subsequent regeneration and recovery by 5 weeks, although degeneration and regeneration, on a more limited scale, continue throughout adult life [4-61. It is possible that the lack of dystrophin may be reflected in altered contractile properties [7] and fatiguability of
Key words: contractile properties, dystrophin, eccentric exercise, rndx mice, muscle damage. Abbreviations: MRR, maximum rate of relaxation: MRTD, maximum rate of tension development; TA, tibialis anterior; TP, time to peak tension. Correspondence: D r P. Sacco, Department of Medicine, The Rayne Institute, University Street, London WCI E 611, U.K.
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dystrophic muscle or in the susceptibility to damaging exercise and the ability to recover. We have compared the contractile properties and fatigue characteristics of mdx tibialis anterior (TA) muscle with those of normal mice. However, a difficulty in interpreting these results is the fact that dystrophic muscles contain populations of both necrotic and regenerating fibres, and it is possible that any abnormalities may be a reflection of the presence of these fibres. In the second part of the work presented here we have investigated th'e contractile properties of damaged and regenerating normal mouse muscle and compared these values with those of mdx muscle. Exercise in which active muscles are forcibly stretched (negative work or eccentric contractions) can lead to considerable loss of force, fibre degeneration and subsequent regeneration [8-101 even in normal muscles, and it has been suggested that dystrophic muscles may be more susceptible to the damaging effects of eccentric exercise [ 111. The recent speculation that dystrophin is a structural protein, which may serve to stabilize the fibre membrane against the damaging effects of mechanical stresses associated with muscle activity [ 121, is consistent with this hypothesis. In the third part of the work we have tested the hypothesis that the absence of dystrophin causes mdx muscle to be more vulnerable to stretch-induced muscle damage.
MATERIALS AND METHODS Experimental animals Myopathic mice ( m d x )were maintained as a breeding colony from stock donated by Dr G. Bulfield, Department of Genetics, University of Edinburgh. Normal animals were obtained from a colony of C57BL/10 mice, this being the strain in which the mdx mutant arose. Experiments were performed on female animals aged 16-26 weeks (mdx, mean=21.4 weeks) and 16-24 weeks (normal C57, mean= 21.0 weeks).
Force recordings All investigations were carried out using the TA muscle. Animals were anaesthetized by an intraperitoneal injection of chloral hydrate [4.5% (w/v) solution, 0.01 ml/g body weight]. The distal tendon of the TA was cut and the proximal end was tied to a Statham force transducer with fine silk suture. The sciatic nerve was isolated and cut, and the leg was secured by metal pins through the knee and ankle joints. A bipolar platinum stimulating electrode was positioned in contact with the peripheral stump of the sciatic nerve. Muscles were stimulated via the nerve with square wave pulses of 100 ,us duration and the voltage was adjusted so as to give supramaximal stimulation (5-10 V). An initial tetanic stimulation at 100 Hz for 0.5 s was used to remove any slack which might be present in the tendon attachments. The muscle length was then adjusted to give maximum twitch tension without a significant increase in
resting tension. Data were recorded (a) on a storage oscilloscope screen, (b) on a Devices chart recorder and (c) after digitization on a computer database for subsequent analysis. The latter was used to measure the time to peak tension (TP), maximum rate of tension development (MRTD) and maximum rate of relaxation (MRR). Since the MRTD and MRR are related to the absolute force of the contraction, they were normalized for peak force to obtain a value of the percentage change in peak force/ms.
Stimulation protocol A force-frequency relationship was determined for each muscle by stimulating for half a second at 1,40, 60, 80 and 100 Hz. At least 30 s recovery time was allowed between tetani. After 5 min recovery, muscles were subjected to a fatigue protocol consisting of supramaximal stimulation at 40 Hz for 250 ms every second for 3 min. The tetanic force after 3 min of fatigue was expressed as a percentage of the maximum tetanic force; this was usually the initial contraction, but occasionally there was some potentiation during the first 2-5 s, in which case the greatest value was used.
Exercise protocol Eccentric contractions were elicited in the dorsiflexor muscles of the right foot [13], the contralateral muscles serving as non-exercised controls. Mice were anaesthetized with an intraperitoneal injection of 4.5% (w/v) chloral hydrate (0.01 ml/g body weight) and the right knee was clamped firmly to restrict movement to the lower limb. The peroneal nerve was exposed just above the knee and was placed on a small hook electrode. The foot was taped to a perspex holder, which rotated around a pivot at the ankle. The foot holder was attached to the rim of a motor-driven wheel, the rotation of which caused the foot to travel from full flexion to full extension (approximately 100")and back in 600 ms. The TA was stimulated for 300 ms at 100 Hz using 100 ,us square wave pulses of supramaximal voltage beginning just before and continuing during the lengthening phase. The shortening phase was entirely passive. Lengthening contractions were repeated every 5 s for 240 repetitions, after which the incision was closed and the animals were allowed to recover. One, three, six or twelve days after exercise the animals were again anaesthetized, and the strength, contractile properties and fatiguability of exercised and contralateral muscles were determined as described above.
Post-mortem examination After the fatigue run, the mice were killed by a lethal dose of chloral hydrate. TA length was measured in situ in a number of mice. All muscles were carefully excised, weighed and a portion from the belly of the muscle was orientated to give transverse sections before being frozen in isopentane cooled with liquid nitrogen.
Contractile properties and susceptibility to damage of mdx mouse muscle
Histology and histochemistry Cryostat sections were cut at 8 p m and were stained with haematoxylin and eosin and for NADH-tetrazolium reductase.
Morphometry Fibre areas were measured by using semi-quantitative image analysis [14] on transverse sections stained with haematoxylin and eosin. One-hundred fibres were measured from each TA muscle in 10 adjacent fields covering the lateral portion of the muscle. To provide a quantitative estimate of damage the number of 'damage foci'/field were counted under x 400 magnification for every second field across the section (15 fields counted/section). Damage foci were defined as fibres which were infiltrated by mononuclear cells.
229
where the TA muscle represented a significantly smaller portion of body weight (1.5%). Muscles from C57 mice were normal in appearance, fibres with internal nuclei being very rare (Fig. la). Examination of rndx muscles showed a wide variation in muscle fibre size, foci of fibre necrosis and infiltration with mononuclear cells (Fig. l b ) . A notable feature of mdx muscles was that the majority of fibres had one or more internally placed nuclei1 (Fig. l b ) . Staining for NADH-tetrazolium reductase showed similar proportions of oxidative and non-oxidative fibres in normal and rndx muscles. Muscle fibre areas of normal and rndx TA muscles are shown in Fig. 2 and Table 1. Mean fibre areas were very similar for normal and mdx muscles, but the latter showed a greater range of fibre areas, illustrated by the larger SD (Table 1).Thus 17% of the mdx fibres had an area less than 2000 pm2 compared with 10% of normal muscles, whereas 9% of the mdx fibres had an area greater than 6000 pm2 compared with 1% of the normal fibres.
Statistics Values for grouped mean data were compared by using Student's t-test; where not specifically stated, significance was set at the 5% level. Results are expressed as means f SEM.
RESULTS Muscle size and histology Details of animal and muscle weights are given in Table 1. rndx mice were, on average, 20% heavier than normal C57 mice, whereas the individual TA muscles from rndx mice were 80% heavier than those from normal animals. Both types of muscles were the same length, so that the calculated cross-sectional areas of mdx muscles were greater than those of the normal C57 muscles. The mdx TA muscle weights were approximately 2.3% of total body weight, in contrast to the normal group,
Table I. Physical properties of mdx and normal TA muscles. Values are given as meanstsm except for fibre areas, which are given as means tso. Abbreviation: CSA, cross-sectional area. Statistical significance: * P
