Age does not influence muscle fiber length adaptation to increased excursion
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《应用生理学杂志》
Department of Health and Performance Sciences, Georgia Institute of Technology, Atlanta, Georgia 30332-0356
ABSTRACT
Muscle fiber length adaptation to static stretch or shortening depends on age, with sarcomere addition in young muscle being dependent on mobility. Series sarcomere number can also increase in young animals in response to increased muscle excursion, but it is not clear whether adult muscles respond similarly. The ankle flexor retinaculum was transected in neonatal and adult rats to increase tibialis anterior muscle excursion. Sarcomere number in tibialis anterior was determined after 8 wk of adaptation. Muscle moment arm and excursion were increased 30% (P < 0.01) in both age groups. Muscle cross-sectional area was reduced by 12% (P < 0.01) in response to the increased mechanical advantage, and this reduction was unaffected by age. Fiber length change was also unaffected by age, with both groups showing a trend (P < 0.10) for slightly (6%) increased fiber length. Retinaculum transection results in shorter muscle length in all joint configurations, so this trend opposes the fiber length decrease predicted by an adaptation to muscle length and indicates that fiber length is influenced by dynamic mechanical signals in addition to static length.
muscle architecture; retinaculum transection; mathematical modeling
INTRODUCTION
SERIES SARCOMERE NUMBER is an important determinant of skeletal muscle function (1, 20). Force generation at the sarcomere level depends on both length and velocity, and the number of sarcomeres in series determines the relationship between muscle length and velocity and sarcomere length and velocity. The variation in sarcomere number or fiber length contributes more strongly to both the static and dynamic performance of muscle than fiber type (1, 21).
Fiber length is a highly plastic characteristic of muscle, undergoing substantial and rapid changes in response to growth or immobilization (29). This is necessary to maintain optimal force generation in the physiological range of motion. In addition to these quasi-static stimuli, fiber length can adapt to alterations in muscle excursion (2, 13). This latter plasticity may contribute to the correlation between muscle moment arm, which determines muscle excursion, and muscle fiber length (17). It may also contribute to the coordination of moment arm and fiber length for generation of maximal power during locomotion (14).
Previous studies have suggested a sharp contrast in the adaptation of developing and mature muscle to changes in length or excursion. Adult muscles respond to chronic stretch immobilization by rapidly increasing fiber length and to shortened immobilization by rapidly decreasing fiber length (12, 22, 30). Immobilization at a very young age results in reduced fiber length growth in mouse soleus muscle, independent of whether the muscle is stretched or shortened (30). The fiber length reduction can be very nearly relieved by mobilization of the muscle, suggesting that the reduced mobility is the source of sarcomere number reduction. This fiber length reduction is maintained for at least 6 wk in mice, well past the age at which the mature pattern of adaptation would be expected (29). This suggests that the restriction of mobility may establish a completely different pattern of length adaptation that is relieved only on mobilization of the muscle.
The response of muscle to increased excursion has also been suggested to be age dependent (2). When the tibialis anterior (TA) muscles of young rabbits are permitted increased excursion by transection of the ankle flexor retinaculum, the muscles respond by increasing fiber length (13). Adult mouse TA muscles respond to the increased excursion and decreased length of retinaculum transection by rapidly reducing sarcomere number (2). It is difficult to compare these results directly because of species, age, and experimental differences.
These observations suggest that skeletal muscle undergoes a fundamental shift in the mode of length adaptation during early growth. This is a dynamic time in the development of muscle, coinciding with the development of locomotion, changes in nutrition, and establishment of mature molecular phenotype (7, 16, 26). In the mouse, this period also coincides with an enormous increase in soleus sarcomere number, more than doubling within 3 wk, and motion appears to be required for this increase (28). Mature muscle fiber length appears to be strongly influenced by static length signals (2, 30).
Excursion appears to exert a subtle influence on sarcomere number compared with static length. Whereas a static stretch of 30% is capable of generating an increase in fiber length of more than 15% (30), a 40% increase in muscle excursion resulted in only 10% increase in sarcomere number (13), so the gain of the increased excursion appears smaller. It is possible that the excursion signal requires more time to be expressed. Burkholder and Lieber (2) reported that sarcomere number decreased in mice with increased excursion after 2 wk, the same period over which Williams and Goldspink (30) report large increases in stretch immobilized muscle. In contrast, the studies that have reported increased muscle or fiber length after retinaculum transection in growing rabbits have not investigated periods shorter than 3 mo (6, 13). Interpretation of the few retinaculum transection studies is complicated by the variety of species, durations, and treatments.
The purpose of the present study was to determine whether the differences in adaptation to increased excursion can be attributed to the age of the animal at the time of manipulation or to other factors, including species and experimental duration.
METHODS
Adult (~9-10 wk old, 200-225 g) and juvenile (10 days old, ~14 g) Wistar rats (Harlan, Indianapolis, IN) were randomly divided into control and transection groups. The adult age was chosen because these animals are skeletally and reproductively mature and have a well-established locomotor pattern. The young age was chosen to be before the age at which the change in adaptation mode has been reported, yet with animals large enough to facilitate the surgical procedure. These animals are substantially younger and less mobile that the rabbits used by Koh and Herzog (13) or Crawford (6). The rat species was chosen to facilitate surgery in the young animals yet be readily comparable to the existing body of sarcomere number regulation literature, the most substantial portion of which is based on mice.
Rats undergoing retinaculum transection were sedated with an intraperitoneal cocktail of Rompun (10 mg/kg), ketamine (90 mg/kg), and acepromazine (1.5 mg/kg). With the aid of a dissecting microscope, a small (2-mm) incision was made on the lateral surface of the foot, exposing the flexor retinaculum (crural ligament). The ligament was transected with fine scissors, and the TA tendon was elevated from its normal path, resulting in a more direct line to the insertion site. The tendon path was visibly altered throughout the range of motion, but the slight deviation at full plantar flexion is unlikely to allow substantial shortening of the muscle in this position. The wound was then closed with cutaneous glue (Nexaband, Phoenix, AZ). Transected rats were permitted normal cage activity immediately on recovery. The entire procedure was typically completed in 3-5 min. Animals behaved normally immediately on recovery, indicating the minimally traumatic nature of this intervention. Animals were visually monitored periodically throughout the duration of the experimental period and were behaviorally indistinguishable from controls. Cage activity and gait kinematics were not quantitated. Activity of cage housed animals is normally quite low and has previously been shown to be unaffected by this intervention (13). Gait kinematics are also minimally impacted by this intervention (2, 13) and would contribute little to the analysis. All procedures were approved by the institutional animal care and use committee and were performed in accordance with the Guide for the Care and Use of Laboratory Animals.
At the conclusion of the 8-wk experimental period, animals were killed by CO2 asphyxiation, and the TA was exposed. Absence of the flexor retinaculum and free mobility of the tendon was confirmed. The retinaculum in very young animals was prone to regrowth or fibrotic overgrowth, and only animals with clearly released TA tendons were included in the transection group. Passive muscle length was measured in situ with the ankle fully plantar flexed (~150°) and fully dorsiflexed (~25°) by use of a pair of dial calipers. Muscles were then harvested, pinned under slight tension, and fixed overnight in 4% paraformaldehyde in PBS. The entire distal TA tendon was carefully collected from some specimens, and it was length measured.
Muscles were rinsed in PBS and cleaned of extraneous tissue before weighing. Muscle length was measured with dial calipers under a dissecting microscope. Small fiber bundles (5-50) were teased from the lateral, medial, and deep regions of each muscle and mounted on glass slides. Fiber bundle length was measured with dial calipers, and sarcomere length was measured by laser diffraction in at least three regions of each fiber bundle. Series sarcomere number was calculated for each bundle by dividing fiber bundle length by the average of its sarcomere lengths. Fiber length for the whole muscle was then calculated by multiplying the average bundle sarcomere number by optimal sarcomere length (2.5 μm; Ref. 25). Thus there was one final measurement of fiber length for each muscle, which was a multiple of sarcomere number. Physiological cross-sectional area (PCSA) was calculated according to the formula
where 1.06 is the density of muscle (mg/mm3; Ref. 18) and S is sarcomere number. The term "0.0025 mm · S" is fiber length normalized to a sarcomere length of 2.5 μm and corrected for any variations in muscle length that occurred as a result of fixation at slightly different sarcomere lengths. The normalization to a sarcomere length of 2.5 μm corresponds to the optimal sarcomere length in rats (25).
Estimates of muscle force production were made by use of a mathematical model (1). This generalized model uses muscle architectural properties to estimate sarcomere length and velocity from muscle length and velocity. Force production was then estimated by using the sarcomere force-length and force-velocity relations. Muscle power was calculated by multiplying estimated isokinetic force by shortening velocity. Because of the hyperbolic decline in force with increasing velocity, peak power occurs at a shortening velocity of ~0.3 Vmax. Vmax was taken to be 10 l/s, and maximum isometric muscle stress was 250 kN/m2, with optimal length being 2.5 μm (5). Moment arm was estimated from muscle excursion, with full plantar flexion assumed to be 160° and full dorsiflexion to be 25°. Peak dorsiflexion velocity was estimated as 1,500°/s, on the basis of a 130-ms swing time (19) and sinusoidal motion profile.
Measurements were screened for normality and compared by two-way ANOVA with significance level set to 0.05. Between group differences were determined by unpaired t-test. Statistical trends were recognized at P < 0.10. Retrospective statistical power calculations were based on an expected difference equal to the minimum detectable difference. This was felt to be a conservative estimate, because the sample size permits resolution of ~0.5 standard deviations, a level that is almost certainly smaller than the biologically relevant threshold. No difference between groups was noted only when statistical power exceeded 90%. This distinction is necessary because several of the results of this work are similarities, rather than differences, and it is important to distinguish between "similar" and "not different." There are four possible statistical categories for a comparison: for P < 0.05, the comparison is significantly different; for P < 0.10, a trend; for power > 90%, statistically identical; for P > 0.10; and for power < 90%, statistically indeterminate. Results are reported as means ± SD unless otherwise noted. Statistical differences and trends are supported by the P value, i.e., the chance of a type I error or of incorrectly rejecting the null hypothesis of equality. Statistical identity is supported by statistical power, i.e., the probability of correctly accepting the null hypothesis of equality. Power is 1 , where is the probability of type II error or of incorrectly accepting the null hypothesis.
RESULTS
Surgical transection of the flexor retinaculum resulted in a 30% increase (Table 1; P < 0.01) in TA at the completion of the experiment that was identical between age groups (power > 94%). It is likely that this increase was greater in the first few days posttransection, because a pseudoretinaculum was generally present at termination. Muscles of both adult and juvenile animals responded to this increase with a trend (P < 0.1) toward increased fiber length (Table 1), which was not influenced by age (power > 93%). Age was a strong influence on overall fiber length, with muscles in younger animals displaying fibers that were 15% longer (Table 1; P < 0.01), without regard for surgical treatment. PCSA was reduced 12% by retinaculum transection (Table 1; P < 0.01), and this was not affected by age (power > 91%). Tendon lengths were measured in six adult control (14.1 ± 1.1 mm) and four adult transected animals (14.1 ± 0.6 mm) and were found to be identical (power >95%).
Sarcomere lengths (Table 1) were extrapolated from measurements in the fixed muscle by scaling to muscle lengths measured at the extremes of motion. Estimated sarcomere excursion was increased by 34% (P < 0.01) after retinaculum transection, independent of age (power > 92%). This increase was a mathematical consequence of the measured increase in muscle excursion, on which the extrapolation depends. The increased excursion was not associated with systematically altered maximum or minimum in vivo lengths, although a trend was found for maximum length to be slightly increased (P = 0.06).
The functional implications of these morphological changes were estimated by mathematical modeling. The functional estimates demonstrate the contrasting effects of the trend for fiber length increase and the decrease in PCSA. Neither statistical identity nor difference in the magnitude of predicted peak muscle power (Table 1) could be established. The joint velocity at which peak power would be achieved did decline by 24% (Table 1; P < 0.01), as a result of the great increase in moment arm. Had there been no adaptation, peak power would have remained constant, and the velocity of peak power would have declined by 30%. To restore the angular velocity of peak power production (1,300°/s), fiber length would have had to increase by 33%.
Despite the decline in PCSA, maximal isometric joint torque was predicted to increase by more than 15%, as a result of the increase in moment arm. This change is significant in adults (P < 0.01 ) and a trend in juveniles (P < 0.08). Using an estimated dorsiflexion velocity of 1,500°/s, torque capacity during locomotion was estimated to decline by 16%, indicating that the increase in moment arm may not be sufficient to compensate for the decrease in PCSA during locomotion.
DISCUSSION
The objective of this study was to determine whether juvenile and mature muscle respond differently to surgical increase in muscle excursion. The response of fiber length, PCSA and muscle excursion were nearly identical in the two age groups and argues strongly against age influencing the increase in fiber length induced by retinaculum transection.
Although age did not influence the experimental adaptation, there were clear morphological differences between the age groups. Most significant of those differences may be the longer fiber length and shorter sarcomere length of the younger muscles. This finding is somewhat surprising, given that the adult group is only 2 mo older than the juvenile group. Although sarcomere number and fiber length have been reported to be 5-10% lower in aged animals than adults (11, 15), it seems unlikely that age is the only factor involved in the 13% difference reported here. Myofilament lengths do not change with age (9, 10). The adult population was received directly from the vendor, where they were housed at higher density than the juvenile group, which was raised at the university vivarium. It is conceivable that the differences in the rearing environment or diet contributed to the difference in fiber length between age groups. Nutritional status has been shown strongly to influence fiber length (10, 27).
Joint kinematics were not measured in these animals, but previous work in both mice (2) and rabbits (13) suggests that dorsiflexion velocity and range of motion are only minimally affected by this procedure. A 22% decrease in dorsiflexion velocity would be required to restore near-optimal power generation, which is substantially greater than the (not significant) 7% decrease reported by Koh and Herzog (13) and the 14% decrease reported by Burkholder and Lieber (2). Optimal power generation during locomotion does not appear to have a strong influence on muscle architecture. This suggests that those systems in which muscles do operate near optimal power reflect tuning of the nervous system and selection of an optimized style of locomotion, rather than adaptation of the musculature. It is possible that the trend for increased fiber length combines with a decrease in dorsiflexion velocity and does restore peak power generation. This would also support the notion that the nervous system adjusts to meet muscular performance more effectively than muscle structure adjusts to meet functional demands.
The trend for fiber length increase after retinaculum transection parallels results seen in long-term studies in rabbits (6, 13). The transection of the retinaculum permits the TA to take a more direct path from the tibial origin to the metatarsal insertion, thus slightly reducing the muscle length in plantar flexion. Furthermore, the greater moment arm causes the released muscle to shorten more during motion so that the reduction in muscle length is even greater in dorsiflexion. If the muscle adapts solely to a static length or passive tension signal, then sarcomere number should be decreased by this preparation, as was seen in the previous short-term study (2) and many other studies (9, 23, 28), because length and passive tension are reduced by retinaculum transection. The finding that fiber length and sarcomere number tend to increase indicates that there is a nonstatic mechanical signal influencing fiber length. This in vivo model cannot distinguish among the likely signals of excursion, velocity, and power, but in vitro both excursion and velocity have been shown to influence smooth muscle growth (4).
The results of the mathematical model were intended to permit evaluation of the relative effects of the significant (12%) atrophy and the trend for fiber length increase (6%). In vivo function of the muscle depends on the PCSA, the velocity of shortening, the muscle moment arm, and neural recruitment. Ignoring the latter, retinaculum transection directly alters the muscle moment arm, and both PCSA and fiber length are found to change in response. If these changes are driven by functional demands, then some aspect of function should be similar in the control and transected groups. The mathematical model predicts an equivocal change in the magnitude of peak power production, a slight decrease in the angular velocity of peak power generation, and a substantial increase in the magnitude of isometric torque (Table 1). The velocity of peak power generation is quite close to the estimated speed of dorsiflexion (1,500°/s), although this may be a coincidence. Thus the model results suggest that adaptation of the muscle preserves peak power output.
The existing reports of sarcomere number adaptation to retinaculum transection suggest two aspects to the response. Immediately after transection, the muscle appears to respond primarily to the reduction in length or passive tension, reducing sarcomere number to restore optimal sarcomere length (2). This period may last as long as 4 wk in mice (unpublished observations). In parallel, a slower process appears to add sarcomeres to restore sarcomere excursion or velocity (present study; Refs. 6, 13). The latter process does not appear to be influenced by the age of the animal and appears to occur equally well in rats and rabbits. Interestingly, the classic Williams and Goldspink immobilization studies (28-30) fall within the initial time period of 4 wk. The age-dependent, bifurcate response of the mouse soleus to immobilization, in which immobilization of the young muscle inhibits normal sarcomere addition, remains intriguing. It is possible that the postnatal development of this, and possibly other, muscles is somehow dependent on mechanical input. Passive mechanical stimulation is known to initiate several signaling pathways in vitro (8, 24).
Sarcomere excursion was greatly increased after retinaculum transection, but this change was not reflected by coordinated changes in estimates of either minimum or maximum sarcomere length. A trend was found for maximum sarcomere length to be increased after transection, whereas minimum sarcomere length was numerically and statistically identical. The rest posture of the rat maintains the feet dorsiflexed, which may contribute to the relative conservation of short sarcomere length. This suggests that the static length signal of resting or neutral sarcomere length may be more important to long-term plasticity than infrequent stretches during locomotion. It also suggests that the range of sarcomere length may be preserved by the adaptation. Estimation of in vivo sarcomere lengths by extrapolation from a single measurement is notoriously difficult (3), and these observations should be viewed with suspicion.
In summary, the results of this study indicate that age does not influence the long-term trend for increased fiber length in TA in response to an increase in muscle excursion. Increasing muscle excursion tends to increase muscle fiber length, although not as dramatically as sustained passive stretch. The trend for fiber length increase opposes the adaptation that would be expected from passive tension, which is reduced by retinaculum transection. The regulation of fiber length appears to involve mechanical signals in addition to passive tension, although adaptation to changes in excursion may occur on a different time scale.
ACKNOWLEDGEMENTS
Surgical space was kindly provided by Dr. T. Richard Nichols. Animals were housed by the Emory University Department of Physiology.
FOOTNOTES
Address for reprint requests and other correspondence: T. J. Burkholder, Health & Performance Sciences, Georgia Institute of Technology, 281 Ferst Dr., Atlanta, GA 30332-0356 (E-mail: thomas.burkholder@hps.gatech.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 8 February 2001; accepted in final form 1 August 2001.
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ABSTRACT
Muscle fiber length adaptation to static stretch or shortening depends on age, with sarcomere addition in young muscle being dependent on mobility. Series sarcomere number can also increase in young animals in response to increased muscle excursion, but it is not clear whether adult muscles respond similarly. The ankle flexor retinaculum was transected in neonatal and adult rats to increase tibialis anterior muscle excursion. Sarcomere number in tibialis anterior was determined after 8 wk of adaptation. Muscle moment arm and excursion were increased 30% (P < 0.01) in both age groups. Muscle cross-sectional area was reduced by 12% (P < 0.01) in response to the increased mechanical advantage, and this reduction was unaffected by age. Fiber length change was also unaffected by age, with both groups showing a trend (P < 0.10) for slightly (6%) increased fiber length. Retinaculum transection results in shorter muscle length in all joint configurations, so this trend opposes the fiber length decrease predicted by an adaptation to muscle length and indicates that fiber length is influenced by dynamic mechanical signals in addition to static length.
muscle architecture; retinaculum transection; mathematical modeling
INTRODUCTION
SERIES SARCOMERE NUMBER is an important determinant of skeletal muscle function (1, 20). Force generation at the sarcomere level depends on both length and velocity, and the number of sarcomeres in series determines the relationship between muscle length and velocity and sarcomere length and velocity. The variation in sarcomere number or fiber length contributes more strongly to both the static and dynamic performance of muscle than fiber type (1, 21).
Fiber length is a highly plastic characteristic of muscle, undergoing substantial and rapid changes in response to growth or immobilization (29). This is necessary to maintain optimal force generation in the physiological range of motion. In addition to these quasi-static stimuli, fiber length can adapt to alterations in muscle excursion (2, 13). This latter plasticity may contribute to the correlation between muscle moment arm, which determines muscle excursion, and muscle fiber length (17). It may also contribute to the coordination of moment arm and fiber length for generation of maximal power during locomotion (14).
Previous studies have suggested a sharp contrast in the adaptation of developing and mature muscle to changes in length or excursion. Adult muscles respond to chronic stretch immobilization by rapidly increasing fiber length and to shortened immobilization by rapidly decreasing fiber length (12, 22, 30). Immobilization at a very young age results in reduced fiber length growth in mouse soleus muscle, independent of whether the muscle is stretched or shortened (30). The fiber length reduction can be very nearly relieved by mobilization of the muscle, suggesting that the reduced mobility is the source of sarcomere number reduction. This fiber length reduction is maintained for at least 6 wk in mice, well past the age at which the mature pattern of adaptation would be expected (29). This suggests that the restriction of mobility may establish a completely different pattern of length adaptation that is relieved only on mobilization of the muscle.
The response of muscle to increased excursion has also been suggested to be age dependent (2). When the tibialis anterior (TA) muscles of young rabbits are permitted increased excursion by transection of the ankle flexor retinaculum, the muscles respond by increasing fiber length (13). Adult mouse TA muscles respond to the increased excursion and decreased length of retinaculum transection by rapidly reducing sarcomere number (2). It is difficult to compare these results directly because of species, age, and experimental differences.
These observations suggest that skeletal muscle undergoes a fundamental shift in the mode of length adaptation during early growth. This is a dynamic time in the development of muscle, coinciding with the development of locomotion, changes in nutrition, and establishment of mature molecular phenotype (7, 16, 26). In the mouse, this period also coincides with an enormous increase in soleus sarcomere number, more than doubling within 3 wk, and motion appears to be required for this increase (28). Mature muscle fiber length appears to be strongly influenced by static length signals (2, 30).
Excursion appears to exert a subtle influence on sarcomere number compared with static length. Whereas a static stretch of 30% is capable of generating an increase in fiber length of more than 15% (30), a 40% increase in muscle excursion resulted in only 10% increase in sarcomere number (13), so the gain of the increased excursion appears smaller. It is possible that the excursion signal requires more time to be expressed. Burkholder and Lieber (2) reported that sarcomere number decreased in mice with increased excursion after 2 wk, the same period over which Williams and Goldspink (30) report large increases in stretch immobilized muscle. In contrast, the studies that have reported increased muscle or fiber length after retinaculum transection in growing rabbits have not investigated periods shorter than 3 mo (6, 13). Interpretation of the few retinaculum transection studies is complicated by the variety of species, durations, and treatments.
The purpose of the present study was to determine whether the differences in adaptation to increased excursion can be attributed to the age of the animal at the time of manipulation or to other factors, including species and experimental duration.
METHODS
Adult (~9-10 wk old, 200-225 g) and juvenile (10 days old, ~14 g) Wistar rats (Harlan, Indianapolis, IN) were randomly divided into control and transection groups. The adult age was chosen because these animals are skeletally and reproductively mature and have a well-established locomotor pattern. The young age was chosen to be before the age at which the change in adaptation mode has been reported, yet with animals large enough to facilitate the surgical procedure. These animals are substantially younger and less mobile that the rabbits used by Koh and Herzog (13) or Crawford (6). The rat species was chosen to facilitate surgery in the young animals yet be readily comparable to the existing body of sarcomere number regulation literature, the most substantial portion of which is based on mice.
Rats undergoing retinaculum transection were sedated with an intraperitoneal cocktail of Rompun (10 mg/kg), ketamine (90 mg/kg), and acepromazine (1.5 mg/kg). With the aid of a dissecting microscope, a small (2-mm) incision was made on the lateral surface of the foot, exposing the flexor retinaculum (crural ligament). The ligament was transected with fine scissors, and the TA tendon was elevated from its normal path, resulting in a more direct line to the insertion site. The tendon path was visibly altered throughout the range of motion, but the slight deviation at full plantar flexion is unlikely to allow substantial shortening of the muscle in this position. The wound was then closed with cutaneous glue (Nexaband, Phoenix, AZ). Transected rats were permitted normal cage activity immediately on recovery. The entire procedure was typically completed in 3-5 min. Animals behaved normally immediately on recovery, indicating the minimally traumatic nature of this intervention. Animals were visually monitored periodically throughout the duration of the experimental period and were behaviorally indistinguishable from controls. Cage activity and gait kinematics were not quantitated. Activity of cage housed animals is normally quite low and has previously been shown to be unaffected by this intervention (13). Gait kinematics are also minimally impacted by this intervention (2, 13) and would contribute little to the analysis. All procedures were approved by the institutional animal care and use committee and were performed in accordance with the Guide for the Care and Use of Laboratory Animals.
At the conclusion of the 8-wk experimental period, animals were killed by CO2 asphyxiation, and the TA was exposed. Absence of the flexor retinaculum and free mobility of the tendon was confirmed. The retinaculum in very young animals was prone to regrowth or fibrotic overgrowth, and only animals with clearly released TA tendons were included in the transection group. Passive muscle length was measured in situ with the ankle fully plantar flexed (~150°) and fully dorsiflexed (~25°) by use of a pair of dial calipers. Muscles were then harvested, pinned under slight tension, and fixed overnight in 4% paraformaldehyde in PBS. The entire distal TA tendon was carefully collected from some specimens, and it was length measured.
Muscles were rinsed in PBS and cleaned of extraneous tissue before weighing. Muscle length was measured with dial calipers under a dissecting microscope. Small fiber bundles (5-50) were teased from the lateral, medial, and deep regions of each muscle and mounted on glass slides. Fiber bundle length was measured with dial calipers, and sarcomere length was measured by laser diffraction in at least three regions of each fiber bundle. Series sarcomere number was calculated for each bundle by dividing fiber bundle length by the average of its sarcomere lengths. Fiber length for the whole muscle was then calculated by multiplying the average bundle sarcomere number by optimal sarcomere length (2.5 μm; Ref. 25). Thus there was one final measurement of fiber length for each muscle, which was a multiple of sarcomere number. Physiological cross-sectional area (PCSA) was calculated according to the formula
where 1.06 is the density of muscle (mg/mm3; Ref. 18) and S is sarcomere number. The term "0.0025 mm · S" is fiber length normalized to a sarcomere length of 2.5 μm and corrected for any variations in muscle length that occurred as a result of fixation at slightly different sarcomere lengths. The normalization to a sarcomere length of 2.5 μm corresponds to the optimal sarcomere length in rats (25).
Estimates of muscle force production were made by use of a mathematical model (1). This generalized model uses muscle architectural properties to estimate sarcomere length and velocity from muscle length and velocity. Force production was then estimated by using the sarcomere force-length and force-velocity relations. Muscle power was calculated by multiplying estimated isokinetic force by shortening velocity. Because of the hyperbolic decline in force with increasing velocity, peak power occurs at a shortening velocity of ~0.3 Vmax. Vmax was taken to be 10 l/s, and maximum isometric muscle stress was 250 kN/m2, with optimal length being 2.5 μm (5). Moment arm was estimated from muscle excursion, with full plantar flexion assumed to be 160° and full dorsiflexion to be 25°. Peak dorsiflexion velocity was estimated as 1,500°/s, on the basis of a 130-ms swing time (19) and sinusoidal motion profile.
Measurements were screened for normality and compared by two-way ANOVA with significance level set to 0.05. Between group differences were determined by unpaired t-test. Statistical trends were recognized at P < 0.10. Retrospective statistical power calculations were based on an expected difference equal to the minimum detectable difference. This was felt to be a conservative estimate, because the sample size permits resolution of ~0.5 standard deviations, a level that is almost certainly smaller than the biologically relevant threshold. No difference between groups was noted only when statistical power exceeded 90%. This distinction is necessary because several of the results of this work are similarities, rather than differences, and it is important to distinguish between "similar" and "not different." There are four possible statistical categories for a comparison: for P < 0.05, the comparison is significantly different; for P < 0.10, a trend; for power > 90%, statistically identical; for P > 0.10; and for power < 90%, statistically indeterminate. Results are reported as means ± SD unless otherwise noted. Statistical differences and trends are supported by the P value, i.e., the chance of a type I error or of incorrectly rejecting the null hypothesis of equality. Statistical identity is supported by statistical power, i.e., the probability of correctly accepting the null hypothesis of equality. Power is 1 , where is the probability of type II error or of incorrectly accepting the null hypothesis.
RESULTS
Surgical transection of the flexor retinaculum resulted in a 30% increase (Table 1; P < 0.01) in TA at the completion of the experiment that was identical between age groups (power > 94%). It is likely that this increase was greater in the first few days posttransection, because a pseudoretinaculum was generally present at termination. Muscles of both adult and juvenile animals responded to this increase with a trend (P < 0.1) toward increased fiber length (Table 1), which was not influenced by age (power > 93%). Age was a strong influence on overall fiber length, with muscles in younger animals displaying fibers that were 15% longer (Table 1; P < 0.01), without regard for surgical treatment. PCSA was reduced 12% by retinaculum transection (Table 1; P < 0.01), and this was not affected by age (power > 91%). Tendon lengths were measured in six adult control (14.1 ± 1.1 mm) and four adult transected animals (14.1 ± 0.6 mm) and were found to be identical (power >95%).
Sarcomere lengths (Table 1) were extrapolated from measurements in the fixed muscle by scaling to muscle lengths measured at the extremes of motion. Estimated sarcomere excursion was increased by 34% (P < 0.01) after retinaculum transection, independent of age (power > 92%). This increase was a mathematical consequence of the measured increase in muscle excursion, on which the extrapolation depends. The increased excursion was not associated with systematically altered maximum or minimum in vivo lengths, although a trend was found for maximum length to be slightly increased (P = 0.06).
The functional implications of these morphological changes were estimated by mathematical modeling. The functional estimates demonstrate the contrasting effects of the trend for fiber length increase and the decrease in PCSA. Neither statistical identity nor difference in the magnitude of predicted peak muscle power (Table 1) could be established. The joint velocity at which peak power would be achieved did decline by 24% (Table 1; P < 0.01), as a result of the great increase in moment arm. Had there been no adaptation, peak power would have remained constant, and the velocity of peak power would have declined by 30%. To restore the angular velocity of peak power production (1,300°/s), fiber length would have had to increase by 33%.
Despite the decline in PCSA, maximal isometric joint torque was predicted to increase by more than 15%, as a result of the increase in moment arm. This change is significant in adults (P < 0.01 ) and a trend in juveniles (P < 0.08). Using an estimated dorsiflexion velocity of 1,500°/s, torque capacity during locomotion was estimated to decline by 16%, indicating that the increase in moment arm may not be sufficient to compensate for the decrease in PCSA during locomotion.
DISCUSSION
The objective of this study was to determine whether juvenile and mature muscle respond differently to surgical increase in muscle excursion. The response of fiber length, PCSA and muscle excursion were nearly identical in the two age groups and argues strongly against age influencing the increase in fiber length induced by retinaculum transection.
Although age did not influence the experimental adaptation, there were clear morphological differences between the age groups. Most significant of those differences may be the longer fiber length and shorter sarcomere length of the younger muscles. This finding is somewhat surprising, given that the adult group is only 2 mo older than the juvenile group. Although sarcomere number and fiber length have been reported to be 5-10% lower in aged animals than adults (11, 15), it seems unlikely that age is the only factor involved in the 13% difference reported here. Myofilament lengths do not change with age (9, 10). The adult population was received directly from the vendor, where they were housed at higher density than the juvenile group, which was raised at the university vivarium. It is conceivable that the differences in the rearing environment or diet contributed to the difference in fiber length between age groups. Nutritional status has been shown strongly to influence fiber length (10, 27).
Joint kinematics were not measured in these animals, but previous work in both mice (2) and rabbits (13) suggests that dorsiflexion velocity and range of motion are only minimally affected by this procedure. A 22% decrease in dorsiflexion velocity would be required to restore near-optimal power generation, which is substantially greater than the (not significant) 7% decrease reported by Koh and Herzog (13) and the 14% decrease reported by Burkholder and Lieber (2). Optimal power generation during locomotion does not appear to have a strong influence on muscle architecture. This suggests that those systems in which muscles do operate near optimal power reflect tuning of the nervous system and selection of an optimized style of locomotion, rather than adaptation of the musculature. It is possible that the trend for increased fiber length combines with a decrease in dorsiflexion velocity and does restore peak power generation. This would also support the notion that the nervous system adjusts to meet muscular performance more effectively than muscle structure adjusts to meet functional demands.
The trend for fiber length increase after retinaculum transection parallels results seen in long-term studies in rabbits (6, 13). The transection of the retinaculum permits the TA to take a more direct path from the tibial origin to the metatarsal insertion, thus slightly reducing the muscle length in plantar flexion. Furthermore, the greater moment arm causes the released muscle to shorten more during motion so that the reduction in muscle length is even greater in dorsiflexion. If the muscle adapts solely to a static length or passive tension signal, then sarcomere number should be decreased by this preparation, as was seen in the previous short-term study (2) and many other studies (9, 23, 28), because length and passive tension are reduced by retinaculum transection. The finding that fiber length and sarcomere number tend to increase indicates that there is a nonstatic mechanical signal influencing fiber length. This in vivo model cannot distinguish among the likely signals of excursion, velocity, and power, but in vitro both excursion and velocity have been shown to influence smooth muscle growth (4).
The results of the mathematical model were intended to permit evaluation of the relative effects of the significant (12%) atrophy and the trend for fiber length increase (6%). In vivo function of the muscle depends on the PCSA, the velocity of shortening, the muscle moment arm, and neural recruitment. Ignoring the latter, retinaculum transection directly alters the muscle moment arm, and both PCSA and fiber length are found to change in response. If these changes are driven by functional demands, then some aspect of function should be similar in the control and transected groups. The mathematical model predicts an equivocal change in the magnitude of peak power production, a slight decrease in the angular velocity of peak power generation, and a substantial increase in the magnitude of isometric torque (Table 1). The velocity of peak power generation is quite close to the estimated speed of dorsiflexion (1,500°/s), although this may be a coincidence. Thus the model results suggest that adaptation of the muscle preserves peak power output.
The existing reports of sarcomere number adaptation to retinaculum transection suggest two aspects to the response. Immediately after transection, the muscle appears to respond primarily to the reduction in length or passive tension, reducing sarcomere number to restore optimal sarcomere length (2). This period may last as long as 4 wk in mice (unpublished observations). In parallel, a slower process appears to add sarcomeres to restore sarcomere excursion or velocity (present study; Refs. 6, 13). The latter process does not appear to be influenced by the age of the animal and appears to occur equally well in rats and rabbits. Interestingly, the classic Williams and Goldspink immobilization studies (28-30) fall within the initial time period of 4 wk. The age-dependent, bifurcate response of the mouse soleus to immobilization, in which immobilization of the young muscle inhibits normal sarcomere addition, remains intriguing. It is possible that the postnatal development of this, and possibly other, muscles is somehow dependent on mechanical input. Passive mechanical stimulation is known to initiate several signaling pathways in vitro (8, 24).
Sarcomere excursion was greatly increased after retinaculum transection, but this change was not reflected by coordinated changes in estimates of either minimum or maximum sarcomere length. A trend was found for maximum sarcomere length to be increased after transection, whereas minimum sarcomere length was numerically and statistically identical. The rest posture of the rat maintains the feet dorsiflexed, which may contribute to the relative conservation of short sarcomere length. This suggests that the static length signal of resting or neutral sarcomere length may be more important to long-term plasticity than infrequent stretches during locomotion. It also suggests that the range of sarcomere length may be preserved by the adaptation. Estimation of in vivo sarcomere lengths by extrapolation from a single measurement is notoriously difficult (3), and these observations should be viewed with suspicion.
In summary, the results of this study indicate that age does not influence the long-term trend for increased fiber length in TA in response to an increase in muscle excursion. Increasing muscle excursion tends to increase muscle fiber length, although not as dramatically as sustained passive stretch. The trend for fiber length increase opposes the adaptation that would be expected from passive tension, which is reduced by retinaculum transection. The regulation of fiber length appears to involve mechanical signals in addition to passive tension, although adaptation to changes in excursion may occur on a different time scale.
ACKNOWLEDGEMENTS
Surgical space was kindly provided by Dr. T. Richard Nichols. Animals were housed by the Emory University Department of Physiology.
FOOTNOTES
Address for reprint requests and other correspondence: T. J. Burkholder, Health & Performance Sciences, Georgia Institute of Technology, 281 Ferst Dr., Atlanta, GA 30332-0356 (E-mail: thomas.burkholder@hps.gatech.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 8 February 2001; accepted in final form 1 August 2001.
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