Adaptation in the number of sarcomeres in series in skeletal muscle fibres or fascicles (sarcomere number) may be important in pathologies associated with cerebral palsy (O'Dwyer, Neilson & Nash, 1989) and with bone lengthening (Simpson, Williams, Kyberd, Goldspink & Kenwright, 1995). Adaptations in sarcomere number also have implications for the function of normal muscle: muscle force-length properties (passive and active; Williams & Goldspink, 1978) and force-velocity properties (Spector, Gardiner, Zernicke, Roy & Edgerton, 1980) can be influenced by sarcomere number. Despite the likely importance of sarcomere number adaptation, the factors responsible for such adaptation remain unknown. Specifically, though mechanical signals appear to be important for inducing serial sarcomere addition or deletion (Herring, Grimm & Grimm, 1984; Goldspink, 1985), the nature of these signals remains to be elucidated.
Muscle length (or passive tension) is widely thought to be important in regulating sarcomere number. Immobilizing adult skeletal muscle in stretched or shortened positions produces increases or decreases, respectively, in sarcomere number (e.g. Tabary, Tabary, Tardieu, Tardieu & Goldspink, 1972; Williams & Goldspink, 1978). The adaptations in sarcomere number appear to produce near-optimal sarcomere lengths at the immobilized muscle length. These results have led to the conclusion that the working length of the muscle is important in regulating sarcomere number.
In growing animals, muscle excursion (the change in muscle length required to produce the full range of joint motion) may be important in regulating sarcomere number. In contrast to the results from adult animals, immobilizing growing muscle in a stretched position decreases sarcomere number relative to the contralateral control muscle (Williams & Goldspink, 1971, 1978; Tardieu, Tabary, Tabary & Huet de la Tour, 1977). Such a decrease could be associated with the decreased excursion induced by immobilization.
In addition, a procedure assumed by Crawford (1954, 1961) to increase muscle excursion increased longitudinal muscle growth in young animals (Crawford, 1954, 1961). This procedure involved releasing the tibialis anterior (TA) tendon from its retinacular restraint at the ankle joint in 3- to 4-week-old rabbits (Fig. 1). After 4 months, muscle belly length was 20 % longer for the released compared with the contralateral TA, despite the muscle working at a shorter length for all but the most plantarflexed ankle joint angles. If the increase in muscle length was accounted for by an increase in sarcomere number, these results may suggest a relationship between increased muscle excursion and increased sarcomere number. However, neither muscle excursion nor sarcomere number were measured; thus a relationship between the two remains speculative. In fact, in all studies of sarcomere number adaptation to date, the in vivo mechanical environment of muscle has not been defined. Thus, suggested links between the mechanical environment and sarcomere number regulation in general have only been speculative.
Figure 1. Schematic diagram of releasing the tibialis anterior (TA) from its retinacular restraint at the ankle joint
Tibia and foot shown as continuous lines; foot shown in plantarflexion and dorsiflexion. TA muscle-tendon unit shown as dashed line; length of line between origin and insertion represents TA muscle-tendon unit length in plantarflexion and dorsiflexion. Difference between TA muscle-tendon unit lengths in plantarflexion and dorsiflexion is TA muscle excursion. After TA release, TA muscle-tendon unit length is decreased for all but the most plantarflexed ankle joint angles and TA muscle excursion is increased.
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Another explanation for the increase in muscle length associated with TA release involves the possible decreased force production of the TA. TA release increases the moment arm of the TA at the ankle joint, and thus decreased TA force production may help to achieve near normal torque production about the ankle joint (Koh, 1997). This possibility is supported by the observation that, 4 months after release, the maximum isometric force produced by the released TA is less than that produced by its contralateral counterpart (Crawford, 1961). Chronically decreased force production could be responsible for reduced longitudinal tendon growth; the released tendon was shorter than the contralateral tendon (Crawford, 1954, 1961). This possibility is supported by data suggesting that tension is an important stimulus for longitudinal tendon growth (Tardieu, Blanchard, Tabary & le Lous, 1983; Blanchard, Cohen-Solal, Tardieu, Allain, Tabary & le Lous, 1985; Davison, 1992). Decreased tendon growth would result in a chronically increased working length of the released muscle. A chronically increased muscle length has been shown to be a stimulus for sarcomere number addition (Tabary et al. 1972; Tardieu et al. 1977). Thus, this sequence of events could be responsible for the increase in longitudinal muscle growth associated with release.
The working hypothesis for the present study was that muscle excursion is important in regulating sarcomere number in growing animals. The specific hypotheses of the study were (1) that increased excursion results in increased serial sarcomere addition following TA release in growing animals (primary hypothesis), and (2) that decreased TA force production results in increased serial sarcomere addition following TA release (secondary hypothesis). The primary hypothesis was tested by determining whether sarcomere number and in vivo muscle excursion were increased following TA release. Cage activity and in vivo ankle joint kinematics were also recorded to determine whether altered animal or joint activity patterns could be related to sarcomere number adaptation. The secondary hypothesis was tested by determining whether in vivo TA force production was decreased following TA release, and whether increasing the in vivo force production of the released TA (via ablation of the synergistic extensor digitorum longus (EDL)) inhibited the expected increase in sarcomere number associated with release. The present investigation appears to be the first study of sarcomere number adaptation in which the in vivo mechanical environment of muscle has been defined.
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Previous studies have suggested that muscle excursion may be important in regulating sarcomere number in growing animals (Crawford, 1954, 1961; Williams & Goldspink, 1971, 1978). However, changes in the in vivo mechanical environment of the muscle (excursion, force production, joint kinematics, animal activity) were not measured in these studies; thus this hypothesis could not be evaluated.
The primary hypothesis of this study was that increasing excursion results in increased serial sarcomere addition following TA release in growing animals. TA release resulted in increased muscle excursion, and in increased sarcomere number in both superficial and deep fascicles of the TA. The increase in sarcomere number occurred despite the working length of the muscle being reduced for all but the most plantarflexed ankle joint positions (Fig. 1); decreased working length has been associated with decreased sarcomere number for immobilization experiments using adult muscle (Tabary et al. 1972; Williams & Goldspink, 1978).
TA release did not appreciably affect rabbit mass or tibia length, nor cage activity over a 24 h period. These results suggest that increased serial sarcomere addition in the released TA was not the result of changes in animal growth or activity. In addition, TA release did not appreciably affect the ankle joint kinematics of the experimental leg during hopping on a treadmill, suggesting that the increased serial sarcomere addition was not due to a chronic change in ankle joint kinematics. The architecture of the soleus muscle (both sarcomere number and PCSA) is highly sensitive to changes in the mechanical environment (Lieber, 1992); the absence of an effect of TA release on soleus architecture provides further evidence that chronic changes in joint kinematics or hindlimb loading were not responsible for the increased serial sarcomere addition in the released TA. Finally, the mean sarcomere number and mean muscle excursion for all of the experimental groups of this study showed a linear relationship. Taken together, these results support the hypothesis that increased excursion results in increased serial sarcomere addition following TA release in growing animals.
The percentage increase in muscle excursion was larger than the percentage increase in sarcomere number for the released TA. This may simply indicate that muscle excursion does not regulate sarcomere number in a one-to-one manner. Another explanation for the discrepancy between the increases in muscle excursion and sarcomere number is that the excursion over which the TA is normally active may not be increased by release as much as the total TA excursion. However, the estimated TA muscle excursion during force production in treadmill hopping for the release group (1.74 ± 0.30 cm) was 40 % greater than that for the control group (1.24 ± 0.11 cm). Thus, the muscle excursion during force production in hopping was increased by the same percentage as total muscle excursion.
Two further possible explanations for the greater increase in excursion than in sarcomere number are: (1) that muscle excursion does not accurately reflect excursion at the sarcomere level (the latter is likely more important for mechanical signal transduction in the muscle cell), possibly because of sliding of in-series fibres within a fascicle past each other, and (2) that muscle excursion is not increased as much immediately post release in 4-week-old rabbits as after 12 weeks of growth. To examine these possibilities, the increase in sarcomere excursion with release was estimated in a separate group of 4-week-old rabbits (following Muhl, Grimm & Glick, 1978). For six rabbits, the left TA was fixed in formalin in full plantarflexion and the right TA was fixed in full dorsiflexion; three rabbits had bilateral TA releases, and the other three served as controls. Released muscle excursion (1.59 ± 0.10 cm) was greater than control muscle excursion (1.23 ± 0.10 cm) and sarcomere excursion for the released muscle (1.24 ± 0.03 μm) was greater than that for control muscle (0.98 ± 0.08 μm). The similar percentage increase in muscle (29 %) and sarcomere (27 %) excursion with release suggests that muscle excursion reflects sarcomere excursion fairly well, and that in-series fibre sliding does not appreciably affect passive sarcomere length changes. These measurements in fixed muscles from 4-week-old rabbits showed a smaller percentage increase in muscle excursion than the fluoroscopic measurements after 12 weeks of growth (29 %versus 40 %). If these measurements represent a real increase with age in the effect of release on muscle excursion, the stimulus for serial sarcomere addition may not be as great as would be indicated by the increase in muscle excursion after the 12 week growth period (40 %), but would still be greater than the percentage increase in sarcomere number.
The secondary hypothesis of this study was that decreased TA force production results in increased serial sarcomere addition following TA release. TA release decreased the in vivo force production of the TA for a variety of activities that required both small and large force magnitudes. Decreased maximum in situ isometric force and PCSA for the released TA support the argument that force production was chronically decreased in the released TA.
Chronically decreased force production of the released TA may not have provided a sufficient stimulus for normal longitudinal tendon growth, as external tendon, proximal aponeurosis, and distal aponeurosis lengths were shorter than control. A link between tension and longitudinal tendon growth is supported by previous observations that longitudinal tendon growth is increased when growing muscle is immobilized in a stretched position, and that this increase can be attenuated by denervating the immobilized muscle (Blanchard et al. 1985). In addition, decreased tendon growth has been associated with surgical shortening of bone; a chronic decrease in tension may be important in this model as well (Tardieu et al. 1983).
Decreased longitudinal tendon growth would chronically increase the working length of the released TA. Since chronically increased muscle length has been shown to be a stimulus for serial sarcomere addition (Tabary et al. 1972; Tardieu et al. 1977), this sequence of events could be responsible for the increase in serial sarcomere addition observed.
Although partial ablation of the EDL increased in vivo force production in the released TA in the present study, increasing the force production of the released TA did not inhibit the increased growth in sarcomere number associated with release. Thus the secondary hypothesis that decreased TA force production was responsible for increased serial sarcomere addition was not supported by these results.
The increased minimum angle of the ankle joint during hopping for the release plus ablation group compared with the release-only group may have been due to a decreased ability to dorsiflex the foot when EDL force production is removed. The increased force production of the TA may not have been enough to compensate for EDL ablation (the contribution of the EDL to dorsiflexion is unknown, as EDL forces were not measured). However, the change in kinematics appeared not to affect TA serial sarcomere addition, as TA sarcomere number was not different for release plus ablation and release-only groups.
Partial ablation of the EDL did not increase longitudinal tendon growth as predicted by the secondary hypothesis. Ablation did appear to decrease external tendon length (by 3 mm) and showed a trend of increasing distal aponeurosis length (by 3 mm). Thus, ablation seemed to alter the relative distribution of internal and external tendon lengths. In summary, upon examination of all the groups of this study, no clear relationship between TA force production and longitudinal tendon growth could be discerned.
Adaptations in sarcomere number associated with immobilization of adult muscle appear to produce near-optimal sarcomere length at the immobilized muscle length (Williams & Goldspink, 1978). Thus, such adaptations appear to allow optimum isometric force production at the immobilized muscle length. In the present study, increasing the excursion of the growing TA produced an increased sarcomere number in the released TA compared with control. Without such an increase in sarcomere number, the increased excursion of the released TA would increase the amount and rate of shortening that individual sarcomeres would have to undergo to produce a given amount of ankle dorsiflexion. This would decrease the force potential of the TA muscle, based on the force-length (Gordon, Huxley & Julian, 1966) and force-velocity relationships (Hill, 1938) of skeletal muscle. Thus, the increase in sarcomere number associated with TA release may restore the functional capabilities of the muscle. In support of this idea, Crawford (1961) has shown that the plateau of the force-length relationship is indeed wider for released compared with control TA muscles.
Satellite cell proliferation and fusion may be important in adaptive longitudinal growth of muscle fascicles. Moss & Leblond (1971) have demonstrated that satellite cells are the source of myonuclei during growth. Williams & Goldspink (1971) presented electron microscopic evidence for satellite cell fusion at the end of growing muscle fibres. Cyclic stretch of muscle cells has been shown to release insulin-like growth factor 1 (IGF-1; Perrone, Fenwick-Smith & Vandenburgh, 1995), which, in turn, stimulates proliferation and differentiation of satellite cells (Allen & Rankin, 1990). Thus increased excursion following release could increase the rate of proliferation and differentiation of satellite cells, which could be involved in increased serial sarcomere addition.
Cyclic stretch (which could be considered excursion) has been shown to increase DNA, RNA and protein synthesis and accumulation in cultured cells of different types (e.g. muscle: Vandenburgh, Hatfaludy & Shansky, 1989; tendon: Banes et al. 1995; endothelial: Awolesi, Sessa & Sumpio, 1995). For muscle cells, cyclic stretch increases the growth in length and diameter of cultured myotubes (Vandenburgh et al. 1989); however, the influence of cyclic stretch on sarcomere number in myotubes has not been investigated. Although controlled comparisons of static versus cyclic strain appear not to have been made for muscle cells, cyclic strain appears to be better than static strain for preventing bone loss and producing new bone formation in vivo (Lanyon & Rubin, 1984).
The series-fibred architecture of the TA provides different possibilities for increasing sarcomere number in fascicles spanning the distance between proximal and distal aponeuroses. Fascicle sarcomere number (or fascicle length) could be increased by (1) sliding of fibres past each other during adaptation without sarcomere addition, (2) addition of sarcomeres only to the ends of the fibres at the proximal and/or distal aponeurosis without such fibre sliding, or (3) addition of sarcomeres to all fibres with concomitant sliding of fibres. These possibilities remain unexplored and would be intriguing issues for future study.
The interpretation of the data for this study is limited by the lack of information about the mechanical environment at time points other than those immediately prior to the killing of the animal. The possibility exists that differences in the mechanical environment between groups changed over the growth period. For example, excursion was increased more in the measurements immediately prior to death (40 %) than in the measurements immediately after release (29 %). However, in both measurements excursion was significantly increased, and the difference between measurements does not substantially affect the interpretation of the data. In addition, visual observation of the rabbits suggested that joint kinematics did not change appreciably over time (each rabbit was observed at least once per week). The observation that changes in cross-sectional area of the muscle, as well as the maximum in situ isometric muscle force, paralleled changes in in vivo force production supports the argument that force production was chronically decreased in the released TA. Hence the possibility that a change in the mechanical environment over time would affect the interpretation of the results is considered unlikely.
The interpretation of the data for this study is also limited by the lack of knowledge of unmeasured factors that could influence sarcomere number regulation. For example, the surgical procedure for release could have produced unknown systemic factors that may have influenced serial sarcomere addition. The age-matched control and sham-operated animals should control for the effects of growth and the surgical procedures (apart from TA release). However, the results may have been influenced by unknown interactions between muscle growth and TA release. Nonetheless, the results are pertinent to understanding the regulation of sarcomere number in growing animals.
In conclusion, the results of this study support the working hypothesis that muscle excursion is important in regulating sarcomere number following TA release in growing animals. Characterization of the in vivo mechanical environment of the TA allowed direct correlation of excursion and sarcomere number, and allowed the exclusion of altered cage activity, ankle joint kinematics and TA force production as possible factors contributing to altered serial sarcomere addition. The cellular mechanisms that regulate sarcomere number also remain an exciting area for future study.