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. 2003 Apr 25;549(Pt 3):877–888. doi: 10.1113/jphysiol.2002.038018

In vivo and in vitro heterogeneity of segment length changes in the semimembranosus muscle of the toad

A N Ahn 1, R J Monti 1, A A Biewener 1
PMCID: PMC2342988  PMID: 12717006

Abstract

Many studies examine sarcomere dynamics in single fibres or length–tension dynamics in whole muscles in vivo or in vitro, but few studies link the various levels of organisation. To relate data addressing in vitro muscle segment behaviour with in vivo whole muscle behaviour during locomotion, we measured in vivo strain patterns of muscle segments using three sonomicrometry crystals implanted along a fascicle of the semimembranosus muscle in the American toad (Bufo americanus; n = 6) during hopping. The centre crystal emitted an ultrasonic signal, while the outer crystals received the signal allowing the instantaneous measurement of lengths from two adjacent muscle segments. On the first day, we recorded from the central and distal segments. On the second day of recordings, the most distal crystal was moved to a proximal position to record from a proximal segment and the same central segment. When the toads hopped a distance of two body lengths, the proximal and central segments strained −15.1 ± 6.1 and −14.0 ± 4.9 % (i.e. shortening), respectively. Strain of the distal segment, however, was significantly lower and more variable in pattern, often lengthening before shortening during a hop. From rest length, the distal segment initially lengthened by 2.6 ± 2.0 % before shortening by 6.5 ± 3.2 % at the same hop distance. Under in vitro conditions, the central segment always shortened more than the distal segment, except when passively cycled, during which the segments strained similarly. When the whole muscle was cycled sinusoidally and stimulated phasically in vitro, the two adjacent segments strained in opposite directions over much (up to 34 %) of the cycle. These differences in strain amplitude and direction imply that two adjacent segments can not only produce and/or absorb varying amounts of mechanical energy, but can also operate on different regions of their force–length and force–velocity relationships when activated by the same neural signal. Understanding regional differences in contractile dynamics within muscles is therefore important to linking our understanding of sarcomere behaviour with whole muscle behaviour during locomotion.

Many studies examine sarcomere dynamics in single fibres or length–tension dynamics in whole muscles in vivo or in vitro, but few studies link the various levels of organisation. Single fibre experiments reveal the basics of contraction dynamics associated with myofilament interactions, while whole muscle studies examine muscle function with respect to animal behaviour. To begin to link our understanding of in vitro sarcomere behaviour with in vivo muscle behaviour during locomotion, we measured in-series segment strains along a single fascicle within a leg muscle of the toad Bufo americanus both in vivo during locomotion and subsequently in vitro under controlled conditions.

Variable strain along a muscle (i.e. sarcomere heterogeneity) has been most commonly noted in single fibres under various conditions (Huxley & Peachey, 1961; Julian & Morgan, 1979; Edman & Reggiani, 1984; Burton et al. 1989; Horowitz & Pollack, 1993). In general, when single fibres are stretched, end sarcomeres shorten to lengthen the central sarcomeres to even greater lengths during maximally stimulated contractions (Julian & Morgan, 1979; Edman & Reggiani, 1984). Although less commonly observed in fibres at lengths corresponding to the ascending limb or plateau region of the their force-length relation, the behaviour of the sarcomeres at these lengths is more variable than when the fibre is held at long lengths. Occasionally, all segments shorten homogeneously along the fibre (Cleworth & Edman, 1972; Mutungi & Ranatunga, 2000). Sometimes, shortening and lengthening regions are dispersed along the fibre randomly (Burton et al. 1989). Yet in other studies, the central segments shorten while the end segments elongate or remain isometric (Edman & Reggiani, 1984; Mutungi & Ranatunga, 2000).

In contrast to studies of single fibres, studies of length changes in whole muscles mainly attribute tendon or aponeurosis compliance as the source of internal sarcomere shortening in whole muscles. During fixed-end contractions, studies in whole muscle show that the central segments of muscle can shorten up to 28 % due to the lengthening of the in-series tendon (Griffiths, 1991; Kawakami & Lieber, 2000). Even during cyclical contractions that simulate walking, the central segments shorten while the whole muscle-tendon unit lengthens demonstrating the compliance in the tendon (Griffiths, 1991). More recently, in vivo measurements of strain in human leg muscles using ultrasound also show the compliant nature of aponeurosis and tendon during walking and counter-movement exercises (Fukunaga et al. 2001; Kawakami et al., 2002). Using cine-magnetic resonance imaging (MRI) on humans, in vivo passive and low level, isometric contractions (5 and 15 % maximum voluntary contraction) result in uniform shortening along anterior fascicles and non-uniform shortening along centreline fascicles of the biceps brachii due to the presence of a central, internal aponeurosis (Pappas et al. 2002).

In the present study, we examine the behaviour of multiple, adjacent segments along the semimembranosus (SM) muscle of toads during hopping (in vivo) and subsequently while isolated with imposed stimulation and length conditions (in vitro). We test the hypothesis that fascicle segments strain similarly along the muscle. Since the SM muscle operates on the plateau region of its force-length relation during jumping in Rana pipiens (Lutz & Rome, 1994), we would expect the sarcomeres to contract homogeneously (Cleworth & Edman, 1972).

METHODS

Animals

Adult American toads (Bufo americanus) were collected and housed at the Concord Field Station in Bedford, MA, USA. All animals (n = 12; mass 37.2 ± 12.8 g; mean ± s.d.) were fed crickets dusted with calcium and vitamin powder and had free access to water. The animal room was maintained at 20–22 °C with a reverse 12:12 h dark:light photoperiod. All experiments were performed at room temperature (22 °C) unless otherwise noted. All experiments were performed in accordance with the Animal Care and Use guidelines of Harvard University (ACUP 20–13).

Sonomicrometry

Sonomicrometry recordings of muscle segment length change are based on measurements of the transit time of a 5 MHz ultrasonic pulse that travels from an emitting to a receiving piezoelectric crystal (for thorough reviews of the use of sonomicrometry to measure muscle segment length changes in vivo and in vitro, see Griffiths, 1987; Biewener et al. 1998; Olson and Marsh, 1998). For the present study, a single emitting crystal was positioned between two receiving crystals (Fig. 1). The sonomicrometer unit (model 120–1001; Triton Technology, Inc.) converted the timing of ultrasonic signals transmitted between the crystals to a voltage signal. The output of the sonomicrometer unit was also viewed during surgery with an oscilloscope (Tektronix 2235A 100 MHz) to assure maximum signal strength when implanting and aligning the crystals. We corrected the output signals by a factor of 1.027 because we assumed a sound pulse propagation velocity of 1540 m s−1 for vertebrate skeletal muscle (Goldman & Richards, 1954; Goldman & Heuter, 1956; Hatta et al. 1988), whereas the length calibration of the sonomicrometer system is based on a pulse velocity of 1500 m s−1 for water. The data were also corrected for a 5 ms phase delay introduced by filters within the Triton sonomicrometer. Segment length signals were filtered using a 58–62 Hz notch filter to remove 60 Hz interference and a second-order low-pass filter with a 50 Hz cut-off frequency. Changes in muscle stiffness during contraction that might alter the speed of sound transmission and hence measurements of muscle length using this technique, have been found to be small (∼1.3 %; Griffiths, 1987; Hatta et al. 1988) and thus unlikely to influence the relative differences in length change observed between adjacent, similarly activated fascicle segments in the present study. All negative strain values represent shortening, while all positive strain values represent lengthening of the muscle or muscle segments.

Figure 1. Segment lengths in the semimembranosus muscle.

Figure 1

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A, dorsal view of an anaesthetised toad. The thick black lines indicate the implant sites for the two outer receiving and the one middle emitting, sonomicrometry crystals in the SM muscle to measure central and distal segment lengths. EMG electrodes are not shown. B, a schematic diagram of the SM muscle showing the distribution of muscle fibre lengths and internal aponeuroses within the muscle. Three sono crystals (a, b and c) were used to measure central and distal segments. The proximal segment (dashed arrow) could not be measured simultaneously with the other two segments. c′ indicates the location of the implantation site during in vivo hopping on the second day when proximal and central segments were measured. Although from its external appearance this muscle appears to be a simple, parallel-fibred strap muscle, an internal aponeurosis and two tendinous inscriptions were discovered after careful dissection. The orange lines show the internal tendinous inscriptions that divide the muscle into three discrete sections, while the yellow line represents an internal aponeurosis on the dorsal surface of the muscle. Extreme care was taken to ensure the sonomicrometry crystals were implanted along the fascicles that span the entire length of the muscle, as indicated by the arrows at either end of the muscle.

In vivo recordings

Surgical procedures

Six American toads (mass, 36.9 ± 8.4 g) were anaesthetised by immersion in a buffered tricaine methanesulfonate solution (MS-222; 1 g l−1). While anaesthetised, an incision was made through the skin of the dorsal upper right leg to reveal the muscle of interest, the semimembranosus (SM) muscle. The SM muscle is bi-articular, spanning both hip and knee joints. However, the moment arm, and thus the moment this muscle can exert, at the knee is very small (Lutz & Rome, 1994; Kargo & Rome, 2002). The SM muscle, therefore, functions mainly as a hip extensor (Fig. 1A). We selected this muscle because of its seemingly simple architecture. The SM comprises parallel fibres and is relatively free of visible aponeuroses. Like many leg muscles, however, the SM muscle is slightly asymmetrical and tapered in shape, containing some fibres that do not extend along the entire length of the muscle. Although strap-like, its origin from the posterior surface of the ischium is broader than its insertion onto the tibiofibula (Duellman & Trueb, 1986). Therefore, extreme care was taken to implant the crystals and electrodes along fascicles that span the muscle from its origin to insertion (arrows in Fig. 1B). After exposing the muscle, three small pockets were created within the muscle along a fascicle using sharp, pointed forceps. These pockets were created by delicately teasing apart the fascia between muscle fibres in order not to damage the contractile elements. After sonomicrometry crystals (1.0 mm; Sonometrics Corp., London, Ontario, Canada) were implanted into the pockets, the opening to the pocket (at the perimyseal surface) was then closed with 6–0 silk suture. Since the crystals fit snugly into the pockets, resulting from the intrinsic elasticity of the fascicles and surrounding connective tissue, crystal movement within the pocket (i.e. relative to the fascicle) was considered to be negligible. Moreover, the passive length changes of the fascicle segments measured by the sonomicrometry crystals were similar to the measurements obtained for the whole muscle-tendon using the muscle ergometer (see Results below). After closing the crystal pockets, 6–0 silk suture was used to anchor the crystals and their lead wires into fixed positions and orientations (Fig. 1) which prevented any movement of the wires relative to the crystals or local fascicles. Two pairs of bipolar electrodes (offset twist hook; Loeb & Gans, 1986) were constructed from insulated silver wire (0.1 mm diameter; California Fine Wire Co.) to record electromyographical (EMG) activity from the respective muscle segments along the fascicle. The skin incision was then closed with 4–0 silk suture. External to the animal, the sonomicrometry and EMG wires were braided together to avoid electrical cross-talk. The braided wires were sutured to the back of the animal to prevent the animal from tangling or pulling the wires. The animals were allowed to rest for at least 2–4 h to recover fully from anaesthesia before making in vivo experimental measurements.

In vivo data collection

While hopping on a treadmill (21 × 60 cm working space), toads were recorded with high-speed digital video (Fig. 2A–D; 125 frames s−1; Redlake Motionscope). Central and distal segments of the SM muscle were recorded on the first day (similar to Fig. 1A). After the animals were kept overnight at 11°C, they were re-anaesthetized on the second day with the MS-222 solution before the most distal sonomicrometry crystal was moved to a proximal location along the same fascicle (Fig. 1B). Thus, subsequent recordings were made from the proximal and central segments of the same muscle. Moreover, the central segments recorded from both days were obtained from the same pair of untouched crystals (Fig. 1B). EMG signals from the hopping animals were amplified 100 times at a bandwidth of 100 to 3000 Hz (Grass P5 series AC pre-amplifiers). Voltage changes representing muscle length changes and EMG signals were acquired (AxoScope 8.0) at 4 KHz on a computer while the animals hopped on a treadmill. Hop start time, stop time, and distance were determined from the simultaneous digital video recordings of the trials. We used the hop start and stop times as the reference for the EMG burst timing because these times were the most general parameters that can also be used to compare with other studies.

Figure 2. Hopping in the American toad.

Figure 2

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A–D, digital video frames showing lateral and dorsal views of a typical toad hop. The first frame (A) shows the beginning of the hop (i.e. the onset of movement at time 0), while the last frame (D) shows the end of the hop (i.e. when the toad comes to a stationary resting position). The times at which the hops begin and end are also indicated by the arrows in the graphs. E and F show representative in vivo strain and EMG patterns from all three segments from two different animals during hops of 1 and 1.8 BLs, respectively. Negative strain values represent segment shortening. To show signals from all three segments together, traces of hops of similar distances from the same animal are overlaid (dotted, black line: proximal segment; dashed, blue lines: central segments; continuous, red line: distal segment). The thick traces were acquired together on the first day and the thin traces were acquired together on the second day. From top to bottom, EMG traces are shown as proximal (first day), central (first day), central (second day), and distal (second day).

In vitro recordings

Muscle preparation

Two of the animals used for the in vivo recordings were also used for the in vitro preparations with a total of eight animals used to collect the in vitro data (mass, 37.3 ± 15.9 g). While anaesthetised with the MS-222 solution, three sonomicrometry crystals were implanted into the right SM muscle to determine central and distal segment strains as if the animal were to be used for in vivo hopping experiments (Fig. 1A). During a preparation, only two adjacent segments can be measured simultaneously. Therefore, we examined only the central and distal segments in the isolated muscle experiments because these segments behaved differently in vivo during hopping (Fig. 2 and Fig. 3). After full recovery from anaesthesia, animals were double pithed to avoid effects of MS-222 on the neuromuscular system. In vivo SM muscle length was measured with the hip and knee joints set to 90 deg, which approximates the mid-point of the ranges of motion of the joints. The SM muscle was carefully dissected without damage to the aponeuroses and tendinous insertions into the hip and knee joints. The proximal end of the musculo-tendon unit originates on the ischium, which was held fixed in the preparation by a surgical clip. The surgical clip was tied securely with a 2–0 multifilament polyamide (nylon) suture to a pin fixed within a 2.7 × 11.5 × 2.2 cm well that was machined in a 10 × 19 × 5 cm block of Plexiglas. The distal end of the muscle-tendon (MT) unit inserts onto the proximal tibia just below the knee. The proximal portion of the tibia, to which the muscle inserts, was clamped with a surgical clip. After the muscle was carefully aligned and positioned so that no twisting occurred during contractions, the surgical clip was then tied with 2–0 nylon suture securely onto the lever arm of a servomotor (Aurora Scientific, Inc.; model 305B). The servo motor lever was positioned to be aligned with the pin, which anchored the proximal end of the MT unit. The servo motor was bolted onto a linear manipulator (Velmex, Inc; A25 series), which adjusted the length of the muscle to its resting length as determined in vivo. Oxygenated amphibian Ringer solution (Carolina Biological Supply) was maintained at 25 °C and circulated through the well with a pump for the duration of the experiment.

Figure 3. Peak segment strain as a function of hop distance.

Figure 3

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The magnitude of peak shortening in the proximal segment (open bars) is similar to that seen in the central segment (hatched bars). In contrast, the central segment lengthened first (positive filled bars), then shortened (negative filled bars), but to a lesser magnitude than the other segments during hopping. Values are means ± s..d. (n = 4–6 animals).

The strain and stimulation conditions were chosen after observations from swimming and hopping video trials. A cycle frequency of 3 Hz was a commonly observed and maximal frequency in the animals during swimming. The ±5 % strain amplitude was within the in vivo range of strain amplitude used by the animal. Finally, we chose a supramaximal stimulation pattern in an attempt to recruit the entire muscle. The stimulation pattern (200 Hz for 50 ms) was chosen because 200 Hz exceeded the maximum in vivo EMG frequencies during hopping and swimming. Even though the in vivo activation duration lasted longer than 50 ms during hopping (see Results), the muscle was stimulated for 50 ms in order to maximally stimulate the muscle (see Fig. 4B), but to not exceed the duration of one-fourth of the cycle.

Figure 4. Segment strain and muscle stress during fixed-end contractions.

Figure 4

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A, a twitch contraction. Even though the ends of the muscle–tendon unit are fixed and ‘isometric’, the individual segments shortened. The central segment consistently shortened more than the distal segment. B, a fixed-end contraction using a supramaximal burst of stimulation (200 Hz for 50 ms). During the near tetanic contraction, the muscle segments also shortened while the ends of the muscle-tendon unit were held fixed. These contractions were obtained from the same muscle shown for the cyclical contractions (Figs 5 and 7).

In vitro data collection

We used a computer program (Labview; National Instruments) that controlled whole muscle length and stimulation of the nerve, while acquiring muscle force and whole muscle length data from the servo motor, as well as segment length signals from the sonomicrometry crystals. Although muscle force was recorded from all in vitro trials, these data will be reported in a subsequent paper, because the present paper focuses on fascicle segment length changes in relation to whole muscle length changes. The SM muscle was stimulated through the sciatic nerve with a suction electrode using 0.5 ms square-wave pulses at 200 Hz for 50 ms (supramaximal burst; Grass S48 Stimulator). This supramaximal burst was used to determine the threshold voltage, because the twitch forces were too low. After determining the threshold voltage, an isometric twitch was recorded using a supra-threshold voltage. This supra-threshold voltage was used for all subsequent stimulations, including the fixed-end and cyclical contractions. Force, whole muscle length, segment length, and stimulation signals were acquired at 4 KHz (Labview DAQ system; NI PCI-MIO-16E-4 board). Trials were separated by 2–3 min intervals to minimise both potentiation and fatigue. Trials were stopped when the amplitude of force generated by an isometric contraction declined by more than 10 % of its original force. Passive measurements were obtained after the active properties recorded during the stimulation trials were completed. After removal of the SM muscle from the in vitro muscle bath, all non-muscle tissue was dissected away (e.g. nerve, tendon and bone) and the muscle was weighed with an electronic scale (Sartorius ISO 9002).

For both percentage heterogeneity calculations (Fig. 7) and work loop analyses (Fig. 8), the second cycle from each 3-cycle trial was analysed (Fig. 5). For the present study, percentage heterogeneity was defined as the portion of the cycle, during which adjacent segments along the muscle strained in opposite directions. Instantaneous segment velocities were calculated by differentiating the strain signals, then filtered using a 58–62 Hz notch filter and a second-order low-pass filter with a 25 Hz cut-off frequency. When the product of the instantaneous segmental strain rates was equal to or greater than zero, the two segments were straining homogeneously. When the product of the instantaneous segment strain rates was less than zero, the segments were straining heterogeneously. The sum of the portions of the cycle during which the two segments strained in opposing directions (i.e. when the product of their strain rates was less than zero) divided by the time of the cycle, multiplied by 100. 100 was determined to be percentage heterogeneity.

Figure 7. Percentage of the cycle during which central and distal segments strained heterogeneously as a function of stimulation phase.

Figure 7

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Percentage heterogeneity increased with stimulation phase, maximising at phase 50 (34.0 ± 9.2 %; n = 7; mean ± s..d.). * Statistically different (n = 6 or 7 for each stimulation condition; unpaired t tests; P < 0.05) between the stimulation condition compared to the passive condition. Although the imposed stimulation conditions are not identical to those measured in vivo during jumping (see Methods), stimulation phase 75 most closely approximates the in vivo activation pattern.

Figure 8. Segment work at various stimulation phases.

Figure 8

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Inset graphs show ‘work loops’ with force (N) plotted as a function of strain (%) of each segment at four stimulation phases (see Discussion). The representative work loops are graphed from the data shown in Fig. 5. The axes for the inset ‘work loop’ plots are identical. The (+) and (−) signs indicate net generation or absorption of mechanical energy, respectively.

Figure 5. Strain during cyclical contractions.

Figure 5

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A, strain during a passive cyclical contraction. B–E, strain vs. time during cyclical contractions at four different phases of stimulation: B, phase 0, stimulation at mid-shortening; C, phase 25, stimulation at the beginning of lengthening; D, phase 50, stimulation at mid-lengthening and E, phase 75, stimulation at the beginning of shortening.

Statistics

All reported values represent means ± s..d. All n values indicate the number of animals used to collect those data. Comparisons were tested with either Student's paired or unpaired t tests. When comparing between segments with the same group of animals, we used paired t tests. However, unpaired t tests were used to evaluate comparisons made between groups that consisted of data obtained from different animals. Differences were considered to be statistically significant when P < 0.05.

RESULTS

In vivo muscle patterns during hopping

Sample set

In vivo segment strain and EMG data were obtained from one to six hops per trial, one to five trials per animal, and five animals for both proximal/central and central/distal fascicle segments. Two animals overlapped between these two groups. Unless otherwise noted, a total of 167 hops were analysed from six individuals. For the five individuals in which all three segments were measured, the sum of the segments comprised 70.1 ± 7.2 % of the length of the muscle fibres.

Activity patterns of muscle segments

During hopping, similar activation phases were observed between all three segments of the semimembranosus muscle (Fig. 2). When normalised to the start (0 %) and end of the hop (100 %), activation of the proximal segment began at −0.9 ± 8.4 % (−4.5 ± 27.0 ms), which is similar to the onset of activation for the central segment (1.0 ± 5.7 % or 2.2 ± 17.8 ms; n = 5 for both; P > 0.05; Fig. 2E and F). For trials during which the central and distal segments were recorded, the activation phase for the central segment was −3.4 ± 3.6 % (−9.0 ± 9.8 ms) and −5.1 ± 4.9 % (−13.5 ± 12.8 ms) for the distal segment (n = 5 for both; P > 0.05; Fig. 2E and F). The activity patterns of these segments lasted for 24.3 ± 3.7 % (or 73.0 ± 8.9 ms: central) and 26.5 ± 4.5 % (or 79.1 ± 10.2 ms: distal; P > 0.05). No difference (P > 0.05) in the onset times of the central segments between the first (central/distal) and second (proximal/distal) days of recording was observed. Proximal and distal onset times, recorded on separate days, were also similar (P > 0.05).

Segments started shortening near the beginning of the hop. The proximal segment started shortening 3.0 ± 23.1 ms before the beginning of the hop. The central segment started shortening 3.9 ± 12.6 ms after the beginning of the hop. The distal segment started shortening 26.5 ± 33.7 ms after the beginning of the hop. The onset of segment shortening relative to the beginning of the hop was similar among all three segments (P > 0.05). Strain records for more than 90 % of the hops returned back to 0 before the next hop (Fig. 2E and F). However sometimes, strain at the end of the hop was variable depending on the position of the leg during landing and re-positioning of the leg in preparation for the next hop.

Strain of muscle segments

The proximal and central segments strained more than adjacent distal segments of the semimembranosus muscle in vivo during hopping regardless of hop distance (Fig. 2 and Fig. 3). A subsample of hops closest to one, two and three body lengths (BLs) were compared to examine whether peak strain of the segments varied with hop distance. For hops of 1.1 ± 0.1, 2.0 ± 0.1 and 2.7 ± 0.3 BLs, the proximal segments strained −11.4 ± 4.6, −15.1 ± 6.1 and −15.6 ± 5.3 % (n = 4 or 5 animals), together with central segment (−11.5 ± 5.3, −14.0 ± 4.9 and −16.5 ± 4.8 %; n = 6 animals; P > 0.7 at any given hop distance; Fig. 3). In contrast, the pattern and magnitude of strain in the distal segment, although immediately adjacent to the central segment, differed substantially. The pattern of distal segment strain was more variable and often lengthened before shortening during the hop. Relative to rest length, the distal segment lengthened (3.4 ± 2.6, 2.6 ± 2.0 and 2.9± 2.2 %) before shortening (−4.1 ± 3.4, −6.5 ± 3.2 and −5.2 ± 3.9 %, corresponding to the three hop distances noted above; n = 5 or 6 animals). In all cases, proximal and central segments shortened more than the distal segment (P < 0.05). For all segments, the peak magnitude of shortening strain increased as hop distance increased from one to two BLs (P < 0.05), but did not increase beyond two BLs (Fig. 3). In general, proximal and central segments shortened similarly, while the distal segment strained differently in pattern and less in magnitude.

In vitro segment strains during fixed-end contractions

During fixed-end contractions, the central segment always shortened more than the distal segment, regardless of stimulation pattern (Fig. 4). When stimulated with a twitch pulse, the central segment strained (−9.0 ± 4.5 %) more than the distal segment (−2.3 ± 2.1 %; n = 7; P < 0.05; Fig. 4A). Similarly, when stimulated with a supra-maximal burst (200 Hz for 50 ms), the central segment strained (−23.9 ± 10.0 %) more than the distal segment (−9.5 ± 5.7 %; n = 7; P < 0.05; Fig. 4B). Both central and distal segments shortened less during twitch contractions than during contractions stimulated with a supra-maximal burst (P < 0.05).

In vitro segment strains during passive cyclical length changes

The pattern of central and distal segment strains closely followed the amplitude and pattern of whole muscle- tendon strain during imposed passive cyclical length changes (Figs 5A and 6). Strain magnitude did not differ between segments under this condition. Both central and distal segments shortened similarly (central strain −4.0 ± 2.4 %; distal strain −3.9 ± 0.8 %; P = 0.89) and also lengthened similarly when passively cycled (central strain 6.7 ± 1.0 %; distal strain 6.5 ± 2.6 %; P = 0.87). The matching of the segments with each other and with the whole MT unit during passive oscillations indicates that the measured length changes are not artefactual.

Figure 6. Peak shortening strain as a function of stimulation phase.

Figure 6

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Peak shortening strain increased with stimulation phase, where phase 0 and 50 represent stimulation occurring at mid-shortening and mid-lengthening, respectively. * Statistically different strains between central and distal segments (n = 6 or 7 for each conditions; paired t tests; P < 0.05). □ central segment, ▪ distal segment.

In vitro segment strains during active, cyclical length changes

In contrast to passive length changes, the central segment shortened more than the distal segment at all stimulation phases during imposed cyclical length changes (Fig. 5 and Fig. 6). The muscle fascicle segments always shortened when stimulated, even as the whole MT unit lengthened implying stretching in regions external to the measured segments, including the tendons (Fig. 5B–E). Heterogeneity within the measured segments however, was most dramatic when the muscle was stimulated at mid-lengthening (phase 50; Fig. 5D). As for other stimulation phases, stimulation at mid-lengthening caused both segments to shorten immediately, even though the MT unit was still lengthening. Soon thereafter, the distal segment lengthened to follow the trajectory of the whole MT unit, while the central segment continued to shorten. As the muscle–tendon unit and distal segment began to shorten, the central segment then lengthened (Fig. 5D). For both segments, peak shortening was maximal when stimulation occurred at mid-shortening (phase 0; central strain −29.0 ± 5.5 %; distal strain −14.8 ± 7.4 %; n = 6; Fig. 5B and Fig. 6). In contrast, peak shortening was minimal when stimulation occurred at mid-lengthening (phase 50; central strain −17.9 ± 9.4 %; distal strain −8.1 ± 3.7 %). The magnitude of peak shortening differed significantly between central and distal segments at all stimulation phases (P < 0.05), except at phase 75 (P = 0.052; Fig. 6).

In vitro strain heterogeneity during cyclical length changes

Percentage heterogeneity, or the percentage of segment strain in opposing directions during a single cycle, significantly increased relative to the passive condition at all stimulation phases, except phase 0, when the muscle was stimulated during mid-shortening (Fig. 7). Under passive conditions, heterogeneous patterns of strain between central and distal segments occurred during 1.8 ± 0.7 % of the cycle (n = 7). When stimulated at mid-shortening (phase 0), percentage heterogeneity (7.4 ± 9.0 %; n = 6) did not differ compared with passive oscillations (P = 0.12). When stimulated at any other phase during the cycle, however, percentage heterogeneity increased significantly (P < 0.05) relative to the passive condition and was maximal when the muscle was stimulated during mid-lengthening (phase 50; 34.0 ± 9.2 %; n = 7; Figs 5 and 7).

DISCUSSION

In vivo and in vitro heterogeneity of segment strain

Our results presented here clearly demonstrate that segment strain heterogeneity, where one fascicle segment lengthens while an adjacent segment shortens, occurs in vivo during hopping as well as in vitro during cyclical contractions in the semimembranosus muscle of the toad, B. americanus (Figs 3 and 7). Although the patterns of heterogeneity we observe are not identical between in vivo and in vitro conditions, both conditions show distinct heterogeneity of segment strain along the SM muscle. These results therefore reject the hypothesis that fascicle segments strain homogeneously along the muscle. We find that the central segment of the toad SM muscle shortens in vivo, while the distal segment lengthens (Fig. 2). During hopping, the proximal and central segments of the toad SM muscle shorten similarly in vivo, while the distal segment first lengthens, then shortens less than the other two segments (Figs 2 and 3). Furthermore, during in vitro cyclical contractions of the whole SM muscle-tendon unit, heterogeneity between central and distal segments occurs during up to 34 % of the (sinusoidal) strain cycle (Fig. 7).

In vivo muscle activity and shortening patterns of the semimembranosus muscle

Despite the patterns of segment strain heterogeneity we observe here, the in vivo activation pattern and magnitude of shortening strain observed here in the toad SM muscle approximates that seen in the SM muscles of other anura during jumping. In other frog species, the SM muscle is activated 18–28 ms before shortening begins (Rana pipiens, Lutz & Rome, 1994; Rana catesbeiana, Olson & Marsh, 1998; Bufo marinus, Gillis & Biewener, 2000). In general, the activity pattern for the SM muscle of B. americanus started later, or closer to the onset of muscle shortening, than in these other species. Since the timing of activation was similar between all three segments, the heterogeneous behaviour of the segments we observe cannot be caused by an asynchronous or time-delayed activation of the segments along the muscle. Furthermore, the peak in vivo strain in Bufo americanus (−17 % at 2.7 BLs) is slightly less than that seen during jumping in Bufo marinus (−25 %; 19 - 24 °C; Gillis & Biewener, 2000), Rana catesbeiana (−26 %; 20 °C; Olson & Marsh, 1998) and Rana pipiens (−22 %; 25 °C; Lutz & Rome, 1994). The difference in shortening strains may possibly be due to differences in jump distances, since B. americanus (range: 4 - 22 cm; average 12 cm) jumped shorter distances than B. marinus (range: 15–45 cm; Gillis & Biewener, 2000), R. catesbeiana (range: 15 - 120 cm; Olson & Marsh, 1998), and R. pipiens (average 67 cm; Lutz & Rome, 1994). These previously recorded strain values in other species of frogs, however, are more comparable with the shortening measured in the central segment when stimulated with supramaximal bursts during fixed-end contractions (−24 %; Fig. 4) and cyclical contractions when stimulation occurred at the beginning of shortening (strain −24 % at phase 75; Fig. 5E).

Architectural influences on segment strain heterogeneity in whole muscles

The construction of single fibres into a whole muscle may contribute to much of the difference between segment strain seen in single fibres and whole muscles (Buchtal & Kaiser, 1949; Casella, 1950). The SM muscle of the toad is a parallel-fibred muscle with a broader, proximal origin and a narrower, distal insertion. This tapered shape of the muscle may indicate asymmetries of the perimysium, or collagen, that hold the multiple fibres together within the muscle (see Methods; Duellman & Trueb, 1986). Moreover, fibres that do not span the entire length of the muscle insert into an internal aponeurosis located at the distal portion of the semimembranosus muscle (yellow line in Fig. 1B). Unfortunately, the mechanical effects of variable collagen content or asymmetrical architecture of a whole muscle on strain heterogeneity remain largely unknown (Purslow & Trotter, 1994). However, the possibility exists that variable stiffness or compliance in the perimysium or internal aponeurosis along a muscle influences the strain patterns of the individual segments along that muscle. The high degree of heterogeneity occurring at the tapering, or distal end of the toad SM muscle suggests that muscles which taper at both ends may experience symmetrical patterns of heterogeneity at both ends of the muscle. Although the collagenous matrix surrounding fibres may be a large determinant of the structural mechanics of the whole muscle, it possibly plays little role in the mechanics of single fibres except at the myotendinous junctions (Huijing, 1999, Monti et al. 1999; Kawakami & Lieber, 2000).

The mechanical properties of aponeurosis and tendon have been shown in many recent studies to play a major role in the strain patterns of the muscles in vivo during locomotion (Roberts et al. 1997; Biewener et al. 1998; Fukunaga et al. 2001). During terrestrial locomotion on the level, the compliance in the tendon and aponeurosis allows the leg muscle fibres in turkeys, wallabies and humans to contract nearly isometrically, even though the whole muscle-tendon unit may undergo substantial length change. The presence of an internal aponeurosis has also been argued to underlie the non-uniform in vivo strain behaviour observed during voluntary contractions of the human biceps brachii (Pappas et al. 2002). Thus, in addition to influencing the basic functional properties of muscle (Biewener & Roberts, 2000; Lieber & Friden, 2000) and lateral force transmission between fibres (Monti et al. 1999; Huijing, 1999), muscle architecture may also influence the mechanical strain regime of longitudinal fascicle segments along a muscle.

Variable protein expression along a muscle fibre as sources of strain heterogeneity

In addition to regional differences in fibre architecture and connective tissue matrix, fibre type regionalisation along the proximo-distal axis may also contribute to generating segment strain heterogeneity in muscle (Wang & Kernell, 2001). Although neuromuscular, cross-sectional compartmentalisation has been known to be common in mammalian leg muscles (Ariano et al. 1973; English & Weeks, 1984), proximo-distal regionalisation in fibre type is a newer finding. Unfortunately, proximo-distal fibre type regionalisation is difficult to predict for untested muscles because this regionalisation is highly variable among leg muscles within an animal and among the three mammalian species examined (Wang & Kernell, 2001). This proximo-distal regionalisation most likely results from variable protein expression along a fibre (Wang & Kernell, 2001).

Isoform variation in muscle proteins has also been shown to occur along the muscle fibre (Edman et al. 1988, Lutz et al. 2002; Wilkins et al. 2001) and may thus contribute to segment strain heterogeneity. Specifically, myosin heavy chain (MHC) isoforms have been correlated with differing segment force-velocity characteristics along the length of isolated muscle fibres (Edman et al. 1988; for reviews, see Schiaffino & Reggiani, 1996 and Bottinelli & Reggiani, 2000). Although variation in titin isoforms along a fibre has not been examined, these isoforms can exhibit different mechanical properties with differentially spliced immunoglobin (Ig)-like domains along the titin protein (Watanabe et al. 2002). This variation in MHC, or possibly titin, expression can occur because each myonucleus regulates gene expression only within its own surrounding portion of a fibre (Pavlath et al. 1989).

Implications for muscle modelling

In vivo and in vitro heterogeneity of segment strain suggests that in-series sarcomeres along a muscle operate on different portions of their force–length and force– velocity relationships simultaneously during a contraction. Since the strain pattern of a sarcomere or a muscle directly determines muscle force and power output, future mechanical models of muscle dynamics may need to examine the sensitivity of sarcomere strain heterogeneity on the output of the model. Muscle models could incorporate variable architecture or variable fibre type (i.e. force–velocity properties) and titin mechanics (i.e. visco-elastic properties of the contractile element) along the muscle to examine their effects on segment strain heterogeneity and its subsequent effects on muscle force output. However, additional examinations of the effects of architecture on force output and the characteristics of the elastic structures between fibres are also required in order to improve the input parameters for muscle models. Nevertheless, it seems clear that in vivo measurements of heterogeneous segment strain in skeletal muscle indicate that the generation of muscle force may be more complicated than that described by a single force–length– velocity relationship.

Implications for the neural control of movement

Strain heterogeneity within a pool of recruited muscle fibres indicates that a single neural signal can result in diversity of mechanical power output within a muscle. Past work has clearly shown that the stimulation pattern to a muscle influences its mechanical output (Josephson, 1985, 1999; Stevens, 1988). Differences in strain patterns between adjacent segments, such as those observed here along the toad SM muscle (Fig. 5), indicate that muscle work and power output from segments of a single fascicle may also differ (Fig. 8). When analysed using the ‘work loop’ technique (Josephson, 1985), the work generated or absorbed by the different segments within the toad SM muscle also differs from that observed for the whole muscle-tendon unit. It is important to note that a key limitation of our understanding and approach is that the work patterns depicted in Fig. 8 rely on an external measure of whole muscle-tendon force linked to localised measures of fascicle strain. Actual local forces generated by these segments may, in fact, differ in magnitude and pattern from external forces transmitted by the whole muscle-tendon. Thus, their localised work-loop patterns may be different from those depicted in Fig. 8. Cross-connections between fibres may allow in-series muscle segments to transmit different forces. Nevertheless, the manner in which localised regions of strain heterogeneity within a muscle affect regional patterns of muscle force and work, and how these are integrated to yield whole muscle performance, remains largely unknown. To the extent that heterogeneous patterns of strain, force and work do occur within muscles, they will have important implications for motor recruitment in relation to whole muscle architecture.

Conclusion

In an attempt to begin to integrate studies of sarcomeres and whole muscles, we examined the behaviour of muscle segments along a whole muscle under in vivo and in vitro conditions. In vivo and in vitro measurements of segment strain both show in-series heterogeneity in the semimembranosus muscle of the toad, B. americanus. Our results are similar to those found in single fibres (Edman & Reggiani, 1984; Mutungi & Ranatunga, 2000). However, the differences we observe between strain heterogeneity in single fibres versus the toad SM muscle examined here indicate that the architectural construction of individual fibres into an asymmetrical whole muscle can influence the strain pattern during dynamic contractions, despite its seemingly gross anatomical simplicity. Differential expression of protein isoforms (i.e. MHC and titin) may also contribute to heterogeneity. Segment strain heterogeneity can result in variable mechanical work output along the fibre and also demonstrates that sarcomeres along the fibres simultaneously operate at different portions of their force–length and force–velocity relations. Our results also have implications for the motor control strategies used by animals to co-ordinate muscle forces and generate movement. Just as a given neural signal can result in power production, isometric force generation or energy absorption amongst different locomotory muscles (Dickinson et al. 2000; Ahn & Full, 2002), a given motor command can also result in variable behaviour between segments within a single muscle. Understanding regional differences within muscles under in vivo and in vitro conditions is important to integrating our understanding of sarcomere function within individual fibres with the contractile performance of whole muscles during locomotion.

Acknowledgments

We thank Drs R. K. Josephson and J. G. Malamud for developing the LabView software used to stimulate and control the length of the muscle in vitro (copies available upon request by contacting rkjoseph@uci.edu or aahn@oeb.harvard.edu). We also thank Dr R. L. Marsh for assistance in examining the detailed architecture of the SM muscle and two anonymous reviewers for helpful comments to the manuscript. Funding was provided by NIH F32 AR47741 to A.N.A. and NIH F32 AR08646 to R.J.M.

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