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

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.

Figure 1. Segment lengths in the semimembranosus muscle

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.

Figure 2. Hopping in the American toad

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).

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

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).

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

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).

Figure 5. Strain during cyclical contractions

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.

Figure 8. Segment work at various stimulation phases

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

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 6. Peak shortening strain as a function of stimulation phase

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.