Sustained performance by red and white muscle fibres from the dogfish Scyliorhinus canicula

The contractile performance of fast-twitch white fibres and slow-twitch red fibres that make up the myotomal muscle of fish have been extensively studied from the point of view of their role in swimming of intact fish and the underlying biochemical and energetic processes. However, most of these observations were made during a few cycles of movement and stimulation of fully rested fibres. This strategy is appropriate for the white muscle fibres, which are known from studies of swimming fish, to be used during sprint or burst performance. But since red fibres power sustained performance, the first few contractions can only tell us the beginning of their story. Here we report an investigation of the power output of muscle fibres from the dogfish, Scyliorhinus canicula, when performing long series of contractions with sinusoidal movement. Corresponding series of isometric tetani were also done to see how movement influences progressive changes in mechanical performance.

We found as expected that red fibres maintained performance better than white fibres. This was partly due to force during stimulation declining less in red fibres. The other factor was a striking difference in how relaxation changed during the series of contractions. In white fibres the time required to relax to 10% of maximum force increased progressively, whereas with red fibres progressively less time was required for force to relax to 10% of maximum force.

Experiments were done on bundles of either red or white fibres from the myotomal muscle of dogfish, Scyliorhinus canicula (Linnaeus). The fish were supplied by the Marine Biological Association (Plymouth, UK) and kept in circulating artificial sea water (density 1.26 g ml⁻¹) at about 12°C for at least several days before use. The fish were killed in accordance with Schedule 1 of the UK Animals (Scientific Procedures) Act 1986. The fibre bundles were dissected under elasmobranch saline, which contained (in mmol l⁻¹): NaCl, 292; KCl, 3.2; CaCl₂, 5.0; MgSO₄, 1.0; Na₂SO₄, 1.6; Hepes, 5.0; urea, 483; and tubocurarine, 1.5 mg l⁻¹. The saline was equilibrated with oxygen. The same saline was used during the experiments at 12°C. All fibre bundles were dissected from the same region along the length of the fish, 0–4 cm caudal to the end of the body cavity. The myosepta at the ends of the fibre bundle were held in aluminium foil T-shaped clips. The preparation was mounted in a Perspex bath between a combined motor and force transducer (Cambridge Technology, Inc., Watertown, MA, USA; model 300B) and a fixed hook. The saline was circulated continuously through the bath and was maintained at approximately 12°C. The muscle fibre bundle was electrically stimulated (Digitimer, MultiStim System-D330; Welwyn Garden City, Herts, UK). A program written in TestPoint (Keithley Instruments, Theale, Berks, UK) controlled stimulation and motor arm position, and recorded (at 1 kHz) force, length and stimulation.

The relationship between force and stimulus strength during tetanic stimulation was investigated in each fibre bundle to establish the supra-maximal stimulus strength, which was used in the rest of the experiment. The length–tension relationship was investigated in each fibre bundle to identify the fibre length (L₀) at which tetanic force was maximal.

In preliminary experiments we established the conditions of movement and stimulation that elicited maximum power from most red fibre bundles during the first cycles in a series of sinusoidal movements. These conditions were: frequency of movement 0.75 Hz, peak to peak amplitude 1 mm (about 12% L₀) centred on L₀, and stimulation lasting for 33% of each cycle of movement (stimulus duty cycle=33%) with shortening starting 0.107 s after the start of stimulation (phase=−8%). Stimulus frequency within the tetanus was 30 Hz (interval between stimuli=0.0333 s). The same pattern of movement and stimulation was imposed on bundles of white fibres, so that performance with identical contraction conditions could be compared.

These ‘initial maximum power conditions’ were applied to each fibre bundle during a series of contractions lasting up to 10 min (450 cycles of movement and stimulation) to examine how well power output was maintained. Records of force were also made without stimulation, and the resting force record was subtracted from the total force during stimulation to give active force. The maximum resting force was produced when the fibre bundles were at maximum length and amounted to 25.7±2.8 mN mm⁻² (mean ± s.e.m.; 14 red fibre bundles) and 13.3±4.3 mN mm⁻² (five white fibre bundles). At the time when active force reached its maximum value, the resting force was 0.171±0.014 (14 red fibre bundles) and 0.069±0.022 (five white fibre bundles) of total force. The higher resting force in red fibre bundles was consistent with the larger amount of connective tissue between the fibres, which was obvious during dissection.

Power was evaluated as the work/duration of movement, where work is the integral of active force with respect to length change. We report the power output from the muscle based on active force during the shortening part of the cycle, power input to the muscle based on the active force during the stretch part of the cycle, and net power which is power output minus power input.

In experiments on isometric contractions this same stimulus pattern was used (cycle frequency 0.75 Hz and stimulus duty cycle 33%) for up to 450 cycles, but muscle length was kept constant.

Fig. 1 illustrates how relaxation was measured. As an overall measure of relaxation, we report the force decline from the start of relaxation to 10% of the maximum force and the time required for this decline in force. At the start of each series of contractions, relaxation included a period of time during which force declined approximately linearly, referred to here as the linear phase (Curtin and Edman, 1989). This phase was followed by a highly non-linear decline in force. We report the force decline in the linear phase and the duration of the linear phase. The start of relaxation was defined as either 0.472 s after the first stimulus pulse (0.472 s=time of last stimulus pulse + the time corresponding to one stimulus interval=0.4387+0.0333 s) or the time at which force stopped increasing, which ever occurred later. The time corresponding to the end of the linear phase was found by fitting one or two lines to the observed force during relaxation using Excel Solver. The fit included points between the start of relaxation and an estimate, adjusted as appropriate, of the end of the linear phase. The end of the linear phase was identified as the time at which the residuals (observed force minus fitted value) became greater than the noise in the observations as illustrated in Fig. 1.

Force decrease and the time required for this force decrease are reported for the linear phase and for overall relaxation. The average rates of change (force decrease/time required) for the linear phase and for overall relaxation are reported with the average rates expressed relative to the corresponding value in the first contraction of the series.

During the series of contractions of white fibres progressively less relaxation (decline in force) occurred before the stretch phase of the movement cycle. When the extent of relaxation before the stretch phase of the movement cycle was ‘small’, no attempt was made to measure it. ‘Small’ is defined here as less than 20% of the maximum force. The record labelled 36th in Fig. 2F is an example of such a record where there was very little decline of force between the end of stimulation and the start of the stretch.

At the end of the experiment, the fibre bundle length was set to L₀. Red fibre bundles were stained with Evans Blue to distinguish intact from damaged fibres (Lou et al., 2002) and fixed in formalin. White fibres were fixed in alcohol which made damaged fibres appear opaque. Damaged fibres, myosepta and other non-fibre tissue were removed and discarded. Fibre length at L₀ was measured, and fibres were dried and weighed on a Cahn microbalance.

Power is expressed relative to the mass of the fibres to take account of differences in performance due to differences in muscle size. The average wet masses were 3.621±0.366 mg for the 14 red fibre bundles, and 2.225±0.53 mg for the five white fibre bundles used in the experiments with sinusoidal movement. Isometric force is expressed relative to CSA to take account of differences in performance due to differences in muscle size. The average CSA values were 0.357±0.007 mm² for the four red fibre bundles and 0.241±0.043 mm² for the five white fibre bundles used in the isometric experiments.

Unless stated otherwise values reported are means ± 1 s.e.m.

Fig. 2 shows results for contractions with sinusoidal movement of red (A–D) and white (E–H) fibre bundles. The records of force produced by a bundle of red fibres during the 1st, 23rd and 448th cycle of the series of contractions are shown superimposed in Fig. 2B. The force during the shortening part of the cycle declined during the series, and correspondingly the power during shortening fell to about 50% of its initial value as shown by the open symbols in Fig. 2D. By contrast, during the stretch part of the cycle the force and power remained low for the entire series of cycles (solid symbols in Fig. 2D). As will be described below, relaxation (the decline in force after the end of stimulation) to a low value was always complete and required less time as the series progressed. The changes in force during the series are also clear in the work loops (force versus length change) shown in Fig. 2C, where the arrow shows the direction of the loop. For each cycle of movement the area enclosed within the loop is the net work done by the muscle fibres. The average net power during each cycle of movement is the product of the net work and the frequency of movement. Fig. 3A,B shows the average net power per cycle of movement for the 14 red fibre bundles. There were three phases in the time-course of power decline during the series: (1) a relatively rapid decline in power output down to about 60% of the maximum power between the 1st and approximately 30th cycles; (2) next, there was a pause in the decline or a small rise in power output, which varied among fibre bundles, between approximately the 30th and 100th cycles, and (3) the final phase was a very gradual decline to a steady state power output during the rest of the series.

The same pattern of movement and stimulation (Fig. 2E) was used with bundles of white fibres to allow comparison of performance under the same conditions. Force records from the 1st, 28th and 36th cycle are shown superimposed in Fig. 2F. The force during stimulation and shortening decreased as happened with the red fibres, and thus the power during shortening decreased as shown by the open symbols in Fig. 2H. However, unlike the red fibres, relaxation of force to a low value took progressively more time as the series progressed (Fig. 2F) as will be described in more detail below. As a consequence of the prolongation of relaxation, force during the stretch part of the cycle increased during the series of contractions (Fig. 2F,G). Thus the power during the stretch part of the cycle increased during the series and eventually matched the power during the shortening part of the cycle (Fig. 2H). The superimposed work loops (Fig. 2G) show that both of these effects contributed to making the area enclosed by the loops smaller; progressively less force was produced during shortening, and in addition more force was exerted during stretch and relaxation. Thus this particular white fibre bundle produced very little net work or power by the 36th cycle. Fig. 3A,B shows how the average net power output of five white fibres bundles changed during the series of contractions. The net power declined relatively slowly over the course of about 20 cycles, then dropped more steeply, reaching zero in every fibre bundle by 50 cycles.

The power output results for the 14 red and five white fibre bundles are compared in Fig. 3A,B. In Fig. 3A the net power is expressed in units of W kg⁻¹ (wet mass on the left axis and dry mass on the right axis). Initially the net power output by white fibres is about three times greater than that of the red fibres, but it declines much more quickly, and by 35 cycles the net power output of the red fibres is greater than that of the white fibres. In Fig. 3B the net power in each cycle is expressed as a fraction of the maximum power output by that particular muscle. This graph emphasizes how much better the red fibres are at maintaining what power they can produce. Red fibres were still producing more than half of their maximum net power after 448 cycles of movement, about 10 min of continuous performance. The net power output by every white fibre bundle had declined to zero by between 25 and 50 cycles, which is at most about a minute of performance.

To get some insight into how much change in muscle length contributes to the changes in mechanical performance, isometric experiments were done on a separate group of muscle fibre bundles. In these experiments length was kept constant while applying the same stimulation pattern as described above (duty cycle 33%, 0.44 s tetanus in every 1.33-s cycle).

Fig. 4 shows force records for red (A–C) and white (D–F) fibre bundles during isometric contractions. The stimulation pattern is shown in the top panels. Fig. 3B shows superimposed force records for a red fibres bundle for the 1st, 28th, 58th and 448th tetanus and shows that the maximum tetanic force declines substantially as the series progresses. Fig. 4C shows the corresponding records with force expressed relative to the maximum force in the tetanus. The rate of rise of relative force at the start of stimulation did not change much during the series. By contrast, after the end of stimulation, force relaxed to a low value sooner as the series of contractions progressed.

The maximum isometric force produced by white fibres declined substantially as the series progressed as can be seen from the superimposed records of force in Fig. 4E; in this respect, red and white fibres behaved similarly. The kinetics of force rise and relaxation of white fibres shown in Fig. 4F are, however, very different from those of red fibres. Relative force rises slowest in the 1st contraction of the series; this occurred in four of the five white fibre bundles. Most strikingly, relaxation of white fibres takes progressively longer during the series, which is opposite to the change that occurred in red fibres.

The isometric forces for all the red and white fibres are summarized and compared in Fig. 5. Specific force (peak force/cross-sectional area) was greater for white fibres as shown in Fig. 5A. In Fig. 5B the forces are expressed relative to that in the first tetanus of the series which shows that the time-course of the decline of peak isometric force was similar for red and white fibres during the first 50 tetani.

As mentioned earlier, relaxation, the decline in force after the end of stimulation, changed for both red fibres and white fibres during the series of contractions under both mechanical conditions: with sinusoidal movement and isometric conditions. Figs 6 and 7 show example records of force for the first contraction and a contraction late in the series. The accompanying graphs summarize the decreases in force during the initial linear phase of relaxation and the durations of this phase, along with overall measures of relaxation: extents of force decrease from the start of relaxation to 10% of maximum force and the times required. For the red fibres (Fig. 6), the extent of force decrease during the linear phase increased during the contraction series and this change was larger in the contractions with sinusoidal movement than with isometric conditions. However, the most striking change was the decrease in the time required for overall relaxation (force decline from the start of relaxation to 10% of maximum force), which occurred for both mechanical conditions. The time for overall relaxation of red fibres dropped early in the series so that with sinusoidal movement, relaxation was almost complete (only 10% of maximum force remaining) by the time the stretch phase of the movement cycle started. It should be noted that a similar pattern was seen for red fibres under isometric conditions, showing that the decrease in the time required for overall relaxation does not require active shortening by the fibre. For white fibres (Fig. 7), the most striking change during the series of contractions was the increase in the time required for both the linear phase of relaxation and for relaxation overall. After 10 contractions with sinusoidal movement there was an increasing amount of force remaining when the stretch phase of the movement cycle started. The increases in the time required for relaxation were also seen in the isometric series.

Fig. 8 shows the average rates of force change during the linear phase (A) and during overall relaxation (B; start of relaxation to 10% maximum force). Here they are expressed relative to the corresponding value for the first contraction in the series to emphasize the progressive changes during the series of contractions. For the red fibres, both rates (linear and overall) get faster, whereas for white fibres, both rates get slower during the series of contractions with movement and during the series of isometric contractions.

The results confirm that rested white, fast-twitch fibres can produce more power and more isometric force than red, slow-twitch fibres. This is consistent with earlier studies of rested fibres from dogfish and a number of animals under isometric conditions and during movement, including controlled-velocity or controlled-force conditions (e.g. Altringham and Johnston, 1982; Bone et al., 1986; Curtin and Woledge, 1988; Barclay et al., 1993; Lou et al., 2002) and during sinusoidal movement (e.g. Curtin and Woledge, 1993a; Curtin and Woledge, 1993b; Ellerby et al., 2001a; Ellerby et al., 2001b). It is well established for other animals that the difference between contractile performances of rested fast- and slow-twitch fibres are highly correlated with the identity of their myosin isoform content (Schiaffino and Reggiani, 1994).

We find a bigger difference in power output than in isometric force output when red and white fibres are compared. This can be explained by the fact that power reflects filament sliding velocity as well as the capacity to produce force, and the maximum velocity of shortening of white fibres is about twice that of red fibres (Lou et al., 2002).

Our main aim was to extend the comparison of dogfish fast- and slow-twitch fibres from single contractions to their performance during long series of contractions. We found that for a series of about 50 cycles of contraction for which we have data, the decline in isometric force production was about the same in red and white fibres (Fig. 5), but the decline in power output was much greater for white than red fibres (Fig. 3).

We discuss first the force production during stimulation. In white fibres the capacity to produce force during shortening declines more quickly than the isometric force (compare Fig. 2F and Fig. 4E). The difference between the power series and isometric series for white fibres presumably reflects the higher energetic cost of power output than the production of isometric force by white fibres (Curtin and Woledge, 1991). In red fibres the capacity to produce force during shortening and during isometric contraction changes similarly (compare Fig. 2B and Fig. 4B). A number of factors contribute to the difference between white and red fibres. There is a greater mismatch between ATP demand and supply in white fibres; the volume fraction of mitochondria within the white fibres is only 4% of that in red fibres (Bone et al., 1986). Factors that affect the series with sinusoidal movement include the higher initial power output and lower efficiency of white fibres (Curtin and Woledge, 1993a; Curtin and Woledge, 1993b), which means that each unit of work has a higher ATP cost in white fibres than red fibres.

Another and novel difference between white and red fibres is the way relaxation (force decline after the end of stimulation) changes during the series of contractions: white fibres relax more slowly whereas red fibres relax more quickly as the series of contractions progresses (Fig. 8). This behaviour is seen with both types of contractions. In the contractions with sinusoidal movement, relaxation occurs largely during shortening. Shortening is known to increase the rate of relaxation (Caputo et al., 1994; Lou et al., 1998). However, shortening is not required for the enhanced rate of relaxation of red fibres; it also occurs in the series of isometric contractions of red fibres.

Slowing of relaxation is a characteristic feature of fatigue of fast, white fibres and has been extensively documented (Edwards et al., 1975; Dawson et al., 1980; Gillis, 1985; Curtin, 1986; Curtin and Edman, 1989; Allen et al., 1995; Allen et al., 2008). It has the consequence for isometric conditions that losses in the total force–time integral due to declining maximum force can be compensated for to some extent by gains due to more force during relaxation. However, during contractions that involve shortening and lengthening, mimicking those during locomotion, the slowing of relaxation of the white fibres curtails net power output for a complete cycle of movement (Fig. 2G,H and Fig. 3A,B). Similar prolongation of relaxation and decline in net power output has been observed in white fibre bundles from frog muscle performing long series of contractions with sinusoidal movement (Syme and Tonks, 2004).

We have found that in contrast to white fibres, the relaxation of red fibres becomes faster during a series of contractions (Fig. 8). To our knowledge this is the first demonstration of relaxation becoming faster as force declines (fatigue). Experiments on mouse soleus muscle using a protocol with sinusoidal movement and stimulus parameters similar to those used here resulted in a significant slowing of relaxation that reduced power output considerably (Askew et al., 1997). Other experiments on mammalian fibres have shown that fast- and slow-twitch fibre relaxation does behave differently during a series of fatiguing contractions, with fast fibre relaxation slowing and soleus slow-twitch fibre relaxation remaining unchanged (Bruton et al., 2003; Lunde et al., 2006). However, the behaviour of red fibres from dogfish is more extreme than this in that relaxation actually gets faster.

The progressively faster relaxation of red fibres during the series of contractions is a significant factor allowing power output to be sustained by the red fibres during long series of contractions. This effect is largely responsible for red fibres eventually out-performing white fibres so that specific power produced by red fibres (watts per unit mass of muscle) is greater than that of the white fibres after about 50 cycles of contraction.

Changes in force during both stimulation and relaxation most likely reflect changes in metabolite concentrations arising directly from contraction. ATP, ADP, inorganic phosphate (P_i) and H⁺ participate in the actomyosin cycle and thus can be expected to affect force.

Although changes in hydrogen ion concentration ([H⁺]) are moderated by intracellular buffers and mechanisms such as facilitated diffusion of lactate out of the fibre, intracellular acidification is often implicated in the force reduction during fatiguing protocols. However, we have previously shown that there is no acidification in white fibres from dogfish in a series of contractions (Curtin et al., 1997). This phosphorus nuclear magnetic resonance spectroscopy study of series of contractions of white fibres showed that intracellular pH changes in the alkaline direction and recovers without ever becoming more acid than the resting value. Thus, mechanisms other than intracellular acidification must be responsible for the reduction in force and power reported here for white fibres.

The creatine kinase reaction keeps the [ATP] and [ADP] relatively constant until the phosphocreatine supply is nearly exhausted. Thus [P_i] is likely to change more than other metabolites during the contraction protocols used here and P_i probably has a major part in affecting the behaviour of white and red fibres reported. In addition to its involvement in the crossbridge cycle, P_i has a number of effects on processes involved in activation by Ca²⁺ (reviewed by Allen et al., 2008). For example, increasing [P_i] (1) reduces the Ca²⁺ sensitivity of the filaments, in other words reduces force produced at each free Ca²⁺ concentration, (2) reduces the rate of Ca²⁺ reuptake by the sarcoplasmic reticulum (SR), and (3) reduces the rate of Ca²⁺ release (amount per action potential) from the SR via the ryanodine receptor because of CaP_i precipitation within the SR.

Although increasing [P_i] has been shown to reduce the force produced by maximally Ca²⁺-activated permeabilized fibres from many muscles and species (reviewed by Cooke, 2007), there are two reasons that this particular mechanism has only a small role here. First, we have recently shown that when activated at normal physiological temperature of 12°C, force produced by maximally activated permeabilized white fibres from dogfish is little affected by [P_i] in the range 5 to 25 mmol l⁻¹ (Holohan et al., 2007; Holohan et al., 2010). Second, with the relatively brief tetani used here, the rested white fibres barely reach a plateau of force, and red fibres certainly do not, indicating that the fibres are not maximally Ca²⁺-activated. Therefore it seems more probable that force declines in the first several contractions in the series because the increase in [P_i] reduces the Ca²⁺ sensitivity of the filaments (Millar and Homsher, 1990; Martyn and Gordon, 1992), such that progressively less force is produced at a particular sub-maximal concentration of Ca²⁺ around the filaments.

Later in both the isometric series and in the power experiments where the mechanical performance of the red and white fibres diverge, the [P_i] probably also diverges with P_i continuing to accumulate in white fibres, but stabilizing in the red fibres. We have previously shown that recovery processes that return P_i to phosphocreatine are very slow in white fibres from dogfish, requiring approximately 1 h at 12°C (Curtin et al., 1997; Lou et al., 2000). Furthermore, mitochondria constitute only about 1% of white fibre volume but 25% of red fibre volume (Bone et al., 1986). Thus it seems likely that [P_i] increases in both fibre types during the series and continues increasing in white fibres, but in red fibres reaches an almost constant level because production of Pi from phosphocreatine and ATP hydrolysis is balanced by the use of P_i to rebuild these metabolites.

It is well established from studies of fast muscle fibres from frog that during relaxation there are longitudinal movements of the striations or marked fibre segments, due to shortening of some sarcomeres and lengthening of others (Cleworth and Edman, 1969; Huxley and Simmons, 1970; Cleworth and Edman, 1972; Huxley and Simmons, 1973; Curtin and Edman, 1989). These movements start at the end of the linear phase of relaxation. Furthermore interventions, including fatigue, that delay the end of this phase also reduce the rate of force decline in this phase (Curtin and Edman, 1989). We show here that the pattern of relaxation of the dogfish white fibres is similar to that of frog in that the linear part lasts longer (Fig. 7) and is slower (Fig. 8) in the fatigued state. It thus seems likely that sarcomere movements also occur in the white dogfish fibres and are delayed during the series of contraction; the delay being due to a combination of spatially more uniform free Ca²⁺ and more uniform crossbridge behaviour. Allen and colleagues have recently reviewed the processes involved in the well documented slowing of relaxation, which include changes in Ca²⁺ release and uptake as well as myofilament reactions (Allen et al., 2008). It could be that sarcomere movements also occur in red fibres and that they start earlier as the series of contractions progresses. Evidence about sarcomere uniformity in red fibres is needed to clarify this point.

What mechanisms might be responsible for the white and red fibres of dogfish behaving in the opposite manner: white relaxing more slowly and red more quickly as the series of contraction progresses? The opposite changes in relaxation of red and white fibres could be due to P_i accumulation reducing the Ca²⁺ sensitivity of force in both fibres types, coupled with a difference in Ca²⁺ binding to parvalbumin. Parvalbumin (PA) is a small molecular-mass Ca²⁺-binding protein that can enhance the rate of relaxation (Gillis, 1985). There is an increasing body of evidence for its relevance to relaxation of teleost fish muscle fibres. For example, it is present in high concentration in the super-fast muscle of toadfish swim bladder and a strong case has been made for its functional role in this muscle (Rome, 2006). In addition, recent studies have shown that in myotomal muscle of other fish species PA content and isoform composition are closely related to relaxation kinetics (Brownridge et al., 2009). They vary together during development (Coughlin et al., 2007), and they vary together in a systematic way along the length of the fish (Wilwert et al., 2006).

Of particular relevance to our results is the fact that relaxation slows as PA becomes progressively saturated with Ca²⁺ during tetanic contraction (Hou et al., 1991). We know that the white fibres of dogfish contain PA (J. A. Rall, personal communication). Thus when contractions are repeated as in our protocol, the PA binding sites in white fibres become saturated, and relaxation slows as the amount of Ca²⁺-binding by PA diminishes. We speculate that red fibres in dogfish contain little or no PA or alternatively, little or no PA with Ca²⁺-binding capacity. This would explain why relaxation does not become slower during the series of contractions. We note that red fibres from a number of teleost fish do contain PA (Wilwert et al., 2006; Coughlin et al., 2007). Our hypothesis requires that the PA content (or Ca²⁺-binding characteristic by relevant PA isoforms) within red muscle fibres is yet another of the many differences between the elasmobranch used in our experiments, dogfish (Scyliorhinus canicula L.), and teleost fish; specific evidence about PA in dogfish red fibres is needed.

If Ca²⁺-binding to PA does not occur in red fibres this could explain why red fibre relaxation does not get slower as the contraction series progresses, but what could cause relaxation of red fibres to get faster during the series of contractions? Most changes in the intracellular environment that occur during fatigue act to slow relaxation (Allen et al., 2008). The only suitable candidate for increasing the rate of relaxation is the reduction in Ca²⁺ sensitivity of the filaments as a result of increasing [P_i]. It seems likely that in our experiments this mechanism accelerates relaxation of the red fibres over the first 20 or so contractions because as P_i increases, progressively less force is produced per free Ca²⁺ both during stimulation and during relaxation. In white fibres the increase in [P_i] similarly reduces the Ca²⁺ sensitivity of the filaments, but a reduction in force in relaxation does not occur because it is counteracted by higher free Ca²⁺, due to progressively less Ca²⁺ binding to PA. Later in the series of contractions by red fibres, when [P_i] has presumably stabilized, isometric force, power and relaxation rate also stabilize.

In conclusion, our most novel finding is that red fibres from dogfish relax more quickly as the series of contractions progresses. The difference in relaxation between white and red muscle fibres is a major factor responsible for the rapid drop in power from white fibres that does not occur in red fibres. Since our contraction protocol with sinusoidal movement mimics in vivo muscle performance the functional implications for the intact animal are obvious. The faster relaxation by red fibres during repeated contractions provides additional insight into why these fibres are used to power prolonged swimming, and why white fibres are unsuitable for this task (Bone, 1966).

This work was supported by the Biotechnology and Biological Sciences Research Council (UK). We thank Allia Syed for her expert technical assistance.

 
• CSA

  muscle cross-sectional area

 
• L₀

  muscle length at which isometric force is maximal

 
• M

  dry mass of muscle

 
• PA

  parvalbumin

 
• P_i

  inorganic phosphate

 
• [P_i]

  inorganic phosphate concentration

 
• SR

  sarcoplasmic reticulum