Force-frequency relationship and potentiation in mammalian skeletal muscle.

Repetitive activation of a skeletal muscle results in potentiation of the twitch contractile response. Incompletely fused tetanic contractions similar to those evoked by voluntary activation may also be potentiated by prior activity. We aimed to investigate the role of stimulation frequency on the enhancement of unfused isometric contractions in rat medial gastrocnemius muscles in situ. Muscles set at optimal length were stimulated via the sciatic nerve with 50-micros duration supramaximal pulses. Trials consisted of 8 s of repetitive trains [5 pulses (quintuplets) 2 times per second or 2 pulses (doublets) 5 times per second] at 20, 40, 50, 60, 70, and 80 Hz. These stimulation frequencies represent a range over which voluntary activation would be expected to occur. When the frequency of stimulation was 20, 50, or 70 Hz, the peak active force (highest tension during a contraction - rest tension) of doublet contractions increased from 2.2 +/- 0.2, 4.1 +/- 0.4, and 4.3 +/- 0.5 to 3.1 +/- 0.3, 5.6 +/- 0.4, and 6.1 +/- 0.7 N, respectively. Corresponding measurements for quintuplet contractions increased from 2.2 +/- 0.2, 6.1 +/- 0.5, and 8.7 +/- 0.7 to 3.2 +/- 0.3, 7.3 +/- 0.6, and 9.0 +/- 0.7 N, respectively. Initial peak active force values were 27 +/- 1 and 61.5 +/- 5% of the maximal (tetanic) force for doublet and quintuplet contractions, respectively, at 80 Hz. With doublets, peak active force increased at all stimulation frequencies. With quintuplets, peak active force increased significantly for frequencies up to 60 Hz. Twitch enhancement at the end of the 8 s of repetitive stimulation was the same regardless of the pattern of stimulation during the 8 s, and twitch peak active force returned to prestimulation values by 5 min. These experiments confirm that activity-dependent potentiation is evident during repeated, incompletely fused tetanic contractions over a broad range of frequencies. This observation suggests that, during voluntary motor unit recruitment, derecruitment or decreased firing frequency would be necessary to achieve a fixed (submaximal) target force during repeated isometric contractions over this time period.     

A rapid algorithm for processing digital physiologic signals: Application to skeletal muscle contractions

Quantifying mechanical output is fundamental to understanding metabolism that fuels muscle contraction and more recent attempts to understand signal transduction and gene regulation. The latter requires long-term application of exercise protocols that result in large amounts of data on muscle performance. The purpose of this study was to develop software for automated quantification of skeletal muscle contractions. An in situ mouse sciatic nerve stimulation model was used to produce contractions over a broad range of frequencies and recorded as both digital and analog signals using a PC analog to digital converter board and chart recorder, respectively. Spectral analysis of the noise components formed the basis for designing a smoothing Chebyshev filter. Algorithms implemented in custom software identified twitches and estimated baseline levels from the smoothed signal. The time to peak force, peak force, tension-time integral, and half-relaxation time were determined for each twitch after baseline correction. The automated results were compared to those obtained from manual measurements of the analog signal. Bland–Altman analysis of the parameters computed from digital signals compared with the corresponding measurements by manual planometry demonstrates the agreement of the digital processing algorithm with planometry over a wide range of twitch characteristics. This program may also be used to study the mechanics of other preparations from isolated muscles, human proximal limb performance, and other digital physiologic signals. Adaptation of the filter function is required to apply the analysis to another experimental apparatus with differing noise characteristics. A full version of the program and instructions for its use are available for download at www.rad.msu.edu.     

Estimation of the error between experimental tetanic force curves of MUs of rat medial gastrocnemius muscle and their models by summation of equal successive contractions

More accurate muscle models require appropriate modelling of individual twitches of motor units (MUs) and their unfused tetanic contractions. It was shown in our previous papers, using a few MUs, that modelling of unfused tetanic force curves by summation of equal twitches is not accurate, especially for slow MUs. The aim of this study was to evaluate this inaccuracy using a statistical number of MUs of the rat medial gastrocnemius muscle (15 of slow, 15 of fast resistant and 15 of fast fatigable type). Tetanic contractions were evoked by trains of 41 stimuli at random interpulse intervals and different mean frequencies, resembling discharge patterns observed during natural muscle activity. The tetanic curves were calculated by the summation of equal twitches according to the respective experimental patterns. The previously described 6-parameter analytical function for twitch modelling was used. Comparisons between the experimental and the modelled curves were made using two coefficients: the fit coefficient and the area coefficient. The errors between modelled and experimental tetanic forces were substantially different between the three MU types. The error was the most significant for slow MUs, which develop much higher forces in real contractions than could be predicted based on the summation of equal twitches, while the smallest error was observed for FF MUs – their recorded tetanic forces were similar to those predicted by modelling. The obtained results indicate the importance of the inclusion of the type-specific non-linearity in the summation of successive twitch-like contractions of MUs in order to increase the reliability of modelling skeletal muscle force.     

Muscle weakness following sustained and rhythmic isometric contractions in man

The effects of sustained and rhythmically performed isometric contractions on electrically evoked twitch and tetanic force generation of the triceps surae have been investigated in 4 healthy male subjects. The isometric contractions were performed separately and on different occasions at 30%, 60% and 100% of the force of maximal voluntary contraction (MVC). The area under the maximal voluntary contraction (MVC) force/time curve during the rhythmic and sustained contractions was the same for each experiment. The results showed that following rhythmic isometric exercise there was a small decrease in low (10 and 20 Hz) and high (40 Hz) frequency tetanic tension which was associated with % MVC. However, there was no change in the 20/40 ratio of tetanic forces, MVC or the contraction times and force of the maximal twitch. In contrast, following sustained isometric exercise tetanic forces were markedly reduced, particularly at low frequencies of stimulation. The 20/40 ratio decreased and the induced muscle weakness was greater at 30% than 60% or 100% MVC. The performance of sustained isometric contractions also effected a decrease in contraction time of the twitch and MVC. The results are in accord with previous findings for dynamic work (Davies and White 1982), and show that if isometric exercise is performed rhythmically the effect on tetanic tensions is small and there is no evidence of a preferential loss of electrically evoked force at either high or low frequencies of stimulation following the contractions. For sustained contractions, however, the opposite is true, the ratio of 20/40 Hz forces is markedly reduced and following 30% sustained MVC there is a significant (p less than 0.05) change in the time to peak tension (TPT) of the maximal twitch.     

Effects of stimulation frequency versus pulse duration modulation on muscle fatigue.

During functional electrical stimulation (FES), both the frequency and intensity can be increased to increase muscle force output and counteract the effects of muscle fatigue. Most current FES systems, however, deliver a constant frequency and only vary the stimulation intensity to control muscle force. This study compared muscle performance and fatigue produced during repetitive electrical stimulation using three different strategies: (1) constant pulse-duration and stepwise increases in frequency (frequency-modulation); (2) constant frequency and stepwise increases in pulse-duration (pulse-duration-modulation); and (3) constant frequency and pulse-duration (no-modulation). Surface electrical stimulation was delivered to the quadriceps femoris muscles of 12 healthy individuals and isometric forces were recorded. Muscle performance was assessed by measuring the percent changes in the peak forces and force-time integrals between the first and the last fatiguing trains. Muscle fatigue was assessed by measuring percent declines in peak force between the 60Hz pre- and post-fatigue testing trains. The results showed that frequency-modulation showed better performance for both peak forces and force-time integrals in response to the fatiguing trains than pulse-duration-modulation, while producing similar levels of muscle fatigue. Although frequency-modulation is not commonly used during FES, clinicians should consider this strategy to improve muscle performance.     

The effect of muscle fatigue on the isometric contractile characteristics of skeletal muscle in the cat

The rise time of an isometric twitch, the tetanic tension, the twitch tetanus ratio, the frequency-tension relationship, and the height of the MUAP (motor unit action potential) were measured in fast twitch (medial gastrocnemius) and slow twitch (soleus) muscles of the cat immediately before, in the middle, and immediately after fatiguing isometric contractions at tensions of 30, 50 and 80% of each muscle's initial strength (tetanic tension recorded from the unfatigued muscle). Although the twitch-tetanus ratio was always less for the soleus than for the medial gastrocnemius muscles, the twitch-tetanus ratio for any one muscle was constant throughout the duration of fatiguing isometric contractions at any of the tensions examined. In contrast, the twitch tension and tetanic tension of the muscles were both less after the contractions, the largest reduction occurring for both muscles during contractions sustained at the lowest isometric tensions. The time to peak tension of an isometric twitch was prolonged for both muscles following the contractions. This was associated with a corresponding shift in the frequency tension relationship such that at the point of muscular fatigue, the muscles tetanized at lower frequencies of stimulation than did the unfatigued muscle. In contrast, the amplitude of the MUAP showed only a modest reduction throughout the duration of the fatiguing contractions.     

Tetanic depression in fast motor units of the cat gastrocnemius muscle.

Ability of muscle fibers to generate force is decreased when higher frequency of stimulation of motor units immediately follows lower frequency. This phenomenon called tetanic depression was found in rat medial gastrocnemius. However, it was not clear whether tetanic depression occurred only in rat muscle or it concerns all mammals. This study was conducted on motor units of cat medial gastrocnemius. Analyses were made at three successive trains of stimulation: 30 Hz, 20 and 30 Hz and again 30 Hz (the first pattern) or 40 Hz, 25 and 40 Hz and 40 Hz (the second pattern). In all fast units force generated within the middle tetanus was lower than force generated at the same, but constant frequency of stimulation applied earlier or later. The mean tetanic depression in 30 Hz tetani amounted to 10.9% for fast fatigable (FF) and 15.9% for fast resistant (FR) motor units, whereas in 40 Hz tetani mean values were 5.6% and 7.3% for FF and FR motor units, respectively. In slow motor units tetanic depression was not observed. These results proved the existence of tetanic depression in the feline muscle and indicated that its intensity depends on the fusion of tetanus. It has been concluded, that the tetanic depression is a general property of fast motor units in mammals.     

Nonlinear stiffness--force relationships in whole mammalian skeletal muscles

Small, sinusoidal length changes were superimposed on isometric contractions of fast- and slow-twitch mouse muscles, which were stimulated maximally via their nerves. Stiffness increased with increasing frequency of sinusoidal stimulation, but the relative time course of force and stiffness changes during twitch, tetanic, or partially fused contractions was quite invariant over a range of frequencies in both muscles. Typically, stiffness increases more rapidly than force during contraction and decreases less rapidly during relaxation. This pattern was observed at various temperatures and with various numbers of stimuli. It can be described by a nonlinear relation between stiffness and force with some hysteresis. The presence in the muscle of parallel and series elastic elements, whose stiffness varies with force, may account for the nonlinear relation. This nonlinearity can be used to relate the patterns for summation of force and stiffness observed with brief trains of stimuli under a variety of conditions.     

Tetanic force development of adductor pollicis muscle in anesthetized man.

The tetanic force development of the human adductor pollicis muscle was studied under light anesthesia with nitrous oxide, oxygen, and Demerol, by the use of tetanic stimulation of the ulnar nerve at frequencies ranging from 10 to 100 Hz. The time necessary for the tetanic contraction to reach a plateau was longest at frequencies between 15 and 20 Hz. Fusion of tetanus occurred between 40 and 45 Hz. The mean maximal force of 6.92 kg was developed at a mean frequency of approximately 75 Hz. The maximal force was well maintained up to a stimulation frequency of 100 Hz. The results indicate that in lightly anesthetized man, the maximal force is developed at higher stimulation frequencies than those observed in conscious man and that it is well sustained at higher frequencies.     

Initial force production before sag is enhanced by prior contraction followed by a 3-minute rest period in fast motor units of the rat medial gastrocnemius

Unfused tetanic contractions evoked in fast motor units exhibit extra-efficient force production at the onset of contraction, an effect called “boost”. Boost is diminished in subsequent contractions if there is a short rest period between contractions, but can be re-established with a longer period of rest. We tested the hypothesis that contractile activity and rest could enhance boost-related metrics. Two sets of 3 unfused tetani were evoked 3 min apart in fast fatigable (FF) and fast fatigue-resistant (FR) motor units of the rat medial gastrocnemius. The greatest changes occurred in the first unfused tetanic contractions. Relative to the first contraction in the first set, the first contraction in the second set exhibited higher peak force during boost in a subset of motor units (76% of FF and 48% of FR). Enhanced force during boost was influenced by interaction of slowing of twitch contraction time (up to 20% and 25%, for FF and FR motor units, respectively), half-relaxation time (up to 37% and 49% for FF and FR motor units, respectively), and potentiation of the first twitch (up to 13% and 5% for FF and FR motor units, respectively). Examination of twitches evoked between sets suggested opportunity for greater enhancement of boost with shorter intervening rest periods. The phenomenon of enhanced boost following motor unit activity may interest sports scientists.     

Enhanced Ca2+ transport and muscle relaxation in skeletal muscle from sarcolipin-null mice

Sarcolipin (SLN) inhibits sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) pumps. To evaluate the physiological significance of SLN in skeletal muscle, we compared muscle contractility and SERCA activity between Sln-null and wild-type mice. SLN protein expression in wild-type mice was abundant in soleus and red gastrocnemius (RG), low in extensor digitorum longus (EDL), and absent from white gastrocnemius (WG). SERCA activity rates were increased in soleus and RG, but not in EDL or WG, from Sln-null muscles, compared with wild type. No differences were seen between wild-type and Sln-null EDL muscles in force-frequency curves or maximum rates of force development (+dF/dt). Maximum relaxation rates (-dF/dt) of EDL were higher in Sln-null than wild type across a range of submaximal stimulation frequencies, but not during a twitch or peak tetanic contraction. For soleus, no differences were seen between wild type and Sln-null in peak tetanic force or +dF/dt; however, force-frequency curves showed that peak force during a twitch and 10-Hz contraction was lower in Sln-null. Changes in the soleus force-frequency curve corresponded with faster rates of force relaxation at nearly all stimulation frequencies in Sln-null compared with wild type. Repeated tetanic stimulation of soleus caused increased (-dF/dt) in wild type, but not in Sln-null. No compensatory responses were detected in analysis of other Ca(2+) regulatory proteins using Western blotting and immunohistochemistry or myosin heavy chain expression using immunofluorescence. These results show that 1) SLN regulates Ca(2+)-ATPase activity thereby regulating contractile kinetics in at least some skeletal muscles, 2) the functional significance of SLN is graded to the endogenous SLN expression level, and 3) SLN inhibitory effects on SERCA function are relieved in response to repeated contractions thus enhancing relaxation rates.     

Summation of slow motor unit forces at constant and variable interpulse intervals in rat soleus muscle

Effects of the summation of forces generated by functionally isolated slow-twitch motor units (MU) of the rat soleus muscle were examined in this study. Initially, the twitch, fused tetanic and unfused tetanic contractions evoked by trains of stimuli at variable interpulse intervals were recorded for each MU. Then, two, three or four MUs were co-activated, and the recorded forces were compared to the algebraic sum of the forces of individual MUs. The mean cumulative force of twitches and the mean cumulative force of fused tetani were not statistically different from the respective algebraic sums of forces, which revealed a high degree of linearity in the summation. However, relaxation of the recorded tetanic contractions (either fused or unfused) was faster than that predicted by the linear summation of individual contractions. Moreover, for twitch and tetanic contractions, a tendency to shorten relaxation with an increasing number of co-active MUs was noted. The results indicate that forces of rat soleus slow MUs sum up more linearly than in the respective cat muscle as well as more linearly than for fast MUs in the medial gastrocnemius muscle.     

Experimentally verified mathematical approach for the prediction of force developed by motor units at variable frequency stimulation patterns

During normal daily activity, muscle motor units (MUs) develop unfused tetanic contractions evoked by trains of motoneuronal firings at variable interpulse intervals (IPIs). The mechanical responses of a MU to successive impulses are not identical. The aim of this study was to develop a mathematical approach for the prediction of each response within the tetanus as well as the tetanic force itself. Experimental unfused tetani of fast and slow rat MUs, evoked by trains of stimuli at variable IPIs, were decomposed into series of twitch-shaped responses to successive stimuli using a previously described algorithm. The relationships between the parameters of the modeled twitches and the tetanic force level at which the next response begins were examined and regression equations were derived. Using these equations, profiles of force for the same and different stimulation patterns were mathematically predicted by summating modeled twitches. For comparison, force predictions were made by the summation of twitches equal to the first one. The recorded and the predicted tetanic forces were compared. The results revealed that it is possible to predict tetanic force with high accuracy by using regression equations. The force predicted in this way was much closer to the experimental record than the force obtained by the summation of equal twitches, especially for slow MUs. These findings are likely to have an impact on the development of realistic muscle models composed of MUs, and will assist our understanding of the significance of the neuronal code in motor control and the role of biophysical processes during MU contractions.     

Oxidation/reduction state of cytochrome oxidase during repetitive contractions

There is disagreement regarding whether inadequate O2 determines maximal O2 uptake (VO2max) and lactic acid output (L) during muscular activity. Direct assessment of mitochondrial cytochrome oxidase (cytochrome a-a3) oxidation/reduction (O/R) state should provide an unequivocal answer for this issue. A new near-infrared spectrophotometric method was used to measure the O/R state of cytochrome a-a3 of dog gastrocnemius-plantaris muscle in situ during repetitive isotonic twitch and tetanic contractions. Three contraction frequencies were used for each contraction type in alternating sequence to provide a wide range of VO2 up to VO2max. VO2 and L were measured after 3 and 9 min of a 10-min contraction period, and 15 min were allowed for recovery between contraction periods. VO2 increased with contraction frequency. L was variably increased with contraction frequency at 3 min and uptake usually occurred at 9 min, except at the highest tetanic frequency. The O/R span of cytochrome a-a3 was determined by respiring the animals with 100% N2 to determine the most reduced state. This was followed by respiration with 100% O2, which gave the most oxidized state transiently during recovery. Within this span in muscles at rest, cytochrome a-a3 was 50-80% oxidized. During contractions of both types at all frequencies, cytochrome a-a3 always became more oxidized by an additional 10-20%. These findings should put to rest any arguments that inadequate O2 is a determinant of VO2max or L under the conditions of these experiments: repetitive contractions with free flow in self-perfused muscles and normoxia.     

The muscle sound properties of different muscle fiber types during voluntary and electrically induced contractions.

Soundmyogram (SMG) and electromyogram signals were recorded simultaneously from the relatively fast medial gastrocnemius (MG) and slow soleus (SOL) during voluntary and electrically induced contractions. Using a spike-triggered averaging technique, the averaged elementary sound and corresponding MU spikes were also obtained from about 35 different MUs identified. The rms-SMG of MG increased as a function of force (P < 0.01). On the contrary, these values for SOL increased up to 60% MVC (P < 0.01), but decreased at 80% MVC. The relationship between the peak to peak amplitude of SMG and MU spike indicated significant positive correlations (r = 0.631 to approximately 0.657, P < 0.01). During electrical stimulation at 5 Hz, the SMG power spectral peak frequency (PF) was matched with stimulation frequency in both muscles. At higher stimulation frequencies, e.g., > 15 Hz, only in the MG was SMG-PF synchronized with stimulation frequency; the slow SOL did not show such synchronization. Our data suggest that the SMG frequency components might reflect active motor unit firing rates, and that the SMG amplitude depends upon mechanical properties of contraction, muscle fiber composition, and firing rate during voluntary and electrically induced contractions.     

Monophasic and biphasic relaxation during motor unit tetanic contractions of variable fusion degree

The phenomenon of transition of the monophasic relaxation into biphasic course in the unfused tetanic contractions was studied on functionally isolated motor units of the rat medial gastrocnemius muscle. The sample consisted of 16 FF, 16 FR and 10 S MUs which were stimulated with the same, digitally controlled patterns. The new parameter--QRT/HRT ratio, was introduced as a convenient tool for the classification of the relaxation into monophasic or biphasic. Analysis of tetani evoked at increasing stimulation frequencies revealed similar relationships between the tetanic fusion degree and the shape of relaxation for all three types of motor units investigated. In each MU, the QRT/HRT ratio fell into two distinct ranges related to either monophasic (lower values) or biphasic (higher values) relaxation. The relationship was also found between the shape of relaxation and degree of tetanic fusion--the biphasic course appeared for better fused tetani when fusion index was over the mean of 0.8. Mechanisms of development of the biphasic relaxation were discussed with respect to importance of this parameter in force development and summation of successive contractions into tetanus. Moreover, it was pointed out that adequacy of mathematical modeling of motor unit contractions should benefit from the precise analysis of the mono- or biphasic course of relaxation.     

Length-dependent twitch contractile characteristics of skeletal muscle

The length dependence of force development of mammalian skeletal muscles was evaluated during twitch, double-pulse, and tetanic contractions, and the relation between muscle length and the time-dependent characteristics of twitch and double-pulse contractions were determined. In situ isometric contractions of the rat gastrocnemius muscle were analyzed at seven different lengths, based on a reference length at which the maximal response to double-pulse contractions occurred (Lopt-2P). Twitch and double-pulse contractions were analyzed for developed tension (DT), contraction time (tC), average rate of force development (DT-tC(-1)), half-relaxation time (t50%R), peak rate of relaxation (DT x dtmin(-1)), and 90%-relaxation time (t90%R). Considering the length at which maximal tetanic DT occurred to be the optimal length (Lo-TET), the peak DT for twitch contractions and double-pulse contractions was observed at Lo-TET + 0.75 mm (p < 0.05) and Lo-TET + 0.1 mm (p > 0.05), respectively. When measured at the length for which maximal twitch and double-pulse contractions were obtained, tetanic DT was 95.2 +/- 3 and 99.0 +/- 2% of the maximal value, respectively. These observations suggest that double-pulse contractions are more suitable for setting length for experimental studies than twitch contractions. Twitch and double-pulse contraction tC were 15.53 +/- 1.14 and 25.0 +/- 0.6 ms, respectively, at Lopt-2P, and increased above Lopt-2P and decreased below Lopt-2P. Twitch t50%R was 12.18 +/- 0.90 ms at Lopt-2P, and increased above Lopt-2P and below Lopt-2P. Corresponding changes for double-pulse contractions were greater. Stretching the muscle leads to slower twitch contractions and double-pulse contractions, but the mechanisms of this change in time course remain unclear.     

Diaphragmatic fatigue in the rat

We studied fatigue of rat diaphragm in response to repetitive brief and prolonged electrical stimulation of the phrenic nerve, at 0.2, 1-100 Hz. Low and high frequency of stimulation produced twitch and tetanic contractions in the rat diaphragm. A mean maximum twitch tension of 1.4 +/- 0.1 g was produced at 1 Hz, and a mean maximum tetanic tension of 5.6 +/- 0.3 g was obtained at 100 Hz (means +/- S.E., n = 8). Twitch and tetanic fatigue was produced at all frequencies of stimulations, but with different time scale, or duration, and with different number of stimuli delivered to the muscle. At low rates of stimulation, e.g. 10 Hz, fewer stimuli were needed to fatigue the muscle (3000 in 5 min), whereas at high rates of stimulation, e.g. 50 Hz, more stimuli were needed to fatigue the muscle (6600 in 2.2 min). The amplitude of the tetanic tensions elicited at 10 and 50 Hz, at the end of 5 or 2 min fatiguing stimulation, was 39 +/- 2.7% and 80 +/- 3.1% of their respective control tensions (2.8 +/- 0 2 g and 5.3 +/- 0.5 g, n = 8, P 0.001). It was concluded that fatigue in the rat diaphragm depended on the frequency and duration of stimulation as well as on the number of stimuli delivered to the muscle. Various mechanisms of muscle fatigue are described in the discussion to explain the observations made in the present investigation.     

Structure and function of skeletal muscle in zebrafish early larvae

Zebrafish muscles were examined at an early developmental stage (larvae 5-7 d). Using aluminum clips, preparations (approximately 1.5 mm length, 150 microm diameter) were mounted for force registration and small angle x-ray diffraction. Sarcomeres were oriented mainly in parallel with the preparation long axis. Electrical stimulation elicited fast and reproducible single twitch contractions. Length-force relations showed an optimal sarcomere length of 2.15 microm. X-ray diffraction revealed clear equatorial 1.1/1.0 reflections, showing that myofilaments are predominantly arranged along the preparation long axis. In contrast, reflections from older (2 mo) zebrafish showed two main filament orientations each at an approximately 25 degrees angle relative to the preparation long axis. Electrical stimulation of larvae muscles increased the 1.1/1.0 intensity ratio, reflecting mass transfer to thin filaments during contraction. The apparent lattice volume was 3.42 x 10(-3) microm(3), which is smaller than that of mammalian striated muscle and more similar to that of frog muscles. The relation between force and stimulation frequency showed fusion of responses at a comparatively high frequency (approximately 186 Hz), reflecting a fast muscle phenotype. Inhibition of fast myosin with N-benzyl-p-toluene sulphonamide (BTS) showed that the later phase of the tetanus was less affected than the initial peak. This suggests that, although the main contractile phenotype is fast, slow twitch fibers can contribute to sustained contraction. A fatigue stimulation protocol with repeated 220 ms/186 Hz tetani showed that tetanic force decreased to 50% at a train rate of 0.1 s(-1). In conclusion, zebrafish larvae muscles can be examined in vitro using mechanical and x-ray methods. The muscles and myofilaments are mainly orientated in parallel with the larvae long axis and exhibit a significant fast contractile component. Sustained contractions can also involve a small contribution from slower muscle types.     

Theophylline does not increase maximal tetanic force or diaphragm endurance in vitro

These experiments tested the capacity of theophylline to improve diaphragm strength (maximal force development) and endurance (maintenance of force output during repeated contractions). Rodent diaphragm strips were mounted at optimal length in oxygenated Krebs-Ringer solution (37 degrees C, pH 7.37). Direct stimuli used supramaximal current density, 0.2-ms pulses, and 250-ms tetanic trains. Theophylline (500 mg/ml) increased force development at low stimulation frequencies but did not increase maximal force [25.7 +/- 0.5 for theophylline vs. 26.0 +/- 0.4 (SE) N/cm2 for control (n = 34)]. During repeated submaximal (25-36 Hz) tetanic contractions, theophylline did not affect endurance. During repeated maximal (160 Hz) tetanic contractions theophylline reduced endurance, accelerating the fall of developed force. Theophylline also inhibited recovery of force after endurance trials ended. We conclude that theophylline does not increase maximal tetanic force and can reduce diaphragm endurance in vitro.