Diaphragm muscle fatigue resistance during postnatal development.

Changes in the contractile and fatigue properties of the cat diaphragm muscle were examined during the first 6 wk of postnatal development. Both twitch contraction time and half-relaxation time decreased progressively with age. Correspondingly, the force-frequency curve was shifted to the left early in development compared with adults. The ratio of peak twitch force to maximum tetanic force decreased with age. Fatigue resistance of the diaphragm was highest at birth and then progressively decreased with age. At birth, most diaphragm muscle fibers stained darkly for myofibrillar adenosinetriphosphatase after alkaline preincubation and thus would be classified histochemically as type II. During subsequent postnatal development, the proportion of type I fibers (lightly stained for adenosinetriphosphatase) increased while the number of type II fibers declined. At birth, type I fibers were larger than type II fibers. The size of both fiber types increased with age, but the increase in cross-sectional area was greater for type II fibers. On the basis of fiber type proportions and mean cross-sectional areas, type I fibers contributed 15% of total muscle mass at birth and 25% in adults. Thus postnatal changes in diaphragm contractile and fatigue properties cannot be attributed to changes in the relative contribution of histochemically classified type I and II fibers. However, the possibility that these developmental changes in diaphragm contractile and fatigue properties correlated with the varying contractile protein composition of muscle fibers was discussed.     

Postnatal expression of myosin isoforms in an expiratory muscle--external abdominal oblique.

We studied the postnatal expression of heavy-chain (MHC) and native myosin isoforms in an expiratory abdominal muscle of the rat, the external abdominal oblique (EO). Moreover, we contrasted EO myosin expression with that of the costal diaphragm (DIA) to draw inspiratory vs. expiratory muscle comparisons during development. Examination of MHC gels demonstrated a mature phenotype of slow and adult fast myosin isoforms at an earlier age in the EO (day 60) than in the DIA [day > 115 (adult)]. The mature MHC phenotype of the EO was characterized by a preponderance of MHC 2B, whereas the DIA was characterized by approximately equal portions of MHC slow, MHC 2A, and MHC 2X. During early postnatal development, there was a delay in the expression of MHC 2A in the EO compared with the DIA. However, MHC 2B, expressed later in development in both muscles, was noted in the EO before the DIA. We conclude that 1) the EO mature myosin phenotype is characterized by a preponderance of fast myosin isoforms and 2) the EO and DIA muscles are subject to different temporal patterns of isoform expression during postnatal development.     

Postnatal changes in vagal control of esophageal muscle contractions in rats

AimsReplacement of smooth muscles by striated muscles occurs in the esophagus during the early postnatal period. The aim of this study was to clarify postnatal changes in vagal control of esophageal muscle contractions in rats.Main methodsAn isolated segment of the neonatal rat esophagus was placed in an organ bath and the contractile responses were recorded using a force transducer.Key findingsElectrical stimulation of the vagus trunk evoked a biphasic contractile response in the neonatal esophageal segment. The first and second components of the contractions were inhibited by α-bungarotoxin and atropine, respectively. Ganglion blockers, hexamethonium and mecamylamine, did not affect vagally mediated contractions. The first component gradually enlarged with age in days, whereas the second component declined during the first week after birth. Application of d-tubocurarine or acetylcholine caused an apparent contraction in the esophageal striated muscle at postnatal day 0, but responses to these drugs were not observed at 1 week after birth. The neonatal esophagus expressed the γ-subunit of nicotinic acetylcholine receptors. In contrast, the ε-subunit was dominantly expressed in the adult esophagus.SignificanceThe vagus nerves directly innervate both the esophageal striated muscles and smooth muscles in the early neonatal period. During the process of muscle rearrangement, the property of the striated muscles is altered substantially. The specific features of striated muscles in the neonatal rat esophagus might compensate for immature formation of neuromuscular junctions. Unsuccessful conversion of the striated muscle property during postnatal muscle rearrangement would be related to disorders of esophageal motility.     

Intracellular pH during sequential, fatiguing contractile periods in isolated single Xenopus skeletal muscle fibers.

The purpose of the present study was to test the hypothesis that a preceding contractile period in isolated single skeletal muscle fibers would attenuate the decrease in pH during an identical, subsequent contractile period, thereby reducing the rate of fatigue. Intact single skeletal muscle fibers (n = 9) were isolated from Xenopus lumbrical muscle and incubated with the fluorescent cytosolic H+ indicator 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) AM for 30 min. Two identical contractile periods were performed in each fiber, separated by a 1-h recovery period. Force and intracellular pH (pHi) fluorescence were measured simultaneously while fibers were stimulated (tetanic contractions of 350-ms trains with 70-Hz stimuli at 9 V) at progressively increasing frequencies (0.25, 0.33, 0.5, and 1 contraction/s) until the development of fatigue (to 60% initial force). No significant difference (P < 0.05) was observed between the first and second contractile periods in initial force development, resting pHi, or time to fatigue (5.3 +/- 0.5 vs. 5.1 +/- 0.6 min). However, the relative decrease in the BCECF fluorescence ratio (and therefore pHi) from rest to the fatigue time point was significantly greater (P < 0.05) during the first contractile period (to 65 +/- 4% of initial resting values) compared with the second (77 +/- 4%). The results of the present study demonstrated that, when preceded by an initial fatiguing contractile period, the rise in cytosolic H+ concentration in contracting single skeletal muscle fibers during a second contractile period was significantly reduced but did not attenuate the fatigue process in the second contractile period. These results suggest that intracellular factors other than H+ accumulation contribute to the fall in force development under these conditions.     

Muscle function in children

Electrically evoked mechanical and contractile properties of the triceps surae have been measured in 52 children aged 11 and 14 years, and results compared with previously reported data for adults (Davies and White 1982). The results show that the time to peak tension (TPT), half relaxation time (1/2RT) and supramaximal tension (Pt) of the twitch were not significantly (P greater than 0.1) different in girls and boys and independent of age. The 14-year-old girls and boys were stronger in terms of their supramaximal 10, 20, and 50 Hz tetanic tensions and maximal voluntary contraction (MVC) than their younger counterparts, and both groups of children were significantly (P less than 0.001) weaker than the young adults. However, if standardisation was made for an anthropometric estimate of calf muscle (plus bone) cross-sectional area (CSA), the differences in strength disappeared. Electrically stimulated and voluntary maximal force per unit CSA measured at the knee were 17.1 and 20.5 N X cm2 respectively and independent of sex and age. The loss of force during a 2-min stimulated fatigue test was the same in the children as the adults. The average fatigue indices ranged from 0.52 to 0.72 in the children, compared with 0.68 in the adults. It is concluded that absolute differences in muscle strength in children are a function of muscle mass. The force generating capacity expressed in N X cm2, fatiguability, contraction and relaxation times of the triceps surae would appear to remain unchanged through adolescence and early adulthood.     

Exercise training protects against contraction-induced lipid peroxidation in the diaphragm

Endurance exercise training promotes a small but significant increase in antioxidant enzyme activity in the costal diaphragm (DIA) of rodents. It is unclear if these training-induced improvements in muscle antioxidant capacity are large enough to reduce oxidative stress during prolonged contractile activity. To test the hypothesis that training-related increases in DIA antioxidant capacity reduces contraction-induced lipid peroxidation, we exercise trained adult female Sprague-Dawley (n = 7) rats on a motor-driven treadmill for 12 weeks at approximately 75% maximal O2 consumption (90 min/day). Control animals (n = 8) remained sedentary during the same 12-week period. After training, DIA strips from animals in both experimental groups were excised and subjected to an in vitro fatigue contractile protocol in which the muscle was stimulated for 60 min at a frequency of 30 Hz, every 2 s, with a train duration of 330 m. Compared to the controls, endurance training resulted in an increase (P < 0.05) in diaphragmatic non-protein thiols and in the activity of the antioxidant enzyme superoxide dismutase. Following the contractile protocol, lipid peroxidation was significantly lower (P < 0.05) in the trained DIA compared to the controls. These data support the hypothesis that endurance exercise training-induced increases in DIA antioxidant capacity protect the muscle against contractile-related oxidative stress.     

Tension-time index, fatigue, and energetics in isolated rat diaphragm: a new experimental model.

The tension-time index (TTI) has been used to estimate mechanical load, energy utilization, blood flow, and susceptibility to fatigue in contracting muscle. The TTI can be defined, for a rhythmically contracting muscle, as the product of average force development divided by maximum tetanic force times duty cycle [contraction time / (contraction + relaxation time)]. In this study, the TTI concept was applied to isolated diaphragm via a method that allowed TTI to be clamped at a predetermined value. The hypothesis tested was that, at constant TTI, muscle energetics and the extent of fatigue would vary with stimulation frequency. Isolated diaphragm strips were stimulated at 25, 50, 75, or 100 Hz for 4 min, one per second. Duty cycle was continuously adjusted to maintain TTI at 0.07, which was near the highest TTI tolerated for 4 min, at 20-Hz stimulation. At the end of the fatigue run, muscles were either immediately frozen for determination ATP, creatine, and creatine phosphate concentrations (n = 6) or stimulated for evaluation of low- and high-frequency fatigue (n = 5). Results demonstrated no difference in the extent of fatigue or in the final ATP and creatine phosphate concentrations between groups. Large within-run increases in duty cycle were required at low stimulation frequencies, but only small increases were required at the highest frequencies. The results demonstrate that, at a constant TTI, similar fatigue properties predominate at all stimulation frequencies with no clear distinction between high- and low-frequency fatigue. The method of clamping TTI during fatigue may be useful for evaluating energetics and contractile function between treatment groups in isolated muscle when treatment influences baseline contractile characteristics.     

INVERSE RELATIONSHIP BETWEEN FUNCTIONAL MATURITY AND EXPONENTIAL GROWTH RATE OF AVIAN SKELETAL MUSCLE: A CONSTRAINT ON EVOLUTIONARY RESPONSE

In this study, we investigate whether a tissue-level constraint can explain the general inverse relationship between growth rate and precocity of development in birds. On the whole, altricial (dependent) chicks grow three to four times faster than the less dependent, more able chicks of precocial species of similar adult mass. We suggest that an antagonism between growth and acquisition of mature function in skeletal muscle constrains postnatal growth and development in most species of birds. Altricial species, represented by European starlings in this study, hatch with skeletal muscle having low capacity for generating force but grow rapidly. Conversely, precocial species (northern bobwhite quail and Japanese quail), hatch with relatively mature skeletal muscle, especially in their legs, but grow more slowly. As development proceeds in all species, exponential growth rates decrease as muscles acquire adult levels of function. Among four variables associated with muscle function, exponential growth rate (EGR) was negatively correlated with pyruvate kinase activity (glycolysis), potassium concentration (electrical potential), and dry weight fraction (contractile proteins) in both pectoral and leg muscles but not with citrate synthase activity (aerobic metabolism) in either set of muscles. For pectoral muscle, these variables accounted for 87% of the total variation in EGR in all three species combined despite a twofold difference in growth rates between the starling and quail. EGRs of leg muscle (51% of variation accounted for) were less than predicted by the pectoral-muscle equation in quail during the early part of the postnatal period and in starlings during the late postnatal period. This result would not contradict a growth rate/maturity constraint hypothesis if EGRs were down-regulated for allometric or other considerations.     

Difference in skeletal muscle function in males vs. females: role of estrogen receptor-beta

Male skeletal muscles are generally faster and have higher maximum power output than female muscles. Conversely, during repeated contractions, female muscles are generally more fatigue resistant and recover faster. We studied the role of estrogen receptor-beta (ERbeta) in this gender difference by comparing contractile function of soleus (mainly slow-twitch) and extensor digitorum longus (fast-twitch) muscles isolated from ERbeta-deficient (ERbeta(-/-)) and wild-type mice of both sexes. Results showed generally shorter contraction and relaxation times in male compared with female muscles, and ERbeta deficiency had no effect on this. Fatigue (induced by repeated tetanic contractions) and recovery of female muscles were not affected by ERbeta deficiency. However, male ERbeta(-/-) muscles were slightly more fatigue resistant and produced higher forces during the recovery period than wild-type male muscles. In fact, female muscles and male ERbeta(-/-) muscles displayed markedly better recovery than male wild-type muscles. Gene screening of male soleus muscles showed 25 genes that were differently expressed in ERbeta(-/-) and wild-type mice. Five of these genes were selected for further analysis: muscle ankyrin repeat protein-2, muscle LIM protein, calsequestrin, parvalbumin, and aquaporin-1. Expression of these genes showed a similar general pattern: increased expression in male and decreased expression in female ERbeta(-/-) muscles. In conclusion, ERbeta deficiency results in increased performance during fatigue and recovery of male muscles, whereas female muscles are not affected. Improved contractile performance of male ERbeta(-/-) mouse muscles was associated with increased expression of mRNAs encoding important muscle proteins.     

Changes in isometric contractile properties of extensor digitorum longus and soleus muscles of C57BL/6J mice following denervation

In this study, conducted on mice of the C57BL/6J+/+ strain, we investigated the differential effects of denervation on the isometric contractile properties of the extensor digitorum longus (EDL) and soleus (SOL) muscles. The contractile properties were studied at 1, 28, 84, and 210 days following unilateral section of the sciatic nerve at 12 weeks of age. When isometric tetanus tension was expressed relative to wet weight, the denervated SOL showed an earlier and more pronounced loss in tension generating capacity than the EDL. Both the denervated SOL and EDL showed potentiation of the twitch tension at 28 days postdenervation. The time to peak twitch tension (TTP) and the time to half-relaxation (1/2RT) were prolonged by 28 days postdenervation in both muscles. This trend continued to the oldest age-groups studied in the EDL, but reached an apparent plateau in the SOL at 84 days postdenervation. In response to fatigue, the denervated SOL showed a marked decrease in resistance to fatigue at 1 day but a relatively normal response thereafter, whereas the denervated EDL showed an increase in resistance to fatigue at and beyond the 28-day period. In spite of the fact that the total contraction time of both muscles increased following denervation, the predominantly oxidative SOL remained a slower contracting muscle than the more glycolytic EDL.     

Oxidative capacity and capillary density of diaphragm motor units

Motor units in the cat diaphragm (DIA) were isolated in situ by microdissection and stimulation of C5 ventral root filaments. Motor units were classified based on their isometric contractile force responses and fatigue indexes (FI). The muscle fibers belonging to individual units (i.e., the muscle unit) were identified using the glycogen-depletion method. Fibers were classified as type I or II based on histochemical staining for myofibrillar adenosine triphosphatase (ATPase) after alkaline preincubation. The rate of succinate dehydrogenase (SDH) activity of each fiber was determined using a microphotometric procedure. The location of capillaries was determined from muscle cross sections stained for ATPase after acid (pH = 4.2) preincubation. The capillarity of muscle unit fibers was determined by counting the number of capillaries surrounding fibers and by calculating the number of capillaries per fiber area. A significant correlation was found between the fatigue resistance of DIA units and the mean SDH activity of muscle unit fibers. A significant correlation was also observed between DIA unit fatigue resistance and both indexes of muscle unit fiber capillarity. The mean SDH activity and mean capillary density of muscle unit fibers were also correlated. We conclude that DIA motor unit fatigue resistance depends, at least in part, on the oxidative capacity and capillary density of muscle unit fibers.     

Effects of fatigue and recovery on electromyographic and isometric force- and relaxation-time characteristics of human skeletal muscle

Effects of fatigue produced by a maintained 60% isometric loading on electromyographic and isometric force-time and relaxation-time characteristics of human skeletal muscle were studied in 21 males accustomed to strength training. Fatigue loading resulted in a slight but not significant change in the maximal integrated EMG of a maximal isometric contraction, and a large decrease (20.4 +/- 6.3%, p less than 0.001) in maximal force. Fatigue loading increased (p less than 0.05-0.01) neural activation of the muscles during rapidly produced submaximal isometric forces, but had a considerable adverse effect (p less than 0.001) on the corresponding force-time characteristics. Correlations between the relative changes after fatigue in the IEMG/force ratio at the maximal force level, and in the IEMG/force ratios of the early phases of the force-time curve were not significant, but gradually became significant (p less than 0.01) at higher force levels. The average IEMG of the muscles in the relaxation phase of contraction remained unaltered by fatigue, while a marked deleterious change in the relaxation-time variables (p less than 0.001) occurred concomitantly. During the subsequent 3 min rest period considerable (12.1 +/- 7.0%, p less than 0.001) recovery was noted in the maximal force, with smaller (insignificant or p less than 0.05-0.01) changes in the force-time and relaxation-time variables, while the average IEMG of force production decreased (p less than 0.01-0.001). The present findings suggest that fatigue leading to a worsening in force-time, in maximal force and in the relaxation-time parts of a maximal isometric contraction might take place primarily in the contractile processes.     

Calcium-independent phospholipase A2 modulates cytosolic oxidant activity and contractile function in murine skeletal muscle cells.

Phospholipase A2 (PLA2) activity supports production of reactive oxygen species (ROS) by mammalian cells. In skeletal muscle, endogenous ROS modulate the force of muscle contraction. We tested the hypothesis that skeletal muscle cells constitutively express the calcium-independent PLA2 (iPLA2) isoform and that iPLA2 modulates both cytosolic oxidant activity and contractile function. Experiments utilized differentiated C2C12 myotubes and a panel of striated muscles isolated from adult mice. Muscle preparations were processed for measurement of mRNA by real-time PCR, protein by immunoblot, cytosolic oxidant activity by the dichlorofluorescein oxidation assay, and contractile function by in vitro testing. We found that iPLA2 was constitutively expressed by all muscles tested (myotubes, diaphragm, soleus, extensor digitorum longus, gastrocnemius, heart) and that mRNA and protein levels were generally similar among muscles. Selective iPLA2 blockade by use of bromoenol lactone (10 microM) decreased cytosolic oxidant activity in myotubes and intact soleus muscle fibers. iPLA2 blockade also inhibited contractile function of unfatigued soleus muscles, shifting the force-frequency relationship rightward and depressing force production during acute fatigue. Each of these changes could be reproduced by selective depletion of superoxide anions using superoxide dismutase (1 kU/ml). These findings suggest that constitutively expressed iPLA2 modulates oxidant activity in skeletal muscle fibers by supporting ROS production, thereby influencing contractile properties and fatigue characteristics.     

Fatigue and recovery contractile properties of young and elderly men

The 24 h recovery pattern of contractile properties of the triceps surae muscle, following a period of muscle fatigue, was compared in physically active young (25 years, n = 10) and elderly (66 years, n = 7) men. The fatigue test protocol consisted of 10 min of intermittent submaximal 20 Hz tetani. The maximal twitch (Pt) and tetanic force at 3 frequencies (10, 20 and 50 Hz) were determined at baseline and at 15 min, 1, 4 and 24 h after fatiguing the muscle. Maximal voluntary contraction (MVC) and vertical jump (MVJ) were also assessed. The loss of force during the fatigue test was not significantly different between the young (18 +/- 13%) and elderly (22 +/- 15%). Both groups showed similar and significant reductions of Pt (15%), tetanic force (10 to 35%) and rate of force development (dp/dt) (20%) 15 min and 1 h into recovery. The loss of force was greater at the lower stimulation frequencies of 10 and 20 Hz. Time-to-peak tension was unchanged from baseline during recovery in either group. The average rate of relaxation of twitch force (-dPt/dt) was decreased (p less than 0.05) and half-relaxation time significantly increased at 15 min and 1 h in the elderly but not the young. The findings indicate that after fatiguing contractions, elderly muscle demonstrates a slower return to resting levels of the rate and time course of twitch relaxation compared to the young.     

Chronic hypobaric hypoxia increases isolated rat fast-twitch and slow-twitch limb muscle force and fatigue

Chronic hypoxia alters respiratory muscle force and fatigue, effects that could be attributed to hypoxia and/or increased activation due to hyperventilation. We hypothesized that chronic hypoxia is associated with phenotypic change in non-respiratory muscles and therefore we tested the hypothesis that chronic hypobaric hypoxia increases limb muscle force and fatigue. Adult male Wistar rats were exposed to normoxia or hypobaric hypoxia (PB=450 mm Hg) for 6 weeks. At the end of the treatment period, soleus (SOL) and extensor digitorum longus (EDL) muscles were removed under pentobarbitone anaesthesia and strips were mounted for isometric force determination in Krebs solution in standard water-jacketed organ baths at 25 °C. Isometric twitch and tetanic force, contractile kinetics, force-frequency relationship and fatigue characteristics were determined in response to electrical field stimulation. Chronic hypoxia increased specific force in SOL and EDL compared to age-matched normoxic controls. Furthermore, chronic hypoxia decreased endurance in both limb muscles. We conclude that hypoxia elicits functional plasticity in limb muscles perhaps due to oxidative stress. Our results may have implications for respiratory disorders that are characterized by prolonged hypoxia such as chronic obstructive pulmonary disease (COPD).     

Mechanical and morphological properties of the triceps surae muscle-tendon unit in old and young adults and their interaction with a submaximal fatiguing contraction.

The purposes of this study were to examine (a) whether the morphological properties of the muscle gastrocnemius medialis (GM) contribute to the known enhanced muscle fatigue resistance during submaximal sustained isometric plantar flexion contraction of old compared to young adults and (b) whether a submaximal fatiguing contraction differently affects the mechanical properties of the GM tendon and aponeurosis of old and young adults. Fourteen old and 12 young male subjects performed maximal voluntary isometric plantar flexions (MVC) on a dynamometer before and after a submaximal fatiguing task (40% MVC). Moments and EMG signals from the gastrocnemius medialis and lateralis, soleus and tibialis anterior muscles were measured. The elongation of the GM tendon and aponeurosis and the morphological properties of its contractile element were examined by means of ultrasonography. The old adults showed lower maximal ankle joint moment, stiffness and fascicle length in both tested conditions. The submaximal fatiguing contraction did not affect the force-strain relationship of the GM tendon and aponeurosis of either young or old adults. The time to task failure was longer for the old adults and was strongly correlated with the fascicle length (r(2)=0.50, P<0.001). This provides evidence on that the lower ratio of the active muscle volume to muscle force for the old adults might be an additional mechanism contributing to the known age related increase in muscle fatigue resistance.     

Effect of corticosteroids on diaphragm fatigue, SDH activity, and muscle fiber size.

The influence of dexamethasone on diaphragm (DIA) fatigue, oxidative capacity, and fiber cross-sectional areas (CSA) was determined in growing hamsters. One group received dexamethasone by daily subcutaneous injection for 21 days (D animals), while pair-weight (P) and free-eating controls (CTL) received saline subcutaneously. Isometric contractile properties of the DIA were determined in vitro by supramaximal direct muscle stimulation in the presence of curare. DIA fatigue resistance was determined through repetitive stimulation at 40 pulses/s for 2 min. A computer-based image-processing system was used to histochemically determine muscle fiber-type proportions, CSA, and succinate dehydrogenase activities. The medial gastrocnemius muscle (MG) was used as a limb muscle control, with histochemical studies being performed on both the superficial (s) and deep/red (r) portions. Dexamethasone markedly attenuated the normal increment in body weight over the 3-wk period. DIA fatigue resistance was significantly reduced in the D compared with CTL and P animals. Dexamethasone had no effect on fiber-type proportions of the DIA or MGr (MGs contained only type II fibers). In the DIA, the CSA of type II fibers was reduced 33% in D and 18.5% in P animals compared with CTL. Although no significant atrophy was noted in the type I DIA fibers of either D or P animals, a trend toward significance was noted in D animals compared with CTL. In the MGs, the CSA of type II fibers was reduced 33% in D and 16.5% in P animals compared with CTL. Significant atrophy of type I and II fibers of the MGr was noted in D animals compared with CTL (33.8 and 35% reductions, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationship between numbers and frequencies of stimuli in human muscle fatigue

The effect of stimulus frequency on the rate of muscle fatigue has been studied on dorsiflexor muscles of the human ankle. It was found that significantly fewer stimuli were required to abolish twitch and tetanic torque when the stimuli were delivered at 15 Hz rather than 30 Hz. At both stimulus frequencies twitch torque disappeared before tetanic torque. The difference in numbers of stimuli required for fatigue was not due to impaired excitation of muscle fibers at either of the two frequencies. At both stimulating frequencies, twitch fatigue appeared to be due to a defect in excitation-contraction coupling and/or the contractile machinery.     

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.