W. J. Van Der Laarse

The Development of the Spinal Motor Column in Relation to the Myotomal Muscle Fibers in the Zebrafish (Brachydanio rerio)

SummaryThe neuromuscular system in the trunk of larval and adult zebrafishes was studied by means of light and electronmicroscopical methods. Spinal motoneurons were identified with the horseradish peroxidase retrograde transport method. We correlated the differentiation and growth of the myotomal muscle with the number of motoneurons per spinal cord segment and the size of the motoneuron somata.The adult number of motoneurons is reached in an early larval stage, before the muscle fiber type differentiation in the myotomes is completed. The mean motoneuron size does not bear a clear correlation with the size of the myotomal muscle.In adult zebrafishes we could distinguish the motoneurons which innervate the superficial slow red and the deep fast white muscle fibers on the basis of soma size and position in the motor column. The motoneurons of the red muscle part are small; they are located in the ventrolateral part of the motor column. The motoneurons of the deep fast white fibers are large; they lie near the central canal.

Metabolic changes with fatigue in different types of single muscle fibres of Xenopus laevis.

1. Peak isometric force of single fast (type 1) and slow (type 3) muscle fibres of Xenopus decreased when fibres were stimulated intermittently above their predicted sustainable duty cycle at 20 degrees C. Type 1 fibres could be fatigued to zero force. In most type 3 fibres force did not decrease below 50% of the original (P0) before activation failure, as indicated by irregular contractions. 2. Fibres were rapidly frozen at different force levels and analysed by high‐performance liquid chromatography (HPLC) for ATP, IMP, phosphocreatine (PCr) and creatine (Cr). Lactate was determined enzymatically in type 1 fibres only. The relationships between force and PCr, and between force and ATP during fatigue were, apart from the range of values obtained, the same for both fibre types. When force had fallen to about 60‐80% of original, PCr was fully reduced. At lower force levels, the ATP content‐decreased, and a concomitant rise of IMP content was found. At zero force, ATP had fallen to about 25% of its value in rested type 1 fibres, and up to 200 mumol lactate (g dry weight)‐1 had accumulated. 3. Recovery from fatigue was studied in fibres where force had fallen to 0.6 P0 (both fibre types) and 0.2 P0 (type 1 only). After 1 h of recovery ATP had in all cases returned to the level measured in rested fibres. In fibres fatigued to 0.6 P0, force almost returned to its original value. However, in type 1 fibres fatigued to 0.2 P0, it returned to only 0.3 P0. After 1 h of recovery the PCr/Cr ratio in type 1 fibres was lower (probability, P less than 0.05) than in control fibres, whereas in type 3 fibres it was not significantly different from controls. 4. The relationship between peak force and stimulus frequency, which had a sigmoid shape in fully rested fibres, was drastically changed by fatiguing stimulation. Immediately after fatiguing stimulation of type 1 fibres, force hardly increased with stimulus frequency, corresponding to the observation that calcium efflux from the sarcoplasmic reticulum was decreased at high stimulus frequencies. The force‐frequency relationship of type 3 fibres was the same before and after intermittent stimulation.

ATP formation and ATP hydrolysis during fatiguing, intermittent stimulation of different types of single muscle fibres from Xenopus laevis.

SummaryThis report describes changes of the rate of ATP hydrolysis in single, intact muscle fibres during the development of fatigue induced by intermittent tetanic stimulation. High (type 3) and low (type 1) oxidative muscle fibres dissected from the iliofibularis muscle of Xenopus laevis were studied at 20°C. The rate of ATP hydrolysis was calculated during different time intervals from changes in the content of nucleotides, creatine compounds and lactate, as well as lactate efflux and oxygen uptake. During the first phase of intermittent stimulation, phosphocreatine is fully reduced while the rate of oxygen consumption increases to its maximum, the lactate content increases to a maximum level, and a small amount of IMP is formed; the rate of ATP hydrolysis in type 3 fibres is constant while force decreases, whereas the rate decreases approximately in proportion to force in type 1 fibres. After the first phase, the rate of ATP hydrolysis in type 3 fibres decreases slightly and the fibres reach a steady metabolic state in which the rates of ATP formation and hydrolysis are equal; in type 1 fibres a drastic change of the rate of ATP hydrolysis occurs and a steady metabolic state is not reached. On the basis of the time courses of the metabolic changes, it is concluded that the rate of ATP hydrolysis in type 3 fibres is reduced by acidification and/or a reduced calcium efflux from the sarcoplasmic reticulum, whereas in type 1 fibres inorganic phosphate and/or acidification inhibit the rate initially and ADP is a likely candidate to explain the drastic fall of the rate of ATP hydrolysis during late phases of fatiguing stimulation.

Quantitative histochemistry of three mouse hind-limb muscles: the relationship between calcium-stimulated myofibrillar ATPase and succinate dehydrogenase activities.

SummaryA quantitative modification of Meijers calcium-lead capture method, for the demonstration of calcium-stimulated myofibrillar ATPase activity at physiological pH, is described. A range of myofibrillar ATPase activities has been found among fast muscle fibres in two mouse hind-limb muscles. The myofibrillar ATPase activity of fast muscle fibres is 1.5–3 times higher than the myofibrillar ATPase activity of slow muscle fibres.Myofibrillar ATPase activities and succinate dehydrogenase activities of individual muscle fibres have been determined in serial sections. Activities of the two enzymes are correlated positively in soleus (fast and slow fibres), and negatively in plantaris (almost all fast) and extensor digitorum longus muscle (all fast). However, this correlation is not significant among the oxidative fibres in the extensor digitorum longus. The fibres of the latter muscle cannot be classified satisfactorily into two sub-types.

Calcium-stimulated myofibrillar ATPase activity correlates with shortening velocity of muscle fibres in Xenopus laevis.

SummaryThe iliofibularis muscle ofXenopus laevis is reported to contain five types of fibres which have different force—velocity relationships. Ten fibres of each type were selected on the basis of succinate dehydrogenase activity, cross-sectional area and location in the muscle, in order to assess the validity of the fibre type classification.Maximum calcium-stimulated myofibrillar ATPase activity (Vmax) and apparent Michaelis constant (Km) for ATP were determined for these 50 fibres from serial sections. The values obtained varied according to the type of fibre. Type 1 had the highest and type 5 the lowest values forKm andVmax.In a separate experiment, single freeze-dried fibres were used to determine the relationship between their ATP content and apparentKm for ATP. There was a tendency for high ATP concentrations in fibres with highKm values.When myofibrillar ATPase activity was related to the maximum velocity of shortening of the five fibre types, a significant correlation was found. It is concluded that calcium-stimulated myofibrillar ATPase histochemistry allows an estimate of the maximum shortening velocity of muscle fibres fromXenopus laevis.

Oxygen consumption of single muscle fibres of Rana temporaria and Xenopus laevis at 20 degrees C.

1. Oxygen consumption of contracting single muscle fibres of Rana temporaria and Xenopus laevis was investigated at 20 degrees C. 2. Single fibres of the tibialis anterior muscle of Rana and the iliofibularis muscle of Xenopus were mounted in a chamber containing Ringer solution. The solution was stirred and its partial pressure of oxygen (PO2) was continuously measured polarographically. 3. Steady‐state rates of oxygen consumption (VO2) of single fibres were determined as a function of twitch frequency (0.2‐12 stimuli s‐1, depending on the type of fibre). VO2 increased with twitch frequency until a plateau value (VO2,max) was reached. VO2,max of different fibres ranged from 0.042 to 0.169 nmol O2 s‐1 mg‐1 dry weight in Rana and from 0.045 to 0.412 nmol O2 s‐1 mg‐1 dry weight in Xenopus. Under VO2,max conditions oxygen availability was not the limiting factor. 4. VO2 after injection of the uncoupler carbonyl cyanide m‐chlorophenylhydrazone (CCCP) into the chamber correlated with VO2,max, suggesting that VO2,max is determined by mitochondrial density. This suggestion was confirmed by the observation that a close relationship exists between VO2,max and succinate dehydrogenase activity in three different fibre types of Xenopus. 5. At VO2,max a considerable amount of oxygen was taken up after the twitch train by most fibres, indicating that the oxidative ATP synthesis cannot match ATP hydrolysis. Xenopus muscle fibres with high oxidative capacity did not show this phenomenon. 6. The results are discussed in relation to the occurrence of anoxic cores in muscle fibres and the maximum steady‐state contractile activity attainable by the fibres.

Dependency of the force-velocity relationships on Mg ATP in different types of muscle fibers from Xenopus laevis

MgATP binding to the actomyosin complex is followed by the dissociation of actin and myosin. The rate of this dissociation process was determined from the relationship between the maximum velocity of shortening and the MgATP concentration. It is shown here that the overall dissociation rate is rather similar in different types of muscle fibers. The relation between MgATP concentration and the maximum shortening velocity was investigated in fast and slow fibers and bundles of myofibrils of the iliofibularis muscle of Xenopus laevis at 4 degrees C from which the sarcolemma was either removed mechanically or made permeable by means of a detergent. A small segment of each fiber was used for a histochemical determination of fiber type. At 5 mM MgATP, the fast fibers had a maximum shortening velocity (Vmax) of 1.74 +/- 0.12 Lo/s (mean +/- SEM) (Lo: segment length at a sarcomere length of 2.2 microns). For the slow fibers Vmax was 0.41 +/- 0.15 Lo/s. In both cases, the relationship between Vmax and the ATP concentration followed the hyperbolic Michaelis-Menten relation. A Km of 0.56 +/- 0.06 mM (mean +/- SD) was found for the fast fibers and of 0.16 +/- 0.03 mM for the slow fibers. Assuming that Vmax is mainly determined by the crossbridge detachment rate, the apparent second order dissociation rate for the actomyosin complex in vivo would be 3.8.10(5) M-1s-1 for the fast fibers and 2.9.10(5) M-1 s-1 for the slow fibers. Maximum power output as a function of the MgATP concentration was derived from the force-velocity relationships. At 5 mM MgATP, the maximum power output in fast fibers was (73 +/- 8) mW.g-1 dry weight and (15 +/- 5) mW.g-1 in slow fibers. The Km for MgATP for the maximum power output for the fast fibers was (0.15 +/- 0.03) mM, which is about a factor of 4 lower than the Km for Vmax. The implications of these results are discussed in terms of a kinetic scheme for crossbridge action.

Lactate efflux from fatigued fast‐twitch muscle fibres of Xenopus laevis under various extracellular conditions.

1. Isolated, fast‐twitch, low‐oxidative muscle fibres from the iliofibularis muscle of Xenopus laevis were fatigued by intermittent tetanic stimulation at 20 degrees C in different Ringer solutions and the amount of lactate released was determined. 2. The rate of lactate efflux was constant during 10 min of intermittent stimulation while lactate in the fibres accumulated, and lactate efflux was not hampered by an unstirred layer surrounding the isolated muscle fibre. 3. The rate of lactate efflux at extracellular pH 7.2 was the same as that at pH 7.8, but depended on the type of buffer used; the highest efflux rate (mean +/‐ S.E.M., 7.4 +/‐ 2.2 mumol min‐1 (g dry weight)‐1, n = 8) was observed in bicarbonate‐buffered Ringer solution. This rate was about 2.5 times higher than the rate in phosphate‐buffered Ringer solution (2.9 +/‐ 1.3 mumol min‐1 (g dry weight)‐1, n = 8), indicating that lactate‐bicarbonate exchange is the most important route for lactate extrusion in vivo. 4. The highest rate of lactate efflux corresponds to a rate of glycolytic ATP production which is only about 30% of the oxidative rate of ATP production (calculated from the maximum rate of oxygen consumption determined previously). 5. In the presence of 5 mM alpha‐cyano‐4‐hydroxycinnamate (CHC) the lowest lactate efflux rate (1.5 +/‐ 0.6 mumol min‐1 (g dry weight)‐1, n = 16) was found. This rate was independent of the composition of the Ringer solution. Assuming that 5 mM CHC completely inhibits lactate transporters in the sarcolemma, the rate of lactate efflux in the presence of 5 mM CHC can be explained by passive diffusion, but only if most lactate is extruded via the T‐tubules.

Relationship between myoglobin and succinate dehydrogenase in mouse soleus and plantaris muscle fibres

SummaryThis report describes a quantitative histochemical study of myoglobin in skeletal muscle fibres. The muscle fibres were classified as fast or slow on the basis of their quantitative myofibrillar ATPase histochemistry. A large range of myoglobin absorbance values was found among fast skeletal muscle fibres. This range was relatively small among slow fibres. The concentrations of myoglobin and the activities of succinate dehydrogenase in individual muscle fibres in serial sections are weakly correlated in both the mouse soleus and plantaris muscle. The myoglobin concentration is higher in fast and slow oxidative soleus muscle fibres and the succinate dehydrogenase activity in these fibres is lower than in oxidative plantaris muscle fibres in the same range of cross-sectional area.

Genetic architecture of numbers of fast and slow muscle fibres in the mouse soleus muscle

SummaryThis report analyses the genetic architecture underlying a muscle fibre characteristic. We have counted numbers of fast and slow muscle fibres in the soleus muscle of male mice from different inbred strains and crosses between them (diallel cross). Broad heritabilities of 20 per cent and 73 per cent were found for the number of slow fibres and the number of fast fibres, respectively. Additive genetic variation was present for both phenotypes. Directional dominance was found for number of fast fibres only. This suggests that, in evolutionary history, stabilising selection has operated for intermediate numbers of slow muscle fibres and directional selection for high numbers of fast fibres.