P. C. Diegenbach

Maximum rate of oxygen consumption and quantitative histochemistry of succinate dehydrogenase in single muscle fibres of Xenopus laevis

SummaryThree different types of single living muscle fibre were dissected from the iliofibularis muscle ofXenopus laevis. The fibres were mounted in a glass chamber and their rate of oxygen consumption was determined as a function of twitch frequency at 20‡ C. The rate of oxygen consumption increased with twitch frequency until it levelled off and reached a maximum. The maximum rate of oxygen consumption varied between fibres (0.019 to 0.161 nmol O2 s−1 mm−3) and was reached at different twitch frequencies (<0.2 to 5.7 stimuli s−1). After the determination of the maximum rate of oxygen consumption, the succinate dehydrogenase activity in cross sections of the fibre was determined by means of a quantitative histochemical method. A proportional relationship between the maximum rate of oxygen consumption and the succinate dehydrogenase activity was found. The maximum rate of oxygen consumption and the succinate dehydrogenase activity are also proportional to the volume density of mitochrondria in the three fibre types reported by Smith and Ovalle (1973;J. Anat., Lond.116, 1–24). It is concluded that quantitative histochemistry of succinate dehydrogenase reliably predicts the maximum rate of oxygen consumption of muscle fibres inXenopus laevis and that the maximum rate of oxygen consumption of single muscle fibres is determined by the volume density of mitochondria.

Differentiation of the musculature of the teleost Brachydanio rerio

SummaryThe histological differentiations of myotomes and myosepts in the teleost Brachydanio rerio were studied in relation to function and shape development of the myotomes. The presence of contractile elements, intercellular space, growth by cell proliferation and the collagenous structure of the myosepts were considered as important characteristics.To a certain extent, the first deformations of the somites could be explained with these characteristics.It is suggested that firm attachment of the myosept collagen to the notochord sheath and the asymmetrical growth of the myotomes, might be of importance for the development of the oblique orientation of the muscle fibres. The sequence of the differentiation processes is not the same for all muscle cells. Cells next to the notochord synthetize myofilaments before they become polynuclear, while cells elsewhere in the myotome become polynuclear by fusion before they start to synthetize myofilaments. Some aspects of the histological differentiation of the myotomes in B. rerio were compared with myotome development in the chick, Gallus domesticus.

Resistance to fatigue of single muscle fibres from Xenopus related to succinate dehydrogenase and myofibrillar ATPase activities

This report describes how the resistance to fatigue of a muscle fibre relates to the fibres most important ATP‐producing and ATP‐consuming reactions. Twelve intact single muscle fibres were dissected from lumbrical muscles of Xenopus laevis. Their resistance to fatigue induced by repeated tetanic stimulation was determined, as well as their succinate dehydrogenase activity and calcium‐stimulated myofibrillar ATPase activity. The enzyme activities were determined by means of quantitative histochemistry. It was found that resistance to fatigue correlates with succinate dehydrogenase activity (r = 0.83) and with myofibrillar ATPase activity (r = ‐0.74). The highest correlation was found between resistance to fatigue and the ratio of succinate dehydrogenase to myofibrillar ATPase activity (r = 0.93). It is concluded that muscular fatigue is closely related to cellular energetics.

Identification of motoneurons in the spinal cord of the zebrafish (Brachydanio rerio), with special reference to motoneurons that innervate intermediate muscle fibers

SummaryWe investigated the location and size distribution of motoneurons that innervate red, intermediate and white muscle fibers in the axial musculature of the zebrafish (Brachydanio rerio). Motoneurons were identified by retrograde labeling from the respective myotomal compartments with horseradish peroxidase applied either in small polyacrylamide gel fragments or as pure crystals. We found a spatial relationship between the three myotomal muscle fiber types and the corresponding motoneurons. The white motoneurons are grouped in the dorsal part of the motorcolumn, near the central canal. Motoneurons of the red and intermediate muscle are clustered in the ventral part of the motorcolumn. The average position of the red motoneurons is ventral to that of the intermediate motoneurons. Soma sizes are distributed over wide overlapping ranges, spanning from 41 to 352 μm2 for red and intermediate and from 56 to 894 μm2 for white motoneurons. These data are discussed in relation to the recruitment order of myotomal muscle fibers of different types as revealed by electromyographic recordings in fish, and the so called “size principle” for motoneuron recruitment.

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.

Calibration of Quantitative Histochemical Methods: Estimation of Glycogen Content of Muscle Fibers Using the PAS Reaction

A fairly simple method for calibrating microdensitometric histochemical assays is described. The method is based on paired biochemical and histochemical assays on single freeze-dried skeletal muscle fibers which differ widely in their properties. As an example, the method is applied to investigate the validity of the periodic acid-Schiff (PAS) reaction for the microdensitometric estimation of glycogen content. Some problems that may interfere with the calibration are discussed.

SIZE PRINCIPLE OF STRIATED MUSCLE CELLS

We have investigated the relationship between cross-sectional area (CSA) and maximum rate of oxygen consumption (VO2max, in nmol .mm-3 .s-1) of heart and skeletal muscle cells from different species. VO2max and CSA were determined for single muscle fibres of Xenopus laevis at 20°C and for cardiomyocytes in thin trabeculae dissected from the right ventricle of rats at 38°C. Succinate dehydrogenase activity was determined using a quantitative histochemical method to estimate VO2max in mammalian skeletal muscle fibres. Literature values of volume density of mitochondria were used to estimate VO2max in some mammalian cardiomyocytes. We found that an inverse relationship exists between VO2max (range 1.5 to 0.024 nmol . mm-3 . s-1) and cross-sectional area (0.0002 to 0.018 mm2, respectively): VO2max = constant/CSA, where the value of the constant equals 0.39±0.18 pmol . mm-1 .s-1 (mean ± S.D., n = 14). Such a relationship is predicted by a simple Hill-type model for oxygen diffusion in cilindrical cells, if it is assumed that muscle cells are evolved so that anoxic cores in muscle cells are prevented. This indicates that the product of endurance of muscle cells (which is proportional to VO2max) and force production (which is proportional to cross-sectional area) is limited by oxygen diffusion, and that adaptation of heart and skeletal muscle cells to increased workload is limited by the interstitial oxygen tension.

Muscle fibre typing with sera against myosin and actin: A comparison between enzyme- and immunohistochemical classification

Sera raised against actin and myosin, extracted from white muscle of fish, were used for the immune-histochemical characterization of muscle fibers. It appeared that both, the actin- and the myosin serum are specific for white muscle fibres in fish. Further it was found that in both, the A- and the I-band of the sacromeres, fibre type specific proteins are present. The classification of muscle fibre types obtained with the antisera was compared with the classification obtained with some enzyme histochemical reactions. Muscle fibres that reacted positively with the two sera, also showed a high histochemical myofibrillar ATP-ase activity. The correlation with a low succinate dehydrogenase- and a high lactate dehydrogenase activity was not always found.

Fixation induced variations in quantitative nuclear image features in sections.

Recently quantitative nuclear image features (Q.N.I. features) have become available which have proven to be accurate descriptors of different chromatin patterns. Therefore it can be expected that these features will become increasingly important in diagnostic histopathology. However, if used for routine applications in human pathology, it is to be questioned to which extent Q.N.I. features are able to discriminate between subpopulations of nuclei and to which extent fixation induced variations on Q.N.I. features do exist. In the present study, the discriminating capacity of the Q.N.I. features on the basal and parabasal cell nuclei of human cervical epithelium is investigated. Fixation is varied in concentration, temperature, and time. The results emphasize the excellent discriminating power of the Q.N.I. features and indicate that if a) the concentration is kept between 4 and 6% w/v, b) the temperature of the fixative is room temperature, and c) the fixation time is kept constant at 24 or 36 h, the Q.N.I. features can well be used in histopathology.