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Sodium and calcium components of the action potential in a developing skeletal muscle cell line.

1. Developmental changes in action potential properties were studied in a clonal rat skeletal muscle cell line. 2. Small action potentials were evoked in mononucleate myoblasts. No spike was seen in Na-free saline. A similar spike was evoked in a medium where all NaCl was replaced by LiCl. No spike was evoked when NaCl was replaced by CsCl. 3. Action potentials overshot zero membrane potential in multinucleate myotubes. The action potential was composed of two components, an initial fast spike and a hump on the falling phase or in some cases a distinct second peak. 4. Teh overshoot of the initial fast spike decreased when the external Na concentration was decreased. 5. In saline with 10 mM-Ca the second component often formed a distinct peak following the initial fast spike. A slow regenerative potential was evoked in Na-free media with a depolarizing current pulse. 6. In saline containing BaCl-2 instead of CaCl-2 there was always a second peak, the overshoot of which changed with external Ba concentration. A slow regenerative potential was evoked in Na-free, Ba-saline. The membrane conductance at the peak of the Ba-action potential was larger than in the resting state. 7. In adult rat skeletal muscle, the shape of the action potential was not changed when Ca was replaced by Ba. No action potential was evoked in Na-free Ba-saline or Ba-saline with tetrodotoxin (3 times 10-7 M). 8. The significance of the Ca component in the developing muscle is discussed.

Action Potentials

Postnatal cytochemical development of muscle fibers in segmental tail muscles of the rat.

Postnatal development of extrafusal and intrafusal muscle fibers was examined histochemically in segmental tail muscles of the rat. At birth all fibers show a strong reaction for myosin ATPase, uniformity in diameter, and homogeneity in staining intensity. During the first postnatal week, the muscle fibers undergo gradual hypertrophy and hyperplasia but they all maintain the same intense homogeneous staining pattern for the enzyme. By day 9, further differentiation of the muscle fibers results in the formation of a second intrafusal fiber type while the extrafusal fibers are still relatively homogeneous. Finally, two kinds of extrafusal fiber and a third type of intrafusal fiber can be distinguished by day 21. This histochemical fiber pattern is essentially maintained in the adult. These findings show that fiber type development in rat tail muscles lags behind the usual time course of myogenesis known to occur in more rostral regions of the animal. It also indicates that histochemical differentiation of intrafusal fibers in these muscles does not parallel that which occurs in extrafusal fibers. It is likely that arrival and initial contact of sensory nerve terminals on developing intrafusal fibers at day 7 directly influences their relatively early histochemical heterogeneity.

Adenosine Triphosphatases

Effects of easily chewable diet and unilateral extraction of upper molars on the masseter muscle in developing mice.

The effects of easily chewable diets and unilateral extraction of upper molars on the masseter muscle were studied in developing mice. A liquid diet requiring no mastication suppressed the development of the masseter muscles more than a fine-grained diet, and extraction of unilateral upper molars also caused inhibition of muscle development. Moreover, both unilateral extraction of upper molars and a liquid diet had an additive effect on the suppression of the postnatal development of the masseter muscle, and bilateral suppression of the development of the masseter muscle was induced following unilateral extraction of upper molars. These findings suggest that the sensory input from the sensory endings in the periodontal ligament may also play an important role in the postnatal development of the masseter muscle and that there may be some crossing pathways to convey the sensory input coming from the side of the extracted upper molars to the contralateral motor neurons via the interneuronal circuits.

Animals

Reinnervation of developing rat muscle by non-axotomized motoneurons.

To study the ability of developing motoneurons to reinnervate their denervated muscle, axotomized motoneurons in rat neonates and pups were retrogradely labeled with two fluorescent tracers. Fluorogold (FG), a long-lasting fluorescent dye, was injected into intercostal muscle T8 to retrogradely label the motoneurons that innervated it. Two days later intercostal nerves T7-T9 were cut. The intercostal muscle denervated at birth was reinnervated within 10-20 days, as evidenced by nerve-evoked muscle contraction. Three weeks following axotomy, tetramethylrhodamine isothiocyanate (TRITC) was injected into the same muscle to label the motoneurons that reinnervated it. The motoneurons double-labeled with FG and TRITC were, therefore, axotomized motoneurons that regenerated to reinnervate T8. In neonates, axotomy resulted in a significant reduction in the number of FG-labeled motoneurons, which suggests that axon transection at early postnatal days causes a massive motoneuron death. The percentage of double-labeled motoneurons was significantly smaller than that in non-axotomized rats. TRITC-labeled motoneurons constituted the majority of stained motoneurons; these were located in different nuclei than the intercostal motoneurons. These findings suggest that muscle reinnervation is, at least in part, by motoneurons which originally did not innervate intercostal muscle T8. Unlike axotomy at birth, axotomy performed 2-3 weeks after birth did not result in a significant motoneuron loss. The number of stained motoneurons labeled with both FG and TRITC was significantly smaller, however, than in non-axotomized spinal cords. Our data indicate that in pups only a small percentage of axotomized motoneurons reinnervated the denervated muscle.

Animals

Adult motor patterns produced by moth pupae during development.

Muscle potentials were recorded extracellularly from developing pupae and adults of the saturniid moths Antheraea polyphemus and A. pernyi and the sphingid moth Manduca sexta. During the week prior to the terminal ecdysis, developing moths still enclosed within the pupal cuticle produced motor patterns similar to those recorded from adults during flight and shivering. The pupal patterns had a longer cycle time and were more variable than the adult motor patterns. Characteristic inter-family differences in adult motor patterns were apparent in pupal motor patterns. Development of motor patterns was followed over several days by observing individuals with chronically implanted leads. Early in the pupal period potentials were small and infrequent. The amount of activity gradually increased and became more patterned. As development proceeded adult patterns were produced for increasing lengths of time, although the patterns changed quickly and spontaneously. Restricting the wing movements of A. polyphemus adults increased the cycle time, increased the number of spikes per burst in muscles opposing the restraint, and did not alter the interspike interval within a burst. The flight patterns produced by pharate moths, in which the wings are also immobile, also have a longer cycle time than that of adult flight, but the number of spikes per burst the same and the interspike interval is longer than in adult flight. These observations suggest that the differences between pupal and adult patterns are not necessarily due to the confinement of the wings by the pupal cuticle.

Action Potentials

Ultrastructure of human intramuscular blood vessels in development.

Muscles from human foetuses of nine weeks to nine months development were examined by electron microscopy. Capillaries, arteries and veins are frequent in the human foetal muscle at all stages of development. At nine weeks the vessels have the appearance of capillaries and basement membrane lies around the endothelial cells forming them. The capillaries are of continuous type which do not have apertures in their walls. Tight junctions are seen at some zones of adjacent endothelial cells of the capillaries. Sometimes, pericytes are also seen. At sixteen weeks vessels having the features of veins and of arteries can be identified between the muscle cells. Regarding the arteries, some of the endothelial cells are united to the smooth muscle cells and the intimal elastic lamina is interrupted where these cells approximate. The significance of this junction may be to anchor the intima to the media.

Arteries

Changes in the protein kinase C activity or rat sternomastoid muscle during development and after denervation.

The relationship between the activity of protein kinase C (PKC) and muscle innervation was explored in the rat sternomastoid muscle (SM) from day 18 of gestation (E18) to adult age. Between E18 and birth, PKC activity rose 5-fold, and during the day after birth, diminished to a level characteristic of the mature muscle. The rise chiefly occurred in the neural part of the muscle, in both the membrane and the cytosol fractions. Between E18 and day 5 after birth, the ratios of membrane to cytosol PKC activity rose from 0.5 to 10 and 3 respectively in the neural and aneural parts of the muscle. Denervation of adult SM reduced PKC activity by half in the membrane fraction of the neural part but did not significantly change it in the membrane or cytosol fractions of the aneural parts. These results suggest that innervation plays an important part in determining the level of PKC activity in muscle.

Aging

[Effects of denervation of the masseter nerve and bite raising on the masseter muscle of developing rats].

The effects of masseteric denervation and bite raising on muscle fiber type differentiation were examined in the masseteric muscle of developing rats by histological and histochemical studies. Three-week-old Wistar rats had of their masseteric nerve at right side dissected, and a part of them were bite raised at anterior region one week after denervation for three weeks. The unoperated side and number of sham animals served as control. The animals were killed 3, 7, 10, 14, 21, 28, 35, 49 days later and then their masseter muscles were removed wholly and weighed them. After freezing of superficial and deep masseter muscles serial sections were made and HE staining carried out, ATPase staining and NADH-TR staining. On the photographs of the HE stained specimens, I measured the diameter of the muscle fibers. Gross findings on the denervated group, revealed the lower incisors shifted to unoperated side and on with the bite raising group, it shifted to the operated side. In denervated animals, the wet weight of the masseter muscle had decreased significantly. The masseteric muscle fibers in three-old-week control rats were undifferentiated on ATPase staining, but it became well differentiated on and after four-old-week. The superficial masseter of control mainly composed of type 2B fibers. In the deep masseter, about 10% type 1 and 2C fibers were found in limited area around the muscle spindles, and surrounded with type 2A and 2B fibers. In superficial masseter muscle of the denervated group, the percentage of type 2A fibers increased, and that of type 2B fibers decreased. The type 2C fibers were found from the 21st days after denervation. In deep masseter muscles, the percentage of type 1 and 2A fibers decreased, and that of type 2B and 2C fibers increased. In the denervated bite raising group, the composition of the muscle fiber type approached the control group. These results as above suggested, masseteric denervation causes degeneration of muscle fiber composition, and the possibility that early preventive treatment like bite raising may recover the muscle fiber composition normally.

Animals

The fine structure of developing locomotor muscles of the pelagic tunicate, cyclosalpa affinis (Thaliacea: Salpidae).

Salps are free-swimming tunicates whose peculiar life history renders them ideal for developmental studies. The solitary salp reproduces asexually by budding a stolon containing the complete developmental sequence of the aggregate generation. The ultrastructure of developing locomotor muscle of the aggregate generation of Cyclosalpa affinis was studied. The early muscle contains essentially non-striated myofibrils. However, in transverse sections, , areas indicating early I-band A-bands can be recognized. As development continues, the number of fibrils increases, the Z-line appear, and the fibrils contain more recognizable striations. The fully developed muscle has the caracteristic structure of striated muscle. Longitudinal sections show sarcomeres with irregular and discontinuous (perforated) Z-lines; H-zones are not apparent. No M-lines are seen. Throughout development, the ratio of thin to thick myofilaments is always 2:1, the ratio found in all vertebrate striated muscle. Other finding in C affinis suggest that: (1) multinucleated muscle cells are formed by the fusion of mononucleated cells, (2) membranes of adjacement mononucleated cells destined to fuse form myelin figures, and (3) these myelin figures become closely associated with mitochondria.

Animals

Muscle abnormalities in coeliac disease: studies on gross motor development and muscle fibre composition, size and metabolic substrates.

In 11 children with coeliac disease gross motor development was assessed before and during diet treatment using the gross motor subscale of the Denver developmental screening test. ATP, creatine phosphate (CP), glycogen and lactate concentrations, muscle fibre size and fibre composition were measured in specimens obtained by needle biopsy from the vastus lateralis muscle. Before treatment, gross motor development was delayed. ATP, and to a lesser extent, CP and glycogen concentrations were lowered compared to a control group. After treatment, gross motor development was normal and no differences in ATP, CP or glycogen concentrations were found compared to the control group. Fibre size seemed unaffected by the disease. The percentage of type 1 fibres was significantly lower before treatment, compared to values obtained during treatment and from the control group. Whether these metabolic changes were due to the coeliac disease per se or the inactivity which it causes was not possible to establish. In humans, only altered neurogenic influence on the muscles has been previously shown to give changes in fibre composition.

Adenosine Triphosphate

Ca+2-accumulating components in developing skeletal muscle.

This ultrastructural study on the localization of Ca+2 in developing skeletal muscle indicates that the formation of calcium-accumulating components begins during embryonic development. Both oxalate and pyroantimonate techniques are used to localize Ca+2 in distinct cellular components of chick pectoral and sartorius muscles. Two major sites for Ca+2 accumulation are present in ultrathin sections of embryonic and post-embryonic muscles: the terminal cisternae of the sarcoplasmic reticulum and specific lines in the I-bands. Calcium oxalate-accumulating vesicles are present in the smallest recognizable myotubes at the twelfth day of incubation, but calcium-accumulating components are not seen at myofibrillar I-band sites until the fourteenth to seventeenth days of incubation. The fact that myofibrils first form and later in development accumulate a Ca+2-binding component suggests that this Ca+2-binding component is not necessary for the formation of myofibrils, but is added to myofibrils before hatching to serve a probable regulatory role in contraction.

Age Factors

The importance of competition between motoneurones in developing rat muscle; effects of partial denervation at birth.

1. The number of motor units in developing fourth deep lumbrical muscles was reduced by unilateral partial denervation of the muscle at birth, by cutting the lateral plantar nerve. A minority of the motor axons arrive via the sural nerve, and were thus not cut. Those muscles that contained one motor unit (one-unit muscles) after partial denervation developed in the absence of competition between motoneurones. Muscles with two motor units had little competition. A few four-unit muscles were studied for comparison. 2. Isometric twitch and tetanic tensions of single motor units were recorded in vitro at 60 days of age in response to stimulation of the sural nerve. On average, units in partially denervated muscles generated more tension than normal units. The isometric tension characteristics of the units in the one-unit and two-unit muscles were different from the normal units (e.g. slower contracting and more fatiguable). The units of four-unit muscles had properties similar to those of normal muscles. 3. Fibres of an individual unit were identified by glycogen depletion and S (slow) fibres were identified in cross-section that bound a polyclonal antibody to slow type I myosin. Those fibres that did not bind the antibody were designated F fibres. The units of one-unit muscles had the same total number of fibres and fibre type composition (both S and F fibres in the same unit) as estimated from previous work to exist at birth. The units of two-unit muscles contained the same total number of fibres, but apparently fewer S fibres, though this may have been as a result of incomplete glycogen depletion of some fibres. 4. It is concluded that competition between motoneurone terminals is necessary for the withdrawal of mismatched connections on muscle fibres present at birth; or, alternatively, that such withdrawal cannot take place if it would result in denervation of the muscle fibre.

Animals

Examination of the calcium-modulated protein S100 alpha and its target proteins in adult and developing skeletal muscle.

In this study radioimmunoassay, immunohistochemistry, Northern blot analysis, and a gel overlay technique have been used to examine the level, subcellular distribution, and potential target proteins of the S100 family of calcium-modulated proteins in adult and developing rat skeletal muscles. Adult rat muscles contained high levels of S100 proteins but the particular form present was dependent on the muscle type: cardiac muscle contained exclusively S100 alpha, slow-twitch skeletal muscle fibers contained predominantly S100 alpha, vascular smooth muscle contained both S100 alpha and S100 beta, and fast-twitch skeletal muscle fibers contained low but detectable levels of S100 alpha and S100 beta. While the distribution of S100 mRNAs paralled the protein distribution in all muscles there was no direct correlation between the mRNA and protein levels in different muscle types, suggesting that S100 protein expression is differentially regulated in different muscle types. Immunohistochemical analysis of the cellular distribution of S100 proteins in adult skeletal muscles revealed that S100 alpha staining was associated with muscle cells, while S100 beta staining was associated with nonmuscle cells. Radioimmunoassays of developing rat skeletal muscles demonstrated that all developing muscles contained low levels of S100 alpha at postnatal day 1 and that as development proceeded the S100 alpha levels increased. In contrast to adult muscle S100 alpha expression was confined to fast-twitch fibers in developing skeletal muscle until postnatal day 21. At postnatal day 1, developing contractile elements were S100 alpha positive, but no staining periodicity was detectable. At postnatal day 21, S100 alpha exhibited the same subcellular localization as seen in the adult: colocalization with the A-band and/or longitudinal sarcoplasmic reticulum. Comparison of the S100 alpha-binding protein profiles in fast- and slow-twitch fibers of various species revealed few, if any, species- or fiber type-specific S100 binding proteins. Isolated sarcoplasmic reticulum fractions and myofibrils contained multiple S100 alpha-binding proteins. The colocalization of S100 alpha and S100 alpha-binding proteins with the contractile apparatus and sarcoplasmic reticulum suggest that S100 alpha may regulate excitation and/or contraction in slow-twitch fibers.

Aging

Mechanics and electrolyte composition of arterial smooth muscle in developing dogs.

The effects of age on arterial smooth muscles (SM) mechanics and electrolyte composition were studied during growth and development in six litters of puppies and their mothers from isolated segments of carotid, renal, mesenteric, and iliac arteries. An increase in the maximum wall stress following both norepinephrine (NE) and potassium (K) was found with age at all sites. The ratio of the stress response for K/NE was found to decrease with age. The maximum stress response for both NE and K shifted to lower values of wall strain with age in the same manner that passive stress-strain curves were shifted. The maximum diameter re3 (3 SM activator to SM activator were not significantly altered with age, but they were better maintained at higher transmural pressure in older animals. Chemical analysis indicated "cell" water, Mg, and K contents decreased with age, as did extracellular water, Na, and Cl. Na anhe suggest that changes in SM excitation-contraction coupling occur with age. Passive wall elements appear to interact with the contractile elements in determining wall responses to SM activation.

Aging

Intramembrane charge movement in developing skeletal muscle cells from fetal mice.

The development of intramembrane charge movement was studied in freshly isolated skeletal muscle cells from 13- to 19-day-old mouse fetuses. Charge movement was present in myotubes from 13-day-old fetuses. The relationship between charge movement and membrane potential could be described by a two-state Boltzmann equation. The amount of maximum charge movement (Qmax) increased substantially with the age of the fetuses from 2.84 +/- 0.39 nC/microF (n = 10) at day 13 to 10.01 +/- 0.97 nC/microF (n = 15) at day 19. Nifedipine (1 microM) consistently reduced Qmax by 33 +/- 2% (n = 37) of the control value at each age studied. Increasing the concentration of nifedipine to 20 microM had no further effect, suggesting that the charge movement in developing myotubes consists of at least two components: a nifedipine-sensitive charge movement (Qns) and a nifedipine-resistant one (Qnr). Both Qns and Qnr increased exponentially with a distinct enhancement of rate at day 16.

Animals

Indirect fluorescence of primary and secondary myofibers in developing porcine muscle.

Cytochemical differentiation of two populations of developing skeletal myofibers has been demonstrated in fetal muscle with metachromatic fluorescence of ribonucleic acid and deoxyribonucleic acid by staining fresh frozed cryostat sections of developing porcine skeletal muscle with acridine orange (CL. 46005). Evidence is presented that supports the hypothesis that first-formed myofibers (primary myofibers) serve as a structural framework upon which myoblasts proliferate, fuse in linear sequence and give rise to a second population (secondary myofibers) of myofibers.

Animals