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[Calpains, protein kinase c and development of muscle tissue].

Calpains are Ca2+ -dependent thiol proteases which have been identified in various tissues of eucaryotes, but their physiological function in the cell is uncertain. In the muscle fiber, two types of calpains are present which differ by their calcium sensitivity: calpain 1 and calpain 2, which require for their activity micro and millimolar concentrations of calcium respectively. These calpains are associated with protein kinase C activities in the differentiated fiber. The multinucleate myotube is formed by fusion of mononucleated precursor cells, myoblasts. Calpains have been reported to appear in myoblasts at around the time of fusion. Moreover, an apparent synthesis of 1,2 diacylglycerol, an activator of protein kinase C, was observed during fusion of myoblasts. However, more information is required to incriminate totally protein kinase C and calpains in the mechanism of myoblast fusion.

Animals

Adult forms of the Ca2+ATPase of sarcoplasmic reticulum. Expression in developing skeletal muscle.

Two separate genes encode fast-twitch and slow-twitch/cardiac muscle forms of the Ca2+ ATPase of sarcoplasmic reticulum. Full length Ca2+ ATPase clones have been isolated from adult rabbit fast-twitch, slow-twitch, and cardiac muscles. Segments of these clones containing unique sequences have been used as probes to study developmental changes in Ca2+ ATPase transcripts. The fast-twitch Ca2+ ATPase transcript undergoes developmentally regulated alternative splicing in which a penultimate 42-base pair exon is retained in the adult transcript but is excised in the neonatal transcript. This additional exon shifts the exon encoding the neonatal carboxyl-terminal sequence, -Asp-Pro-Glu-Asp-Glu-Arg-Arg-Lys (Brandl, C. J., Green, N. M., Korczak, B., and MacLennan, D. H. (1986) Cell 44, 597-607) into a nontranslated region and results in the expression of an adult isoform with a carboxyl-terminal -Gly. The neonatal form of the fast-twitch Ca2+ ATPase represents 72% of the fast-twitch Ca2+ ATPase transcripts just prior to birth but only 17% by 14 days of age and 4% in adult fast-twitch muscle. Adult slow-twitch, adult cardiac, and neonatal skeletal muscles express an identical Ca2+ATPase mRNA transcript which is distinct from either of the fast-twitch forms. The slow-twitch/cardiac Ca2+ ATPase is the predominant form expressed in late fetal and early neonatal rabbit skeletal muscle, but this form is lost as the skeletal muscle differentiates into a fast-twitch state. Three or more alternative polyadenylation signals exist for this mRNA in all tissues with the most 3' signal predominating.

Aging

The effect of selective, chronic stimulation on motor unit size in developing rat muscle.

One of the two peripheral nerves which innervate rat lumbrical muscle was stimulated chronically in vivo during the postnatal period of synapse elimination to determine whether the differential stimulation would affect the outcome of the elimination process. Rats were anesthetized for about 4 hr a day for 5 to 6 consecutive days, during which time the sural nerve (or, in other animals, the lateral plantar nerve) was electrically stimulated. Each animal received about 10(6) stimuli. After the last stimulation period, the sizes of motor units in both nerves were estimated from motor unit tension recorded in vitro. We found that, on average, sural motor units were larger than others in animals which had received sural nerve stimulation and smaller than others in animals which had received lateral planter nerve stimulation. These results are consistent with the hypothesis that more active nerve terminals possess a relative advantage in competing for occupancy of the endplate.

Animals

The formation of synapses in striated muscle during development.

1. A study has been made of the formation of synapses in developing striated muscles which receive either a focal (the rat hemidiaphragm) or a distributed (the avian anterior latissimus dorsi) innervation using histological, ultrastructural and electrophysiological techniques.2. In the developing diaphragm only a single synaptic contact was initially established at random along the length of the short (300 mum) myotubes by a single axon; in the developing ALD more than one synaptic contact could be established initially along the length of the long (2500 mum) myotubes by axons, but the distance between these was never less than 170 mum.3. Each synapse established by the initial axonal contact in either the diaphragm or the ALD subsequently received a multiple innervation from further exploring axons in the muscles, and all such additional innervation of muscle cells was constrained to the sites of the initial synaptic contacts; this multiple innervation of synaptic sites was lost in the subsequent 4 weeks.4. It is suggested that the axon forming the initial synaptic contact on myotubes induces a property over an adjacent length of myotube which makes its membrane refractory to synapse formation over this length; this characteristic length is longer for axons forming a focal innervation than it is for those forming distributed innervation.

Acetylcholine

Expression of myosin heavy chain isoforms in developing rat muscle spindles.

The development of muscle spindles, with respect to the expression of myosin heavy chain isoforms was studied in rat hind limbs from 17 days of gestation up to seven days after birth. Serial cross-sections were labelled with antibodies against slow tonic, slow twitch and neonatal isomyosins, myomesin, laminin and neurofilament protein. At 17-18 days of gestation, a small population of primary myotubes expressing slow tonic myosin were identified as the earliest spindle primordia. These myotubes also expressed slow twitch and, to a lesser extent, neonatal myosin. At 19-20 days of gestation a second myotube became apparent; this staining strongly with anti-neonatal myosin. A day later this secondary myotube acquired reactivity to anti-slow tonic and anti-slow twitch myosins. By birth, a third myotube was present; this staining strongly with anti-neonatal myosin but otherwise unreactive with the other antibodies against myosin heavy chains. Three days after birth a fourth myotube, with identical reactivity to the third one, became apparent. Regional variation in the expression of isomyosins, which was present since birth in the two nuclear bag fibers was further enhanced: the nuclear bag staining strongly with anti-slow tonic and antineonatal in the equatorial region and with decreasing intensity towards the poles, whilst with anti-slow twitch the stainability was low in the equatorial and high in the polar region. The nuclear bag fiber showed a homogeneous staining: high with anti-slow tonic, moderate with anti-neonatal, and displayed stainability to anti-slow twitch myosin in the polar regions only. No regional variation was found along the chain fiber/myotube.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals

The influence of muscle length on muscle fibre conduction velocity and development of muscle fatigue.

The influence of muscle (vastus lateralis) length on the muscle fibre conduction velocity (MFCV) and on muscle fatigue was studied in 8 healthy volunteers. In experiment 1, the electromyographic (EMG) responses were evoked by electrical stimulation of the motor point and recorded by a surface electrode array aligned along the muscle fibre direction. The MFCV (determined by cross-correlation) was measured at knee flexions of 5 degrees (full extension), 45 degrees, 90 degrees and 120 degrees with 3 different extension torques. The MFCV declined with increasing muscle length and increased with increasing background torque at knee flexions from 5 degrees to 90 degrees. From 90 degrees to 120 degrees knee flexion of MFCV tended to increase. In experiment 2, the EMG activity at a static fatiguing contraction (80% MVC) was measured at 45 degrees and 90 degrees knee flexion. The EMG was measured until the subject gave up contracting the muscle (endurance). The largest increase in the RMS amplitude and the fastest decreases in the mean power frequency (MPF) and MFCV were found at 90 degrees flexion. The MVC at 45 degrees knee flexion was 35% lower than at 90 degrees and the time until endurance was approximately twice as long for the 45 degrees contraction. The results indicate that muscle length is an important parameter for the propagation velocity of action potentials and for the development of static muscle fatigue.

Action Potentials

Shortening velocity and myosin heavy chains of developing rabbit muscle fibers.

The regulation of vertebrate muscle contraction with respect to the role of the different subunits of myosin remains somewhat uncertain. One approach to gaining a better understanding of the molecular basis of contraction is to study developing muscle which undergoes changes in myosin isozyme composition and contractile properties during the normal course of maturation. The present study utilizes single fibers from psoas muscles of rabbits at several ages as a model system for fast-twitch muscle development. This approach eliminates the inherent problems of interpreting results from studies on whole muscles which usually contain heterogeneous fiber types with respect to contractile properties and isoenzyme composition. Maximum velocity of shortening and tension-generating ability of individual fibers were measured and the myosin heavy chain composition of the same fibers was examined using an ultrasensitive sodium dodecyl sulfate-polyacrylamide gel system. The results indicate that 1) with regard to contractile properties, there is a transitional period from slow to fast shortening velocities within the first postnatal month; 2) a strong, positive correlation exists between the speed of shortening and tension-generating ability of individual postnatal day 7 fibers, suggesting that as more myosin is incorporated in these developing fibers it is of the fast type; and 3) there is a wide variation in maximum velocity of shortening among postnatal day 7 psoas fibers which is also a time when a mixture of heavy chain isoforms characterizes the myosin composition of single muscle fibers.

Aging

Role of nerve and muscle factors in the development of rat muscle spindles.

The soleus muscles of fetal rats were examined by electron microscopy to determine whether the early differentiation of muscle spindles is dependent upon sensory innervation, motor innervation, or both. Simple unencapsulated afferent-muscle contacts were observed on the primary myotubes at 17 and 18 days of gestation. Spindles, encapsulations of muscle fibers innervated by afferents, could be recognized early on day 18 of gestation. The full complement of spindles in the soleus muscle was present at day 19, in the region of the neuromuscular hilum. More afferents innervated spindles at days 18 and 19 of gestation than at subsequent developmental stages, or in adult rats; hence, competition for available myotubes may exist among afferents early in development. Some of the myotubes that gave rise to the first intrafusal (bag2) fiber had been innervated by skeletomotor (alpha) axons prior to their incorporation into spindles. However, encapsulated intrafusal fibers received no motor innervation until fusimotor (gamma) axons innervated spindles 3 days after the arrival of afferents and formation of spindles, at day 20. The second (bag1) intrafusal fiber was already formed when gamma axons arrived. Thus, the assembly of bag1 and bag2 intrafusal fibers occurs in the presence of sensory but not gamma motor innervation. However, transient innervation of future bag2 fibers by alpha axons suggests that both sensory and alpha motor neurons may influence the initial stages of bag2 fiber assembly. The confinement of nascent spindles to a localized region of the developing muscle and the limited number of spindles in developing muscles in spite of an abundance of afferents raise the possibility that afferents interact with a special population of undifferentiated myotubes to form intrafusal fibers.

Animals

An immunocytochemical study of type I muscle fibres in developing human skeletal muscles.

An immunocytochemical study was done on the skeletal muscles of human fetuses (19-36 weeks gestation), infants and adults using a new monoclonal antibody (McAb) ALD-47. The antibody was generated against slow myosin of chicken and is specific for myosin heavy chain (MHC). In human infants and adults the type I muscle fibres are strongly reactive with this McAb and the type II fibres uniformly non-reactive. In the fetuses from 19-20 weeks gestation (in whom the fibre types are not distinguishable by the histochemical myosin ATPase test) a proportion of muscle fibres react specifically with ALD-47. Other muscle fibres at this stage react positively with a fast specific MHC McAb HM-1.2 or are negative to both ALD-47 and HM-1.2 antibodies. These McAbs, thus, identify three distinct fibre populations in the early fetal muscle which by histochemical staining appears homogeneous. The percentage of ALD-47 positive fibres increases in fetuses at later gestational periods; at all stages these fibres lack reactivity with the HM-1.2 antibody. Because of its selective fibre type reactivity in differentiating muscles, the McAb ALD-47 in conjunction with HM-1.2 should be useful in immunoaffinity fractionation and biochemical studies of myosin isoforms in developing human muscles.

Adenosine Triphosphatases

Development of muscle nerve in the teleost fish, medaka.

The entire process of normal development of a muscle nerve to a muscle (middle interradial muscle) in the tail region of the medaka (Oryzias latipes) is briefly reported. The nerve was stained immunohistochemically by using anti-neurofilament protein antibodies or stained by HRP and DiI labeling methods. The muscle was stained immunohistochemically by using anti-troponin T and anti-desmin antibodies. The smallness and transparency of the medaka embryos provide us with an opportunity to examine nerve-muscle development in whole-mount specimens. Our observations suggest that prior to the appearance of the middle interradial muscle a neural pathway has established, extending from the starting point to the 'door step' of the muscle.

Animals