Species- and muscle type-dependence of perinatal isomyosin transitions.

The progressive transition from developmental to adult myosin isoforms during perinatal development was quantified in four muscles (diaphragm, gastrocnemius medialis, masseter and tongue) of four mammals (guinea-pig, hamster, rabbit and rat). It was observed that the timing of transition varied for each muscle, and differed according to the mammal as well. This suggests that the synthesis of adult myosin isoforms may be partly related to the specialized contractile function of a given muscle in a given species.

The same myosin isoforms are found in the female and male sexually dimorphic levator ani muscle of the rat, but their postnatal transitions are not synchronous.

The levator ani of the female adult rat is greatly atrophied in comparison to the same muscle in males. In the present study, the female levator ani was, nevertheless, found to contain type IIb myosin isoforms similar to those contained in the male muscle. These adult type isoforms were, however, synthesized later in the female than in the male levator ani: the half-transition times of the myosin transition curve were 20 days postnatal in the male and 35 days postnatal in the female. The transition curves for castrated and uncastrated male rats were the same. Thus, the presence of male gonadal hormones apparently did not affect the myosin transition.

Opposite regulations by androgenic and thyroid hormones of V1 myosin expression in the two types of rabbit striated muscle: skeletal and cardiac.

The finding that V1 cardiac myosin is expressed in masticatory skeletal muscles of the rabbit provided a unique opportunity for comparing the hormonal regulation of V1 in skeletal and cardiac muscles. Thyroid hormones had no significant effect on the postnatal expression of V1 in masticatory muscles, but increased this expression in cardiac ventricles. In contrast, androgenic hormones reduced V1 expression in masticatory muscles, but did not affect it significantly in cardiac ventricles. Modulation of V1 gene transcription in striated muscle is thus shown here to depend both on the target muscle and on the hormone.

Relationship between muscle myosin isoforms and contractile features in rabbit fast-twitch denervated muscle.

The effects of 8-day-old rabbit fast-twitch gastrocnemius denervation on the type of myosin isoforms and on contractile features (maximum velocity Vmax and contraction time (CT) of the muscle were followed between 15 and 60 days postnatal. The myosin isoforms and the Vmax and CT values of the denervated gastrocnemius displayed large changes during this period. These changes, which led at 2 months postnatal to a muscle displaying the properties of a slow-twitch muscle did not occur in synchrony: complete conversion to slow-type myosin isoforms occurred only at 60 days postnatal, whereas complete conversion to slow-twitch Vmax and CT values occurred as soon as 35 days postnatal. The results address a new question concerning the relationship between muscle myosin and contractile features.

Early days in the research to localize skeletal muscle acetylcholinesterases.

Very early in the study of the mechanism of neuromuscular transmission in skeletal muscles, it was clear that the hydrolysis of acetylcholine by muscle cholinesterases within the time of the refractory period required a very high concentration of the enzyme near the motor terminals. David Nachmansohn and George B. Koelle and their collaborators obtained the first biochemical and histochemical data consistent with this prediction. Now that the various molecular forms of AChE have been satisfactorily described, it is possible to analyse the mechanisms by which they are anchored to the structures of the neuromuscular junction, and in particular, to the synaptic basal lamina.

Reinnervation of denervated extensor digitorum longus of the rat by the nerve of the soleus does not induce the type I myosin synthesis directly but through a sequential transition of type II myosin isoforms.

The fast-contracting extensor digitorum longus (EDL) muscle of 1-month-old rats was denervated and reinnervated by the nerve innervating the slow-contracting soleus muscle. After variable periods of time, the myosin isoform content of the EDL was analyzed by sensitive electrophoretic techniques, which allowed to discriminate between the slow-type I and the three, IIA, (IID or IIX) and IIB, fast-type II myosin isoforms. Compared to the control EDL, which contains predominantly the IIB isoform, the operated muscles contained variable proportions of all the isoforms. Analysis of the results leads us to conclude that reinnervation of EDL induces a sequential transition of myosin isoforms: IIB----(IID or IIX)----IIA----I.

Structure of the subsynaptic sarcoplasm in the interfolds of the frog neuromuscular junction.

After aldehyde fixation of frog muscles, complex organelles, which appear specific to the subsynaptic sarcoplasm, were observed at the neuromuscular junction. These organelles have a cylindrical shape; their diameter ranges generally between 150 and 300 nm, and their length between 500 nm and several micrometres. They are situated between the folds formed by the postsynaptic membrane beneath the nerve terminal branches, and, like these folds and the filament bundles of the interfolds, are orientated perpendicular to the axis of the terminal branches. The organelles are not limited by a membrane, but their cylindrical form is delimited by the interfold filaments which are applied to their surface and which constitute a sort of muff. The majority of the interfolds contain only a single subneural cylinder. On occasion two, three or more may be seen in the same interfold; in this case, the cylinders remain distinct, generally being separated from each other by filaments. Each of these cylinders contain an electron-dense axial strip from which radiate tenuous trabeculae. These trabeculae make contact with the filaments surrounding the cylinder. At both ends the axial strip connects with the plasma membrane. Tubules belonging to the sarcoplasmic reticulum can be seen on the surface of the cylinder: some of these pass through the cylinders, by-passing the axial strip. Several hypotheses concerning the possible functions of these cylinders and of the subneural filaments are discussed.

[Structural changes in motor endplates in frog muscle treated with vinblastine]. / Modifications structurales des terminaisons motrices de muscles de grenouille soumis á l'action de la vinblastine

Two kinds of abnormalities have been observed in motor nerve endings of muscles soaked in vinblastine: the occurrence of paracrystalline structures, which could derive from the precipitation of one or several proteins in the axon terminal, and the presence of large vesicles, often of festooned shape. These abnormal vesicles seem to result from the fusion of synaptic vesicles and could explain the appearence of spontaneous giant potentials which are most probably produced by the release of big packets of ACh.

[Differentiation factors of active zones of presynaptic membranes]. / Facteurs de la différenciation des "zones actives" des membranes présynaptiques

The differentiation of the subsynaptic areas depends very probably on a local influence exerted by the axon terminals; but conversely, as suggested by obervations on the development of neuromuscular junctions of "fast" and "slow" muscle fibres in Anura, complementing the results of previous degeneration experiments on frog muscles, the subsynaptic areas might intervene in the differentiation of "active zones" of presynaptic membranes.

[Dedifferentiation and remodeling of motor endplates following experimental localized lesions of muscle fibers]. / Dédifférenciation et remodelage des plaques motrices consécutifs à des lésions expérimentales localisées des fibres musculaires.

Local anaesthetics, cardiotoxin and mechanical injuries may cause necrosis of muscle fibres while leaving the motor nerve fibres and their terminals intact. With local injuries to mouse muscles carried out by freezing or cutting we made a point of preserving both the nerve terminals and the muscle fibre portions on which these terminals were located. It was thus possible to follow the changes induced at endplates by these lesions. Within two or three days of the freezing or cutting, the muscle fibres underwent very different degrees of regression of the contractile material and T-system. The neuromuscular junctions also underwent changes, principally affecting their postsynaptic portion, in particular the folds of the subneural apparatus. After dedifferentiation of subsynaptic areas, we observed sprouting of the nerve terminal on muscle fibres which survived the amputation of one end and formed actively new myofibrils. This sprouting restored synaptic connections at the original sites, but with new structural features and correlative changes in the distribution of cholinergic receptors and cholinesterases. It is probable that after a phase of involution followed by a phase of recovery, the injured muscle fibres triggered off the nerve terminal sprouting which led to the remodelling of the endplates.

Regeneration of muscles after cardiotoxin injury. I. Cytological aspects.

Regeneration of several adult rat and mouse skeletal muscles was studied after degeneration of muscle fibers had been obtained by the selective action of the cardiotoxin of Naja mossambica mossambica venom. Experimental conditions were set up to ensure minimal damage to satellite cells and also the nerves and blood vessels of the original muscles. As in the other types of experimental regeneration, the structure of the regenerated muscle appeared in many respects different from that of the normal muscle. Moreover the neuromuscular junctions of 'en plaque' type were transformed to 'en grappe' type junctions. Many ultrastructural abnormalities often displayed by these junctions might be linked, at least partially, to the persistence in the regenerating muscle of the original synaptic basal lamina sheaths and their inductive properties.

Effect of testosterone and thyroid hormone on the expression of myosin in the sexually dimorphic levator ani muscle of rat.

During postnatal development, the myosin transition from embryonic and neonatal isoforms to adult isoforms has been shown to occur with half-transition times of about 20 and 32 days in the male and female levator ani muscles, respectively. We show that this difference could not be attributed to the testosterone male hormone, since treatment of newborn females by testosterone did not modify the half-transition time. However, treatment of females by thyroid hormone accelerated the myosin transition of the female muscle, which then occurred at almost the same time as the transition of the male muscle. This suggests that the difference between the half-transition times of the male and female levator ani muscles may be largely attributed to different sensitivities of the male and female muscles to thyroid hormone. This is the first example of sexually dimorphic muscle response to thyroid hormone.

Specific programs of myosin expression in the postnatal development of rat muscles.

The expression of myosin during postnatal development was studied in a dozen muscles of the rat. All muscles displayed the usual sequential transitions from embryonic to neonatal and to adult isomyosins. However, we observed that these transitions did not take place uniformly. Thus, half-transition times for the appearance of the adult intermediate and fast myosin extended from seven days for diaphragm, the most precocious muscle of all those examined, to 23 days for male rat masseter. Besides the large differences between their half-transition times, we noticed that the transition curves displayed different slopes, covering different periods. Differences between muscles mainly affected the neonatal-to-adult transition rather than the embryonic-to-neonatal transition, since the embryonic-type myosin disappeared from all muscles examined except for one, at about the same time, by the end of the first week after birth. In addition, the appearance of slow myosin varied for each muscle and did not follow curves parallel to those for intermediate and fast myosins. These results indicate that each muscle of the rat is subjected to a specific program of myosin isoform transitions during postnatal development.

The effect of denervation on myosin isoform synthesis in rabbit slow-type and fast-type muscles during terminal differentiation. Denervation induces differentiation into slow-type muscles.

The soleus and gastrocnemius medialis of eight-day-old rabbits were denervated and the effects were examined after fifty-two days by biochemical, cytochemical and mechanical methods. The contralateral soleus exhibited the properties of slow-type muscle, namely a predominance of slow-type myosin isoforms and slow-type oxidative fibers, slow twitch and low maximal velocity for shortening. The contralateral gastrocnemius exhibited the properties of fast-type muscle, namely a predominance of fast-type myosin isoforms and fast-type non-oxidative fibers, fast twitch and high maximal velocity of shortening. Denervation of muscles caused the differentiation of the two muscles towards slow-type muscles. Both denervated soleus and gastrocnemius muscles exhibited a predominance of slow-type myosins (either the normal type, made up of slow heavy and light chains, or the hybrid type, made up of slow heavy and regulatory light chains and fast essential light chains), a predominance of slow-type fibers, and slow mechanical properties. Thus, innervation in rabbit appears to be a determining factor for differentiation into fast-type muscle, but it is not necessary for differentiation into slow-type muscle. This conclusion contradicts the findings of previous studies in rat and thus raises new questions concerning the role of nerves in controlling the expression of myosin isoforms and the differentiation of muscle fibers.

Regeneration after cardiotoxin injury of innervated and denervated slow and fast muscles of mammals. Myosin isoform analysis.

The regeneration of adult rat and mouse slow (soleus) and fast (sternomastoid) muscles was examined after the degeneration of myofibers had been achieved by a snake venom cardiotoxin, under experimental conditions devised to spare as far as possible the satellite cells, the nerves, and the blood vessels of the muscles. Three days after the injury, no myosin was detectable in selected portions of the muscles. New myosins of embryonic, neonatal, and adult types started to be synthesized during the following two days. Adult myosins thus appeared more precociously than in development, which implies that the synthesis of myosin isoforms during regeneration does not entirely 'recapitulate' the sequence of myosin transitions observed during normal development. Two weeks after the injury, the isomyosin electrophoretic pattern displayed by regenerated muscles was already the same as that of control muscles; the normal adult pattern was therefore expressed more rapidly in regenerating than in developing muscles. Except for the synthesis of the slow isoform which was generally inhibited in denervated muscles, the same types of myosins were expressed during the early stages of regeneration in denervated as in innervated muscles; long-term denervation prevented however the qualitative and quantitative recovery of the normal myosin pattern.

[Myosin isoforms synthesized during regeneration of fast contraction skeletal muscle regeneration, in the presence of motor nerve and after denervation. Study in adult rats and mice]. / Isoformes de la myosine synthétisées au cours de la régénération de muscles squelettiques à contraction rapide, en présence du nerf moteur et après dénervation. Etude chez le rat et la souris adultes.

Adult rat and mouse fast contracting skeletal muscles were injured by a cardiotoxin. New myosins of embryonic, neonatal and adult types appeared 4 and 5 days after the treatment in both innervated and denervated muscles. Although their structure remained altered, innervated--but not denervated--muscles rapidly recovered a normal isomyosin pattern.

[The regulator role of thyroid hormones in myogenesis. Analysis of isoforms of myosin in muscular regeneration]. / Rôle régulateur des hormones thyrïdiennes dans la myogenèse. Analyse des isoformes de la myosine dans la régénération musculaire.

Fast-twitch muscle regeneration has been studied in experimental hyper- and hypothyroid adult rats. The degeneration of the muscle fibres was achieved through the injection of a snake venom cardiotoxin and the synthesis of new isomyosins was examined 7, 10, 15, and 21 days after the injury. As early as the 7th day after the toxin treatment, that is 3 days after the start of the regeneration, the muscles of hyperthyroid rats do not contain any neonatal myosins and synthesize only adult myosins. In euthyroid rat muscles, neonatal myosins coexist with adult myosins and are no longer present on the 10th day. In hypothyroid rat muscles, both myosin types are still synthesized on the 21st day. Therefore, as for normal myogenesis, hyperthyroidism is shown to favor the synthesis of adult-type myosins and hypothyroidism the synthesis of neonatal-type myosins during regeneration. These results may account, at least in part, for the previously observed differences between the various types of myosins synthesized respectively in postnatal myogenesis and during adult muscle regeneration.

Response to denervation of rabbit soleus and gastrocnemius muscles. Time-course study of postnatal changes in myosin isoforms, fiber types, and contractile properties.

In contrast to general belief, the response of rabbit muscles to denervation is maturation to slow-like type muscles [7]. We report now an investigation by biochemical, morphological, and mechanical studies of the time course effects of muscle denervation on the slow-type soleus and fast-type gastrocnemius to help elucidate the mechanism of maturation of rabbit denervated muscles to slow-like muscles. In both muscles, denervation induced selective progressive atrophy of most fast fibers and hypertrophy of many slow fibers which displayed wide Z-lines; this was accompanied by the appearance of hybrid LC1F- and LC1E-associated slow myosins. The percentage of slow myosins increased with age similarly in the contralateral and denervated soleus. On the other hand, the percentage of slow myosins remained low in the contralateral gastrocnemius, whereas it increased to 95% in the denervated gastrocnemius; in the denervated gastrocnemius, the percentage of slow myosins reached 50% at about 35 days postnatal. At this age, the maximal shortening velocity of the denervated gastrocnemius and its twitch contraction time were already those of a slow-type muscle. This suggests that in addition to myosin, other proteins contributed to the mechanical properties of the denervated gastrocnemius. Transformation of rabbit denervated muscles to slow-like type muscles, which are associated with a lower energy requirement and higher muscle endurance than fast-type muscles, may constitute an adequate model for human neuromuscular pathology.