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The endogenous essential light chain (LC17) of myosin from intestine smooth muscle was replaced with mutated essential light chains prepared using recombinant techniques. Complete exchange was observed with histidine-tagged derivatives of LC17a, LC17b and E122A-LC17a (LC17a and LC17b are the usual constituants of smooth muscle myosin), with small changes in the ATPase activity of reconstituted myosins. Much less exchange was observed with the light-chain derivative lacking the last 12 amino acid residues, demonstrating the importance of this segment, which may act as one arm of a pair of pincers to bind the myosin heavy chain.

We report here, for the first time, the expression of the muscle-specific isoform of the glycolytic enzyme, enolase (EC 4.2.1. 11) (beta enolase), in rabbit skeletal muscles. We have analysed the fast-twitch gastrocnemius and the slow-twitch soleus muscles during normal postnatal development and following denervation. We show that, in rabbit, as already described in rodents, beta enolase gene expression behaves as a good marker of the fast-twitch fibers. In soleus muscle, the beta enolase transcript level is 10-20% of that found in gastrocnemius. Denervation, performed at 8 postnatal days, induces an important drop of beta enolase transcript levels in both developing soleus and gastrocnemius muscles, with a 80% decrease observed 1 week after denervation in the operated muscles, as compared to the corresponding contralateral muscles. Thereafter, the beta enolase transcript level continues to decrease in the fast-twitch muscle, with the beta enolase subunit being detectable only in the atrophic fast-twitch fibers. In contrast, the beta transcript level tends to increase in the denervated slow-twitch muscle, reaching about 50% of that in contralateral soleus, at 7 weeks after surgery. The level of beta enolase transcripts still expressed after denervation seems to stabilize at the same low level in both types of inactive muscles. This suggests that the small fraction of beta enolase expression which is not controlled by the nerve, or by the contractile activity imposed by it, is independent of the muscle phenotype.

1. In this study we investigated whether long-term trimetazidine (anti-ischaemic drug) therapy alters the ventricular myosin heavy chain (MHC) isoform composition in a model of cardiomyopathy. 2. MHC isoforms were analysed in the native state by electrophoresis in a pyrophosphate buffer. Myosin isoform patterns were studied in cardiac muscle from cardiomyopathic hamsters (CMH) of the BIO 14:6 strain during the time course of the disease and compared with those of healthy golden hamsters (F1B). The correlation between myosin profile and Ca2+-activated ATPase activity was determined from 220 days. 3. At the stage of insufficiency (350 days), CMH presented the most abnormal phenotype with 53% V1-24% V3 compared to 79% V1-7% V3 (P<0.001), in F1B. Trimetazidine was administered to cardiomyopathic hamsters from the early stage of active disease (30 days) to the congestive stages (220-350 days). Within 65 days, trimetazidine treatment, in CMH and F1B, reduced V1 to a low level (53% and 62%, respectively), which remained constant throughout the treatment. This level was similar to that in 220 and 350 days-old untreated-CMH. In sharp contrast, a standard calcium blocker, verapamil, administered to CMH in the same conditions resulted in a higher V1 (about 70%) and higher global myosin ATPase activity from 220 days. 4. Previous results in terms of hypertrophy and survival, compared to these results, suggest that verapamil and trimetazidine treatments reveal a dissociation between ventricular hypertrophy and isomyosin distribution. In addition, the shift in favour of V3 may not necessarily be an aggravating factor of the disease but an adaptative compensatory event.

We previously showed that satellite cells isolated from rabbit fast-twitch and slow-twitch muscles presented different behaviours in culture; cells from slow muscle differentiated more quickly and fused into more numerous myotubes than those from fast muscle. Moreover, only slow-muscle derived satellite cells expressed in vitro the slow type I myosin heavy chain isoform (MyHC). We wanted to investigate whether the properties of satellite cells originating from different muscles were under the influence of the adult fibre type on which they were located. For this purpose, we transformed the properties of the adult rabbit fast-twitch semimembranosus accessorius (SMa; approximately 100% type II fibres) and the slow-twitch semimembranosus proprius (SMp; 100% type I fibre) muscles by (1) cross-reinnervating the SMp with the main branch of the fast SMa nerve; or (2) electrical stimulation at 10 Hz of the SMa muscle. We studied their satellite cells in vitro. Five-month cross-reinnervation of the SMp induced a large shift of its MyHC type characteristics towards those of a fast muscle, and three-month electrical stimulation at low frequency transformed the fast-twitch SMa into a slow-twitch muscle, as shown by SDS-PAGE of MyHC. In spite of the transformation of their muscle characteristics, satellite cells in culture kept their original properties. Indeed, as shown by MyoD and myogenin gene expression as markers of fusion, satellite cells isolated from cross-reinnervated and from control SMp began to fuse by eight days of culture, and expressed MyoD and myogenin at that stage. Later they differentiated into numerous myotubes. Satellite cells isolated from electrically stimulated and control SMa presented a similar behaviour in culture: they did not express MyoD and myogenin at eight days, and fused by ten days into only a few myotubes. Moreover, MyHC gene expression showed that, in contrast with slow-muscle derived satellite cells, the type I MyHC gene was not expressed by satellite cells isolated from the stimulated SMa in spite of its homogeneous type I fibre composition. Taken together, these data support the idea that once constituted, muscle fibre types per se do not influence the properties of their associated satellite cells.

The aim of this study was to analyze the effects of treadmill training (2 h/day, 5 days/wk, 30 m/min, 7% grade for 5 wk) on the expression of myosin heavy chain (MHC) isoforms during and after regeneration of a fast-twitch white muscle [extensor digitorum longus (EDL)]. Male Wistar rats were randomly assigned to a sedentary (n = 10) or an endurance-trained (ET; n = 10) group. EDL muscle degeneration and regeneration were induced by two subcutaneous injections of a snake toxin. Five days after induction of muscle injury, animals were trained over a 5-wk period. It was verified that approximately 40 days after venom treatment, central nuclei were present in the treated EDL muscles from sedentary and ET rats. The changes in the expression of MHCs in EDL muscles were detected by using a combination of biochemical and immunocytochemical approaches. Compared with contralateral nondegenerated muscles, relative concentrations of types I, IIa, and IIx MHC isoforms in ET rats were greater in regenerated EDL muscles (146%, P < 0.05; 76%, P < 0.01; 87%, P < 0.01, respectively). Their elevation corresponded to a decrease in the relative concentration of type IIb MHC (-36%, P < 0.01). Although type I accounted for only 3.2% of total myosin in regenerated muscles from the ET group, the cytochemical analysis showed that the proportion of positive staining with the slow MHC antibody was markedly greater in regenerated muscles than in contralateral ones. Collectively, these results demonstrate that the regenerated EDL muscle is sensitive to endurance training and suggest that the training-induced shift in MHC isoforms observed in these muscles resulted from an additive effect of regeneration and repeated exercise.

The hypothesis that the limited adaptive range observed in fast rat muscles in regard to expression of the slow myosin is due to intrinsic properties of their myogenic stem cells was tested by examining myosin heavy chain (MHC) expression in regenerated rat extensor digitorum longus (EDL) and soleus (SOL) muscles. The muscles were injured by bupivacaine, transplanted to the SOL muscle bed and innervated by the SOL nerve. Three months later, muscle fibre types were determined. MHC expression in muscle fibres was demonstrated immunohistochemically and analysed by SDS-glycerol gel electrophoresis. Regenerated EDL transplants became very similar to the control SOL muscles and indistinguishable from the SOL transplants. Slow type 1 fibres predominated and the slow MHC-1 isoform was present in more than 90% of all muscle fibres. It contributed more than 80% of total MHC content in the EDL transplants. About 7% of fibres exhibited MHC-2a and about 7% of fibres coexpressed MHC-1 and MHC-2a. MHC-2x/d contributed about 5-10% of the whole MHCs in regenerated EDL and SOL transplants. The restricted adaptive range of adult rat EDL muscle in regard to the synthesis of MHC-1 is not rooted in muscle progenitor cells; it is probably due to an irreversible maturation-related change switching off the gene for the slow MHC isoform.

In adult rat muscles experimentally exposed to various patterns of activation, expression of myosin heavy chain isoforms changes, but only within a certain adaptive range. It is characteristic and different in fast or slow muscles. This may be due either to different intrinsic properties of the myogenic cells of the two types of muscles or to extrinsic factors. To test these assumptions, either rat soleus or extensor digitorum longus muscles were injured and transplanted to the bed of the extensor digitorum longus muscle. They regenerated and were reinnervated by the extensor digitorum longus nerve. Expression of myosin heavy chain isoforms was demonstrated immunohistochemically and by in situ hybridization, and analysed by SDS-gel electrophoresis. Three months after cross-transplantation, regenerated soleus expressed all adult myosin heavy chain isoforms, including the myosin heavy chain-2B. The latter was detected in about 50% of muscle fibres and contributed about 10-20% of all myosin heavy chains. The same percentage of myosin heavy chain-2B was found in regenerated extensor digitorum longus. In this regard therefore, the adaptive range of the regenerated soleus muscle was not significantly different from that of the extensor digitorum longus regenerating under the same conditions. This indicates that restriction of the adaptive range in a mature soleus muscle is not due to intrinsic properties of its myogenic cells. It is probably imposed by an extrinsic factor leading to irreversible shut-down of individual myosin heavy chain genes. On the other hand, myosin heavy chain-1 expression was significantly greater in the regenerated soleus than in the extensor digitorum longus innervated by the same nerve. Myosin heavy chain-1 and myosin heavy chain-2B were co-expressed in some regenerated soleus muscle fibres.

Recent reports by d'Albis et al. have shown that denervation of 8-day-old rabbit fast-twitch muscle (gastrocnemius) leads to the transformation of the muscle towards a slow phenotype but the changes towards slow-type myosin isoforms and contractile properties of the muscle were temporally uncoordinated. We analyzed the time course of the effects of denervation of the gastrocnemius on the expression of the sarcoplasmic reticulum calcium pump isoforms (SERCA) and on the metabolic state of the muscle. Northern-blot analysis showed a rapid loss of the fast Ca2+ pump isoform (SERCA 1) mRNA from the denervated gastrocnemius which became of the oxidative type. The changes observed were complete as early as 35 days post-natal, i.e at the time when changes in contractile properties were previously observed. Denervation of the slow-twitch soleus led to a 50% decrease in the level of the slow Ca2+ pump isoform (SERCA 2) mRNA and was without effect on the metabolic state of the muscle. These findings extend previous results suggesting that in rabbit, continuous innervation is required for differentiation of fast-twitch muscles but is not an absolute requirement for differentiation of the slow-twitch muscle.

The expression of myosin heavy (MyHC) and light (MyLC) chain isoforms was analyzed after denervation and cross-reinnervation by a fast nerve of the slow-twitch Semimembranosus proprius (SMp) muscle, and after denervation and electrical stimulation at low frequency of the fast-twitch Semimembranous accessorius (SMa) muscle of the rabbit. The control SMp (100% type I fibers) expressed 100% type I MyHC and 100% slow-type (1S', 1S and 2S) MyLC isoforms. Five month denervation did not alter significantly the MyHC expression of the muscle, but induced the expression of a new type 1 MyLC corresponding most probably to an embryonic MyLC. Five-month cross-reinnervation of the SMp by the fast SMa nerve induced a large change of its fiber type properties. As shown by immunocytochemistry, almost all fibers were stained by fast myosin antibody, but a high proportion of them co-expressed slow myosin. This result was in agreement with biochemical data showing that fast MyHC and MyLC isoforms became predominant. The control SMa (nearly 100% type II fibers) expressed almost 100% type II MyHC (70% type IIb and 22% IIx/d) and 100% fast-type (1F, 2F and 3F) MyLC isoforms. Five month denervation of the SMa induced a shift in its MyHC, with 98% type IIx/d and 2% type IIb isoforms, and no change in the proportions of its MyLC. Three month electrical stimulation at 10 Hz of the SMa transformed its fiber type composition. All fibers reacted with the slow myosin antibody and a minor proportion of them were stained by the fast myosin antibody. These observations were in agreement with the biochemical analysis showing a large predominance of the slow-type MyHC and MyLC isoforms. Taken together, these results obtained from rabbit muscles which are normally homogeneous in either fast-twitch or slow-twitch fiber types, further support the idea that the different myosin isoforms, particularly the MyHC, are differentially regulated by motor innervation. Type I MyHC is maintained in denervated SMp muscle, but is not expressed in denervated SMa. Type IIb isoform is the most sensitive to neural influence, as it disappears rapidly in denervated and electrically stimulated fast-twitch SMa muscle, and is barely expressed in cross-reinnervated slow-twitch SMp muscle. In contrast, type IIa and type IIx/d are less dependent upon motor innervation. In addition to the previous studies of d'Albis et al. analysis of these results leads us to conclude that, in the rabbit, sensitivity to motor innervation increases from the glycolytic to the oxydative types of fibers, in the order IIB > IIX/IID > IIA > I.

We present the separation by SDS gel-electrophoresis of the six main myosin heavy chains (MHC) present in rabbit skeletal muscle. The separation of the four adult MHC (1, 2A, 2X/2D, 2B) was compared to that of the corresponding rat MHC as described by Talmadge and Roy [J. Appl. Physiol. 99 (1993) 2337-2340]. We found that many rabbit muscles contained mainly one of the four MHC, in some cases the 2B MHC. In addition, we resolved the embryonic E and perinatal P developmental MHC, which should facilitate muscle differentiation and regeneration studies in the rabbit. An example of application to the study of muscle denervation is given.

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.

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.

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.

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 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.

Transitions from embryonic and neonatal to adult-type-II isomyosins are known to be related to the increase in the thyroid hormone plasma concentration during postnatal development. These transitions have been shown, however, to occur at different times, depending on the muscle, suggesting that each muscle responds differently to the thyroid hormone. We have investigated quantitatively the effects of experimental hypothyroidism and hyperthyroidism on isomyosin transitions from birth until the 45th postnatal day in eight rat muscles: diaphragm, intercostals, gastrocnemius medialis, soleus, plantar muscles of the foot, tongue muscle, levator ani and bulbocavernosus complex, and masseter. Hypothyroidism delayed the isomyosin transitions in all the muscles examined, particularly in the sexually dimorphic muscles (levator ani and bulbocavernosus complex and masseter). However, it did not eventually inhibit isomyosin transitions, indicating that the thyroid hormone was not an absolute requirement for these to occur. Hyperthyroidism had only a slight effect on isomyosin transition in the diaphragm, and accelerated such transitions in the other muscles. The transition curves of all the muscles investigated, except those of the sexually dimorphic muscles, became similar to that of the diaphragm, demonstrating that the various muscles did not display the same sensitivity to the thyroid hormone but were regulated by it in the same way. The isomyosin transitions in the sexually dimorphic muscles remained late in comparison to that in the diaphragm, which suggests a more complex regulation. The effect of hyperthyroidism was not permanent and could be reversed, by interruption of the treatment, to a greater or lesser extent depending on the muscle. In all muscles containing slow-type-I isomyosin, hypothyroidism had no effect on this isomyosin synthesis, whereas hyperthyroidism inhibited it. This inhibition ceased rapidly after the interruption of the treatment.

Changes both in the ATPase myofibrillar profile and in the electrophoretic pattern of myosin isoforms were examined in the mouse dorsal skeletal muscle (longissimus) during postnatal development. In the newborn, only type II C and a few type I fibers were present; differentiation into type II A and II B fibers took place during the 3 weeks following birth. During the same period, a transition from three neonatal isomyosins to four adult isoforms was observed. The two phenomena were related to a marked increase in the serum thyroid hormones levels. Hypothyroidism and hyperthyroidism experiments were performed. Hypothyroidism produced by propylthiouracil treatment of pregnant females and thiourea injections of the litters was shown to induce a complete inhibition of postnatal muscular differentiation. Hyperthyroidism produced by triiodothyronine treatment of the neonate mice significantly accelerated the myosin transition and the switch in the myofibrillar pattern. Our results suggest a primordial role for thyroid hormones in directly regulating the appearance of myosin and fiber adult types and in modulating directly or indirectly the disappearance of the neonatal types.

In the urodelan amphibian Pleurodeles waltlii, spontaneous external metamorphosis was correlated with an increase in the serum level of thyroxine (T4). Within the same period, a change occurred in the myofibrillar ATPase profile of the dorsal skeletal muscle; fibres of larval type were gradually replaced by transitional fibres (type IIC), then by adult fibres of the types I, IIA, and IIB. Likewise, a myosin isoenzymic transition was observed. In larval animals, myosin electrophoresis revealed 3 bands corresponding with isoforms having identical heavy chains (MHC), but different light chains (MLC). In the course of metamorphosis, the 3 larval isomyosins were replaced by 3 isoforms having the adult type MHC and different motility. In a related neotenic species, Ambystoma mexicanum, no spontaneous anatomic metamorphosis occurred; at the time it should theoretically take place, the serum T4 level remained low. The ATPase profile was modified, but transitional fibres that replaced the initial larval types appeared to be persistent, and adult fiber types appeared only in a small amount. Myosin isoenzymic transition was also incomplete, larval isoforms were still distinguished in the neotenic adults. Similar persistence of larval characters was observed in adult Proteus anguinus, a perennibranch that never undergoes anatomical metamorphosis. Experimental hypothyroidian Pleurodeles waltlii displayed no external metamorphosis, only the larval fibre types and isomyosins were detected in those animals. External metamorphosis was induced in Ambystoma mexicanum by a triiodothyronine treatment. A complete myosin isoenzymic transition was observed in metamorphosed animals. These results tend to indicate that a moderate increase in the level of thyroid hormones is sufficient to determine the production of the adult type MHC molecules and the differentiation of the corresponding myofibrillar types in the skeletal dorsal muscle of amphibians, while a marked increase would be necessary for repressing the initial larval feature.

Adult rat, mouse, and guinea-pig masseter muscles display distinct myosin electrophoretic patterns. The rat muscle contains four main forms which by reference to the myosins of the IIB tensor fasciae latae, of the IIA mylohyoid, and of the red and white portions of the sternomastoid muscles, correspond respectively to the intermediate-type and to the three fast-type isoforms. The mouse masseter muscle contains only three main myosins, the intermediate-type and two fast-type isoforms. The guinea-pig muscle also displays only three bands, whose assignment is, however, less straightforward than in the murine species; their electrophoretic mobilities are not strictly the same as those of their homologous forms in rat and mouse. Comparison with the myosins of the tensor fasciae latae and of the sternomastoid muscles of guinea-pig allows their identification as intermediate and fast-type myosins. In addition to these typical adult-type forms, adult murine masseter muscles are observed to contain between zero and 30% of neonatal-type myosins. The comparison of the developmental transitions of myosins in the rat masseter with those in the skeletal muscles of the same animal indicates a delay in the appearance of the adult as well as in the disappearance of the neonatal-type myosins in the masseter muscle. Both the variability in myosin types with the animal species and the atypical presence of neonatal forms in the murine adults suggest that myosin expression in the masseter muscle is subjected to unusual regulations.

In the perennibranchiate Proteus anguinus, larval myosin isoforms were shown to coexist for life with the adult isomyosins that appeared at the end of the larval stage. Analysis of the myofibrillar ATPase profile also revealed that a high percentage of immature fibers persisted in adults. A long-term treatment with large amounts of T3 had no effect on juvenile individuals. Applied to subadult animals it promoted a regression of larval myosin isoforms and a reduction in the percentage of immature fiber types. The regulative effect of T3 in the myosin isoenzymic transition may be delayed and depends on metabolic conditions, which suggests it is indirect.

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.

Myometrial strips from oestrogen-primed rat uterus were exposed to various treatments, isometric contraction was measured, and the extent of myosin light chain phosphorylation determined after rapid freezing in liquid nitrogen. Two-dimensional electrophoresis revealed five spots having the same molecular weight as the light chain, with isoelectric points comprised between 5.15 and 4.95. Two of these spots (pI 5.09 and 5.00) were not present in pure uterine myosin, whether prepared from incubated or nonincubated tissue; they do not represent light chain isoforms or electrophoresis artefacts but rather degradation products appearing during the treatment. Two spots (pI 5.15 and 5.06) were identified as the nonphosphorylated and the phosphorylated forms of the light chain. The fifth minor spot (pI 4.95) may represent a diphosphorylated myosin species. Strips incubated in a normal Ca2+-medium 0.8 mM) exhibited basal contractions and an incorporation of 0.2 mol phosphate per mol light chain. Removal of Ca2+ resulted in almost complete dephosphorylation, coincident with a total relaxation of the muscle. Exposure of the myometrium to carbachol caused tetanic contractions with an increase to 0.5 mol phosphate per mol light chain. Isoproterenol, a beta-adrenergic agonist elevated intracellular cyclic AMP and induced uterine relaxation. Addition of isoproternol to a resting myometrium caused a slight but significant decrease in phosphorylation; its addition prior to carbachol markedly prevented the increase in myosin phosphorylation normally induced by the cholinergic effector. Forskolin (1 microM) increased intracellular cyclic AMP, caused relaxation and a concomitant decrease in basal myosin phosphorylation. Prostaglandin E2-induced elevation in intracellular cyclic AMP was however accompanied by an increase in contraction together with an increase in light chain phosphorylation. The data imply that light chain phosphorylation-dephosphorylation, regulated by Ca2+-dependent mechanisms, is essential for both uterine contraction and relaxation but question the role of cyclic AMP in exclusively mediating relaxation and myosin dephosphorylation in intact myometrium.

Using several electrophoretic procedures, we have compared the forms of myosin and actin in pregnant and non-pregnant uterus of woman, monkey (Macaca fascicularis) and rat. On non-dissociating gels, native myosin of the three species migrates as a single band, of identical mobility independently of the physiological state. Remigration of this band in dissociating conditions shows that it is constituted of two heavy chains of respectively 201 kDa and 205 kDa; the relative proportions of these two bands are different for the three animal species but do not vary during pregnancy. Using two-dimensional gel electrophoresis, we found that the 17-kDa light chain of purified uterus myosin exists under two isoelectric forms, the more acidic one becoming progressively predominant at the end of pregnancy in the human as in the monkey uterus, while we observed no changes in the rat. In two-dimensional gel electrophoresis, actin of human, monkey and rat uterus is present under three isoforms, the most basic one (the gamma form) increasing early in pregnancy in the two primate species but being always the most abundant form in the rat. The ATPase activity of human uterus myosin was found to be similar for the protein extracted from both pregnant and non-pregnant uterus. The changes observed in the 17-kDa light chain and in the actin isoforms might nevertheless participate in the modifications of contractility of the uterus during pregnancy of the primates.

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 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.

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.

In the urodelan amphibian Pleurodeles waltlii, spontaneous anatomical metamorphosis was correlated with an increase in the serum level of thyroxine (T4). It was also accompanied by a change in the myofibrillar ATPase profile of the dorsal skeletal muscle; fibers of larval type were gradually replaced by the adult fiber types I, II A, and II B. Likewise, a myosin isoenzymic transition was observed in dorsal muscle, larval isomyosins were replaced by adult isoforms. In a related species, Ambystoma mexicanum, in which no spontaneous external metamorphosis occurs under standard conditions, the serum T4 level was shown to remain low. During further development, the myofibrillar ATPase profile acquired the adult fiber types, but a high percentage of immature fibers of type II C persisted. Myosin isoenzymic transition was also incomplete; larval isoforms were still distinguished in the neotenic adults. In experimental hypothyroidian P. waltlii, no external metamorphosis occurred; the myofibrillar ATPase profile was of the immature type, and the larval isomyosins persisted. Triiodothyronine induced experimental anatomical metamorphosis in A. mexicanum; only limited changes in the myofibrillar ATPase profile resulted from the treatment, but a complete myosin isoenzymic transition was observed. These results tend to indicate that a moderate increase in the level of thyroid hormone is sufficient to induce the differentiation of adult fiber types, together with the production of adult myosin isoforms in the skeletal dorsal muscle of amphibians, while a pronounced increase would be necessary for repressing the initial larval features.

The isomyosins from dorsal axial muscle, which appear successively through metamorphosis of P.waltlii, are shown to be composed of identical fast-type light chains but of distinct heavy subunits. We observe that this modification goes with a change in ATPase activity as also in the case of mouse. Metamorphosis in amphibian as well as birth in mammalian are thus both accompanied by the synthesis of new myosins of higher catalytic efficiency.