Long-term changes in myosin heavy chain composition after botulinum toxin a injection into rat medial rectus muscle.

PURPOSE: To study long-term changes of extraocular muscles after botulinum toxin (Botx) A-induced paralysis, with special emphasis on myosin heavy chain (MyHC) isoform pattern in muscle fibers. METHODS: Botx A (5 IU) was injected into the ocular medial rectus (MR) muscles of adult rats. After 1, 5, and 8 months muscle cross sections were examined immunohistochemically, histochemically, and morphometrically. MyHC content was analyzed by gel electrophoresis. RESULTS: Paralyzed MR muscles displayed mildly atrophic and hypertrophic muscle fibers and decreased oxidative metabolism, due to decreased succinate dehydrogenase activity. However, muscle morphology was not grossly disturbed. MyHC profile was shifted toward slower isoforms. Electrophoretic analysis showed that the share of MyHCI, and especially of MyHCIIa and MyHCIIx/d, increased several fold, whereas the share of MyHCIIb decreased heavily during the first 5 months. Immunohistochemical analysis generally mirrored the results obtained by electrophoresis. Moreover, specific extraocular MyHC isoform MyHCeom disappeared and could not be detected during the whole experimental period. The portion of MyHCIIb relatively increased 8 months after Botx A injection, although the MyHC profile was still far from normal. CONCLUSIONS: These long-lasting changes in Botx A-paralyzed ocular MR muscles most probably reflect their inability to regain their unique functional characteristics after new motor end plate formation and recovery of muscle contraction.

Myosin heavy chain profiles in regenerated fast and slow muscles innervated by the same motor nerve become nearly identical.

Plasticity of mature muscles exposed to different activation patterns is limited, probably due to restricted adaptive range of their muscle fibres. In this study, we tested whether satellite cells derived from slow muscles can give rise to a normal fast muscle, if transplanted to the fast muscle bed. Marcaine-treated rat soleus and extensor digitorum longus (EDL) muscles were transplanted to the EDL muscle bed and innervated by the 'EDL' nerve. Six months later expression of myosin heavy chain isoforms was analysed by areal densities of fibres, binding specific monoclonal antibodies, and by SDS gel electrophoresis. Both regenerated muscles closely resembled each other. Their myosin heavy chain profiles were similar to those in fast muscles although they were not identical to that in the control EDL muscle. Since not even regenerated EDL was able to reach the myosin heavy chain isoform profile of mature EDL muscle, our experimental model did not permit studying the adaptive capacity of satellite cells in different muscles in its whole extent. However, the results favour the multipotential myoblast stem cell population in rat muscles and underline the importance of the extrinsic regulation of muscle phenotype.

Denervation of rabbit gastrocnemius and soleus muscles: effect on muscle-specific enolase.

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.

Changes in myosin heavy chain profile of mature regenerated muscle with endurance training in rat.

The objective of the present study was to examine the response of fast-twitch muscle to endurance training long after the muscle had regenerated from toxin injury. Seventeen male Wistar rats were randomly assigned to a sedentary (S, n = 10) or a trained group (T, n = 7). Endurance training by treadmill running (5 days week(-1), 30 m min(-1), 7% grade, 2 h day(-1) for 5 weeks) was initiated 5 weeks after myofibre degeneration was induced in the right extensor digitorum longus muscle (EDL) by two injections of 0.2 mL of the unfractionated venom from Naja nigricollis snake. Gel electrophoresis analyses showed that training alone resulted in a 140% increase in type IIX myosin heavy chain (MHC) (P < 0.01) and a slight decrease in type IIB MHC (-14% P < 0.05). Regeneration alone induced an increase in both type IIA and IIX MHC expression (103%, P < 0.05, and 131%, P < 0.01, respectively), and a concomitant decrease in the percentage of type IIB MHC (P < 0.05). The shift from type IIB toward type IIA MHC composition observed in regenerated muscles of T rats resulted not only from an additive, but from a cumulative effect of training and regeneration. Immunohistochemical analysis of MHC content in individual fibres showed similar changes. These data suggest that the impact of endurance training on fast-type MHCs was more marked in mature regenerated muscles than in regenerating ones, and provide evidence of the heightened plasticity of fully regenerated muscles to repeated exercise.

Transformation of slow- or fast-twitch rabbit muscles after cross-reinnervation or low frequency stimulation does not alter the in vitro properties of their satellite cells.

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.

Myosin isoform transitions in four rabbit muscles during postnatal growth.

Four rabbit muscles (i.e. semimembranosus proprius, psoas major, biceps femoris and longissimus lumborum), differing in their fibre type composition in the adult, were investigated during postnatal development. Muscle samples were taken at 1, 7, 14, 21, 28, 35, 49 and 77 days of age. Complementary techniques were used to characterize myosin heavy chain (MHC) isoform transitions, i.e. SDS-PAGE, immunocytochemistry and conventional histochemistry. Good accordance was found between electrophoretic and immunocytochemical techniques. Our results show that rabbit muscles were phenotypically immature at birth. At 1 day of age, perinatal isoform represented 70-90% of the total isoform content of the muscles. Two generations of myofibres could be observed on the basis of their morphology and reaction to specific antibodies. In all muscles, primary fibres expressed slow MHC. In contrast, secondary generation of fibres never expressed slow MHC in future fast muscles, while half of them expressed slow MHC in the future slow-twitch muscle, the semimembranosus proprius. During the postnatal period, all muscles displayed a transition from embryonic to perinatal MHC isoforms, followed by a transition from perinatal to adult MHC isoforms. These transitions occured mainly during the first postnatal month. The embryonic isoform was no longer expressed after 14 days, except in longissimus where it disappeared after 28 days. On the contrary, large differences were found in the timing of disappearance of the perinatal isoform between the four muscles. The perinatal isoform disappeared between 28 and 35 days in semimembranosus proprius and 35 and 49 days in psoas and biceps femoris. Interestingly, the perinatal isoform was still present in 6% of the fibres in longissimus at 77 days, the commercial slaughter age, denoting a great delay in the maturation. Fate of each generation of fibres differed between muscles.

Endurance training affects myosin heavy chain phenotype in regenerating fast-twitch muscle.

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.

Regenerated rat fast muscle transplanted to the slow muscle bed and innervated by the slow nerve, exhibits an identical myosin heavy chain repertoire to that of the slow muscle.

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.

Adaptive range of myosin heavy chain expression in regenerating soleus is broader than in mature muscle.

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.

Sarco(endo)plasmic reticulum Ca2+ pump and metabolic enzyme expression in rabbit fast-type and slow-type denervated skeletal muscles. A time course study.

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.

Diffusion of fluorescently labeled macromolecules in cultured muscle cells.

Myotubes were obtained from culture of satellite cells. They had a sarcomeric organization similar to that of muscle. The diffusion in the direction perpendicular to the fibers of microinjected fluorescein isothiocyanate-dextrans of molecular weight ranging from 9500 to 150,000 was examined by modulated fringe pattern photobleaching. On the time scale of the observation, 10-30 S, all of the dextrans were completely mobile in the cytoplasm. The diffusion coefficients were compared to the values obtained in water. The ratio D(cytoplasm)/D(w) decreased with the hydrodynamic radius R(h) of the macromolecules. The mobility of inert molecules in muscle cells is hindered by both the crowding of the fluid phase of the cytoplasm and the screening effect due to myofilaments: D(cytoplasm)/D(w) = (D/D(w)) protein crowding x (D/D(w))(filament screening). The equation (D/D(w))filament screening = exp(-K(L)RCh) was used for the contribution of the filaments to the restriction of diffusion. A free protein concentration of 135 mg/ml, a solvent viscosity of cytoplasm near that of bulk water, and a calculated K(L) of 0.066 nm(-1), which takes into account the sarcomeric organization of filaments, accurately represent our data.

Expression of myosin isoforms in denervated, cross-reinnervated, and electrically stimulated rabbit muscles.

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.

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.

Muscle creatine kinase-deficient mice. I. Alterations in myofibrillar function.

The regulation of contractile activity in mice bearing a null mutation of the M-isoform of creatine kinase gene, has been investigated in tissue extracts and Triton X-100-treated preparations of ventricular, soleus, and gastrocnemius muscles of control and transgenic mice. Skinned fiber experiments did not evidence any statistical difference in the maximal force or the calcium sensitivity of either muscle type. Rigor tension development at a low MgATP concentration was greatly influenced by phosphocreatine in control but not in transgenic mice as should be expected. In calcium-activated ventricular preparations, although the force developed by each cross-bridge was the same in control and transgenic animals, the rate constant of tension changes appeared to be markedly slowed in transgenic animals. As the ventricular isomyosin pattern was not altered, we suggested that, in transgenic animals, cross-bridge cycling was hindered by a local decrease in the MgATP to MgADP ratio, due to lack of a local MgATP regenerating system. Myokinase activity was not significantly changed while activities of pyruvate kinase or glyceraldehyde-3-phosphate dehydrogenase were found to be increased in transgenic animals. These results show that no fundamental remodelling occurs in myofibrils of transgenic animals but that important adaptations modify the bioenergetic pathways including glycolytic metabolism.

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.

Electrophoretic separation of developmental and adult rabbit skeletal muscle myosin heavy chain isoforms: example of application to muscle denervation study.

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 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.

Rabbit masseter expresses the cardiac alpha myosin heavy chain gene. Evidence from mRNA sequence analysis.

The presence of myosin alpha heavy chain in the rabbit masseter has been previously suggested at the protein level [(1991) Basic App. Myol. 1, 23-34; (1991) Histochem. J. 23, 160-170]. To confirm this finding, we cloned most of the mRNA corresponding to the myosin heavy chain S2 subfragment. PCR analysis and subsequent nucleotide sequence determination of the amplified cDNA demonstrates the presence of a myosin alpha heavy chain mRNA in rabbit masticatory muscles.

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.

A model system of coupled activity of co-immobilized creatine kinase and myosin.

Myosin and creatine kinase were co-immobilized onto Immunodyne films to mimic the behaviour of creatine kinase bound to the M-line of myofilaments. The Mg-ATPase activity of bound myosin was studied by a coupled enzymatic assay, which detects Mg-ADP in the bulk solution by means of pyruvate kinase and lactate dehydrogenase. The competition for Mg-ADP between pyruvate kinase and creatine kinase either free in solution or co-immobilized with myosin was studied at various creatine phosphate concentrations. Bound creatine kinase competed efficiently when present in very low amounts, corresponding to an activity ratio higher than 1:20,000 between creatine kinase and pyruvate kinase and a molar ratio higher than 1:1000 between creatine kinase and myosin. The Mg-ADP produced by myosin ATPase in the vicinity of the film did not diffuse into the bulk solution but, in the presence of creatine phosphate, was recycled into Mg-ATP by the neighbouring creatine kinase. The existence of an unstirred layer near the surface of the film is sufficient to explain the channeling of ADP (or ATP) between co-immobilized myosin and creatine kinase, without direct interaction or 'intimate coupling' between the enzymes. The problem now is to determine the importance of this kind of facilitated diffusion in the myofilaments in vivo.