The influence of denervation of the maturation of the flight muscle of Periplaneta americana.

The metathoracic musculature of the American cockroach Periplaneta americana was denervated by dissecting the nerves originating in the metathoracic ganglion on one side within 2 days after the last moult. The biochemical and morphological differences between normal and denervated musculature were followed two weeks later. Denervation completely prevents the gain in weight, protein content and cytochrome oxidase activity which occur in normal muscles during the two weeks after previous moulting. The content of phosphoarginine in the denervated muscle does not differ from the control muscle and is lower than in young muscles. The ability to resynthesize phosphoarginine postanaerobically is substantially lower in the denervated muscle than in control muscles, or in young muscles immediately after the last moult. The ultramicroscopic structure of the denervated muscle differs from that of the normal muscle: the mitochondria possess less cristae, the matrix between them appears less dense and the sarcoplasmatic reticulum is less developed. Thus the denervated muscle not only fails to mature, but also developes serious structural and biochemical disorders.     

Transplantation and transposition of skeletal muscles into the faces of monkeys

Restoration of normal facial movement after long-term facial paralysis with muscle atrophy has not yet been achieved reliably by either free grafts, in which fibers degenerate and regenerate, or by grafts made with microneurovascular repair, in which most fibers survive. Our purpose was to compare the structural and functional properties of free muscle grafts and continuously perfused muscle flaps transplanted into the faces of monkeys. In adult monkeys, the facial muscles were replaced by either a free graft of a donor muscle from the lower limb or a denervated flap of ipsilateral temporalis muscle. Each graft or flap was reinnervated with the preserved buccal branch of the facial nerve. The control muscles, grafts, and flaps were examined 90 days later for gross appearance, contractile properties, and fiber areas. Compared with muscle flaps, free grafts showed greater adaptability to the new location and innervation and a closer approximation to the structural and functional properties of the original facial musculature.     

Histological evidences of reparative and regenerative effects of beta-adrenoceptor agonists, clenbuterol and isoproterenol, in denervated rat skeletal muscle

The aim of this study was to determine the contribution of beta-adrenoceptor activation in the reconstruction of the structural and functional organization of denervated skeletal muscle. beta-agonists, clenbuterol (1.2 mg/kg body weight) and isoproterenol (2 mg/kg body weight), administration (daily oral administration; maximum 7 days) to normal innervated rats as well as denervated animals caused muscle hypertrophy. An increase in mean fiber diameter confirmed this stimulated growth both in normal innervated and denervated rat gastrocnemius muscle. Examination of muscle nuclei from treated but normal innervated rat gastrocnemius exhibited features like large size, active nucleoplasm and an increase in their number per fiber cross section and per mm mean fiber length indicating towards an elevated biosynthetic activity in tissue in the presence of beta adrenoceptor agonists. Administration of drugs to normal innervated animals resulted in an emergence of central muscle nuclei. The hyperactive and enlarged muscle nuclei ultimately organized themselves into unusually elongated nuclear streaks. beta agonist treatment to denervated rats resulted in amelioration of atrophic state of tissue characterized by hypertrophy of muscle fibers thus lending to a restoration of structural organization of tissue. Bizarre shapes of nuclei in denervated muscle tend to recover to that characteristic to normal innervated muscle in presence of clenbuterol and isoproterenol hydrochloride. All observations were confirmed by administering butoxamine, a beta-adrenoceptor antagonist along with beta-agonists. The results suggests that both clenbuterol and isoproterenol hydrochloride are capable of mimicking normal innervation functions in skeletal muscle and thus play important role in the structural and functional reorganization of tissue. Amelioration of denervation atrophy in rat gastrocnemius in the presence of beta-agonists supports this.     

Comparative Studies of Myosin Rods Isolated by Enzymatic and Non-enzymatic Cleavage of Myosin

Tryptic (LMM Fr 1) and CNBr-treated (LMM-C) rod portion of myosin molecule were prepared from rabbit skeletal muscle myosin. An attempt was made to clarify the difference between LMM Fr 1 and LMM-C as measured by solubility and some physico-chemical techniques. The rigid structure of LMM Fr 1 melted into a random coil as temperature of the solution increased and the value of b₀ and reduced viscosity did not show full recovery upon gradual cooling. The situation of LMM-C, on the other hand, showed the higher thermostability than LMM Fr 1. As stated in our previous paper,¹⁾ we have considered that the rod portion of myosin molecules is substantially thermostable.     

Histological and histochemical studies on the nervous influence on minced muscle regeneration of triceps surae of the rat.

The degree of minced rat muscle regeneration in the absence of nerve fibers was compared with that of normal regenerates between one and 270 days postoperatively. Up to around 30 days, the number of muscle fibers and their morphology were comparable in both normal innervated and denervated regenerates; both showed clear cross striations and peripherally located nuclei. Histochemically, SDH and myofibrillar ATPase (pH=9.4) reactions were positive, but there were no typical signs of fiber types in either case of regeneration. The only consistent difference in the early period was the smaller fiber cross sectional areas in denervated regenerates than in innervated ones. Starting about 40 days, the muscle fibers in innervated regenerates became differentiated into different fiber types (fast-twitch-oxidative-glycolytic, FOG., fast-twitch-glycolytic, FG., slow-twitch-oxidative, SO.) but there were no such activities in denervated regenerates, although their SDH and myofibrillar ATPase reactions remained positive for a long time. Degenerating muscle fibers could no longer be identified in innervated regenerates. In the denervated regenerates, however, muscle fibers underwent atrophic or degenerative changes and were replaced by connective tissue. The complete disappearance of muscle fibers varied with individual regenerates. In some cases, it occurred about 90 days and in others, traces of muscle fibers could still be seen as late as 150 days postoperatively. Thus, nerves seem to be important primarily in the late phase of regeneration; namely, differentiation of fiber types and maintenance of the structural integrity of muscle fibers.     

Evidence for myoblastic potential of satellite cells in denervated muscle

The failure of denervated muscle to undergo effective regeneration, despite reported increases in the number of muscle satellite cells, warranted an investigation of the viability and myoblastic capacity of these cells present in denervated muscle. Four types of satellite cells present in muscle denervated for three weeks are described, based on their ultrastructure and relationship to their principal fiber. The increased number of ribosomes, including helically arranged polysomes; the number of Golgi complexes; the presence of microtubules; the branching subsarcolemmal tubular system; and the appearance of regularly arranged 96 A microfilaments with diffuse electron dense areas are structural features of satellite cells that are similar to those of developing myoblasts in growing and regenerating muscle. The electron microscopic observations suggest that "activated" satellite cells do have myoblastic potential. Possible explanations for the ultimate failure of denervated muscle to regenerate include: 1) the inability of the muscle to produce satellite cells rapidly enough to keep pace with muscle degeneration; 2) a cytotoxic effect produced by the degenerating muscle fiber on the satellite cell; and 3) the inability of satellite cells to form stable, mature multinucleated fibers in the absence of the trophic effect of the nerve.     

Single channel properties of newly synthesized acetylcholine receptors following denervation of mammalian skeletal muscle

We have examined the single channel properties of newly synthesized acetylcholine (ACh) receptors in denervated adult mouse muscle. Patch-clamp recordings were made on freshly isolated fibers from flexor digitorum brevis (fdb) muscles that had been denervated in vivo for periods up to 3 wk. Muscles were treated with alpha-bungarotoxin (alpha-BTX), immediately before denervation, in order to block pre-existing receptors. Denervated fibers exhibited two types of ACh receptor channels, which differed in terms of single channel conductance (45 and 70 pS) and mean channel open time (approximately 7 and 2.5 ms, respectively). In contrast to innervated muscle, where only 3% of the total openings were contributed by the low-conductance channel type, greater than 80% of the openings in the nonsynaptic membrane of denervated muscle were of this type. Importantly, a similar increase in the proportion of low-conductance channels was observed for recordings from synaptic membrane after denervation. These data argue against the proposal that, in denervated muscle, the low-conductance channels undergo continued conversion to the high-conductance type focally at the site of former synaptic contact. Rather, our findings provide additional support for the idea that the functional properties of ACh receptors are governed uniformly by the state of innervation of the fiber and not by proximity to the site of synaptic contact.     

Calcium transport and ATPase activity of sarcoplasmic reticulum in normal and denervated rabbit muscles]

The properties of sarcomplasmic reticulum Ca-pump from normal and denervated rabbit muscles were investigated. Ca+2 ion transport in denervated muscle reticulum was subject to Michaelis-Menten kinetics. The rate of fast Ca2+ outflux from the vesicles was enhanced after denervation; this caused a decrease in the transport efficiency and an increase of the "basic" ATP-ase. At the same time the rate of Ca2+ accumulation and the Ca-ATP-ase transport activity were enhances by a factor of 1.5. Kinetic properties of the denervated sarcoplasmic reticulum proved to be closely related to the features of the excitation-contraction cycle in these muscles.     

Conformational stability of the myosin rod

Chymotryptic cleavage patterns of myosin rods from pig stomach, chicken gizzard, and rabbit skeletal muscle indicate that short (approximately 45 nm) heavy meromyosin subfragment 2 (SF2) is a consistent product of all three rods, whereas long (approximately 60 nm) SF2 is derived only from skeletal muscle myosin. Differential scanning calorimetry was used to follow the thermally induced melting transition of the rods and certain of their subfragments. In 0.12 M KCl, sodium phosphate buffer, pH 6.2-7.6, the light meromyosin (LMM) and SF2 domains of each rod had essentially identical conformational stabilities. Temperature midpoints for the melting transitions were 54-56 degrees C for the two smooth muscle myosin rods and 50-53 degrees C for the skeletal muscle myosin rod. In 0.6 M K Cl buffer, melting transitions for the smooth muscle myosin rods were essentially unchanged, but skeletal muscle myosin rods showed multiphase melting, with major transitions at 43 degrees C and 52 degrees C. The first of these was tentatively attributed to LMM, and the second to SF2. In 0.12 M K Cl buffer, the LMM transition was stabilised so that it superimposed on that of SF2. No melting was observed in any of the rods at physiological temperature. These results indicate that, excluding a possible but only narrow hinge region, the entire myosin rod has essentially uniform conformational stability at physiological pH and ionic strength, and thus that the contractile and elastic properties of the cross-bridge exist in the heavy meromyosin subfragment 1 (SF1) domains of the molecule.     

Expression of native rabbit light meromyosin in Escherichia coli. Observation of a powerful internal translation initiation site

The cDNA-sequence coding for rabbit skeletal muscle light meromyosin (LMM) was placed under the control of the lambda promoter (PL) of an Escherichia coli expression vector. The resulting plasmid pEXLMM74 expressed non-fused rabbit skeletal muscle LMM with yields ranging from 1 to 5% of the total proteins of E. coli. This LMM was specifically recognized by polyclonal antibodies raised against chicken pectoralis muscle myosin. It could be highly enriched from E. coli extracts by using two cycles of high and low ionic strength buffer. The partially purified protein contained a major side-product, with a calculated molecular mass of 59 kilodaltons, that is produced by translation initiation from a site in the coding region of LMM. After deletion of the translation initiation site derived from the expression plasmid, only the 59 kilodalton protein is expressed in E. coli from the resulting plasmid pEXLMM59. Both the 74 and 59 kilodalton proteins were shown to form paracrystals. They were studied by electron microscopy using negative staining and were found to show characteristic striations with an axial periodicity of about 43 nm. By circular dichroism measurement we showed that the purified 59 kilodalton protein is folded mostly as an alpha-helix.     

SOME PROPERTIES OF EMBRYONIC MYOSIN

Myosins from the following sources were purified by diethylaminoethyl-Sephadex chromatography: moytubes grown in vitro for 7–8 days, prepared from pectoralis muscles of 10-day old embryos, and breast and leg muscles from 16-day old embryos. The adenosine triphosphatase activities of these myosins were close to that of adult m. pectoralis myosin. The light chains of the embryonic myosins had the same mobilities in sodium dodecyl sulfate electrophoresis as those in adult pectoralis muscle myosin and were clearly distinguishable from those in myosin from tonic muscle m. latissimus dorsi anterior. The fastest light chain in embryonic muscle myosin—apparent mol wt 16,000—was present in smaller amounts than in adult myosin. The negative staining pattern of paracrystals of embryonic light meromyosin (LMM) was indistinguishable from that of adult fast muscle LMM. The significance of these results for differentiation of various muscle types has been discussed.     

Expression of ACh-activated channels and sodium channels by messenger RNAs from innervated and denervated muscle

Xenopus oocytes were used to express polyadenylated messenger RNAs (mRNAs) encoding acetylcholine receptors and voltage-activated sodium channels from innervated and denervated skeletal muscles of cat and rat. Oocytes injected with mRNA from denervated muscle acquired high sensitivity to acetylcholine, whereas those injected with mRNA from innervated muscle showed virtually no response. Hence the amount of translationally active mRNA encoding acetylcholine receptors appears to be very low in normally innervated muscle, but increases greatly after denervation. Conversely, voltage-activated sodium currents induced by mRNA from innervated muscle were about three times larger than those from denervated muscle; this result suggests that innervated muscle contains more mRNA coding for sodium channels. The sodium current induced by mRNA from denervated muscle was relatively more resistant to block by tetrodotoxin. Thus a proportion of the sodium channels in denervated muscle may be encoded by mRNAs different from those encoding the normal channels.     

Surgical Sympathetic Denervation Increases α1Adrenoceptor-Mediated Accumulation of myo-Inositol Trisphosphate and Muscle Contraction in Rabbit Iris Dilator Smooth Muscle

Sympathetic denervation of the iris muscle produces increases in both the breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2) and in muscle contraction in response to norepinephrine (NE). To shed more light on the biochemical basis underlying this supersensitivity we investigated: the effects of NE on PIP2 breakdown, measured as myo-inositol trisphosphate (IP3) accumulation, and on muscle contraction in normal and denervated rabbit iris dilator; and the effects of denervation on selected biochemical properties of this muscle. The data obtained from these studies can be summarized as follows: The EC50 values (microM) for NE-induced IP3 accumulation in normal and denervated dilators were 14 and 3, respectively. This accumulation of IP3 was blocked by prazosin (1 microM). The EC50 values (microM) for NE-induced contraction for the normal and denervated muscles were 10 and 0.6, respectively. The NE-induced muscle contraction was blocked by prazosin (1 microM). The t1/2 values (s) for IP3 accumulation in normal and denervated muscles were 31 and 11, respectively, and for contraction the values were 19 and 9, respectively. Denervation increased significantly (15-18%) the basal labelling of phosphoinositides from myo-[3H]inositol, but not from 32P or [14C]arachidonic acid. Denervation had little effect on the activities of the enzymes involved in phosphoinositide metabolism. However, the activities of protein kinase C and Ca2+-ATPase increased in the denervated muscle. It is concluded that sympathetic denervation of the iris dilator renders the coupling between alpha1 receptors and PIP2 breakdown into IP3 and 1,2-diacylglycerol (DG) more efficient.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of myosin heavy chain isoforms in regenerating myotubes of innervated and denervated chicken pectoral muscle

Monoclonal antibodies were prepared to stage-specific chicken pectoral muscle myosin heavy chain isoforms. From comparison of serial sections reacted with these antibodies, the myosin heavy chain isoform composition of individual myofibers was determined in denervated pectoral muscle and in regenerating myotubes that developed following cold injury of normal and denervated muscle. It was found that the neonatal myosin heavy chain reappeared in most myofibers following denervation of the pectoral muscle. Regenerating myotubes in both innervated and denervated muscle expressed all of the myosin heavy chain isoforms which have thus far been characterized in developing pectoral muscle. However, the neonatal and adult myosin heavy chains appeared more rapidly in regenerating myotubes compared to myofibers in developing muscle. While the initial expression of these isoforms in the regenerating areas was similar in innervated and denervated muscles, the neonatal myosin heavy chain did not disappear from noninnervated regenerating fibers. These results indicate that innervation is not required for the appearance of fast myosin heavy chain isoforms, but that the nerve plays some role in the repression of the neonatal myosin heavy chain.     

Inhibition of smooth muscle myosin's activity and assembly by an anti-rod monoclonal antibody.

Monoclonal antibodies specific for the rod region can affect smooth muscle myosin's motor properties. Actin movement by phosphorylated myosin was inhibited by an antibody (LMM.4) which binds to the COOH-terminal end of the coiled-coil rod, a region thought to be involved in filament assembly. The actin-activated ATPase activity of the myosin-antibody LMM.4 complex was also reduced 10-fold at actin concentrations that gave maximal turnover rates with filamentous myosin. Metal-shadowing of the phosphorylated myosin-antibody complex at low ionic strength showed small bundles of parallel extended molecules, instead of filaments. Five other anti-rod antibodies had little or no effect on myosin's ability to act as a motor. This is the first demonstration that a muscle myosin's activity is affected by its state of assembly. A common theme that emerges from the studies on both muscle and non-muscle myosins is that assembly into a filamentous structure stimulates the activity of the individual myosin molecules.     

Activity alters muscle reinnervation and terminal sprouting by reducing the number of Schwann cell pathways that grow to link synaptic sites

In partially denervated rodent muscle, terminal Schwann cells (TSCs) located at denervated end plates grow processes, some of which contact neighboring innervated end plates. Those processes that contact neighboring synapses (termed "bridges") appear to initiate nerve terminal sprouting and to guide the growth of the sprouts so that they reach and reinnervate denervated end plates. Studies conducted prior to knowledge of this potential involvement of Schwann cells showed that direct muscle stimulation inhibits terminal sprouting following partial denervation (Brown and Holland, 1979). We have investigated the possibility this inhibition results from an alteration in the growth of TSC processes. We find that stimulation of partially denervated rat soleus muscle does not alter the length or number of TSC processes but does reduce the number of TSC bridges. Stimulation also reduces the number of TSC bridges that form between end plates during reinnervation of a completely denervated muscle. The nerve processes ("escaped fibers") that normally grow onto TSC processes during reinnervation are also reduced in length. Therefore, stimulation alters at least two responses to denervation in muscles: (1) the ability of TSC processes to form or maintain bridges with innervated synaptic sites, and (2) the growth of axons along processes extended by TSCs.     

Neural- and endocrine control of flight muscle degeneration in the adult cricket,Gryllus bimaculatus

Neural- and endocrine mechanisms controlling degeneration of a dorsal longitudinal flight muscle, M112a, have been studied in adult Gryllus bimaculatus (Orthoptera: Gryllidae). Decapitation completely prevented muscle degeneration. Implantation of a pair of corpora allata or injection of juvenile hormone III into decapitated crickets caused muscle degeneration. Denervation of M112a resulted in reduction of muscle mass compared with that in sham-operated crickets. Denervation of M112a in decapitated crickets, however, did not affect muscle mass. Birefringence and ultrastructure of M112a showed an obvious regional difference in the onset of degeneration. Fibrillar structures of M112a always disappeared from the ventral to dorsal part. Distribution of axon terminals of motor neurons and mechanical responses to the motor nerve stimuli showed that M112a is composed of five motor units with similar twitch properties. When M112a was fully denervated, regional differences in degeneration disappeared. Partial denervation resulted in denervated muscle fibers losing birefringence earlier than innervated fibers. These results suggest that juvenile hormone causes breakdown of flight muscles, and neural factors control degeneration of flight muscles to some extent under the presence of the juvenile hormone.     

cDNA cloning and characterization of temperature-acclimation-associated light meromyosins from grass carp fast skeletal muscle

The three types of cDNA clones, previously defined as the 10 degrees C, intermediate and 30 degrees C-types [Tao, Y., Kobayashi, M., Liang, C.S., Okamoto, T., Watabe, S., 2004. Temperature-dependent expression patterns of grass carp fast skeletal myosin heavy chain genes. Comp. Biochem. Physiol. B 139, 649-656], were determined for their 5'-regions which encoded at least the C-terminal half of myosin rod, light meromyosin (LMM), in fast skeletal muscles of grass carp Ctenopharyngodon idella. The deduced amino acid sequence identity was 91.1% between the 10 degrees C and 30 degrees C-types and 91.4% between the 10 degrees C and intermediate-types, whereas a high sequence identity of 97.8% was found between the intermediate and 30 degrees C-types. These three grass carp LMMs all had a characteristic seven-residue (heptad) repeat (a, b, c, d, e, f, g)(n), where positions a and d were normally occupied by hydrophobic residues, and positions b, c and f by charged residues. However, the ratios of hydrophobic residues to the total were higher for the intermediate- and 30 degrees C- than 10 degrees C-type LMM, suggesting that the former both types may form more stable coiled-coils of alpha-helices than the latter type. These differences in the primary structures of LMM isoforms might be partially implicated in differences in the thermostabilities and gel-forming profiles of myosins from grass carp in different seasons reported previously [Tao, Y., Kobayashi, M., Fukushima, H., Watabe, S., 2005. Changes in enzymatic and structural properties of grass carp fast skeletal myosin induced by the laboratory-conditioned thermal acclimation and seasonal acclimatization. Fish. Sci. 71, 195-204; Tao, Y., Kobayashi, M., Fukushima, H., Watabe, S., 2007. Changes in rheological properties of grass carp fast skeletal myosin induced by seasonal acclimatization. Fish. Sci. 73, 189-196].     

Biochemical and electrophysiological properties of antibodies against pure acetylcholine receptor from vertebrate muscles and its subunits from Torpedo in relation to experimental myasthenia

Immunization of rabbits with homogeneous preparations of acetylcholine receptor from denervated muscle of cat and chicken, which contained single or multiple sizes of polypeptides respectively, induced myasthenic-like symptoms. One of the resultant antisera, and the IgG fraction thereof, reduced significantly and irreversibly the amplitude of miniature endplate potentials in murine muscle; the effect was not abolished by heat inactivation of complement. This antiserum also retarded the binding of α-bungarotoxin to a solubilised extract of denervated muscle containing homologous receptor. The other five antibody preparations were unable to affect these miniature potentials but many of them did reduce the binding of α-bungarotoxin to denervated muscle receptor in solution and, in some cases, decreased the effectiveness of the latter in blocking neuromuscular transmission. Although inoculation with each of the four individual subunits from the receptor of Torpedo marmorata electroplax did not produce muscle weakness in rabbits, antibodies to α- or β-polypeptides lowered, to a significant extent, the amplitude of spontaneous synaptic potentials in mouse diaphragm muscle. It is concluded that antibodies with direct blocking actions on the receptor-ion channel complex are not common in such immunized animals and their presence cannot be correlated readily with the induction of physical disability. The majority of the antibody species bind to loci distant from the acetylcholine recognition site. Antiserum from one of the immunized rabbits reacted preferentially with receptor from denervated rather than innervated cat and rat muscle, indicating some dissimilarity.     

Combined effects of tenotomy and denervation on the dynamic properties of soleus muscle of the rat

Isometric contraction time (CT), half relaxation time (1/2 RT), tetanus fusion frequency (TFF) and tetanus: twitch ratio (T : t ratio) were measured in the denervated (D) and tenotomized-denervated (TD) Soleus muscle of the rat. In D muscle there was an apparent speeding effect at the 2nd day after denervation, with a significant decrease of CT, which was followed by the usual slowing process of denervated muscle. In TD muscle, denervation was performed a week after tenotomy. Tenotomy "per se" was ineffective in modifying dynamic properties of muscle, but it accentuated the early shortening of CT caused by denervation, while reducing and delaying the subsequent slowing process. The results are discussed in the light of the hypothesis that muscle disuse has a speeding effect which counteracts the slowing effect of denervation, and/or that tenotomy modifies the effects of denervation by changing the pattern of fibrillation development.