Neural regulation of acetylcholinesterase-associated collagen Q in rat skeletal muscles

Acetylcholinesterase-associated collagen Q is expressed also outside of neuromuscular junctions in the slow soleus muscle, but not in fast muscles. We examined the nerve dependence of muscle collagen Q expression and mechanisms responsible for these differences. Denervation decreased extrajunctional collagen Q mRNA levels in the soleus muscles and junctional levels in fast sternomastoid muscles to about one third. Cross-innervation of denervated soleus muscles by a fast muscle nerve, or electrical stimulation by 'fast' impulse pattern, reduced their extrajunctional collagen Q mRNA levels by 70–80%. In contrast, stimulation of fast muscles by 'slow' impulse pattern had no effect on collagen Q expression. Calcineurin inhibitors tacrolimus and cyclosporin A decreased collagen Q mRNA levels in the soleus muscles to about 35%, but did not affect collagen Q expression in denervated soleus muscles or the junctional expression in fast muscles. Therefore, high extrajunctional expression of collagen Q in the soleus muscle is maintained by its tonic nerve-induced activation pattern via the activated Ca²⁺-calcineurin signaling pathway. The extrajunctional collagen Q expression in fast muscles is independent of muscle activation pattern and seems irreversibly suppressed. The junctional expression of collagen Q in fast muscles is partly nerve-dependent, but does not encompass the Ca²⁺-calcineurin signaling pathway.     

Pattern of arborization of the motor nerve terminals in the fast and slow mammalian muscles.

A silver impregnation method and a morphometric approach were used to define differences existing in the motor nerve terminal branching pattern between a fast-twitch muscle (extensor digitorum longus) and a slow-twitch one (soleus) of the normal adult rat. Because no single measure can describe precisely all geometrical properties (ie both topology and metrics) of the nerve terminals, we evaluated morphologic parameters defining length and angular characteristics in the different terminal segments classified according to their centrifugal order. The main results indicate that the distal free-end segments in the extensor digitorum longus muscle are shorter and less divergent than in the soleus nerve terminals. The endings in the two muscles have different fractal dimensions. Findings are discussed in the context of the hypothetical mechanisms governing motor nerve terminal size and complexity.     

The exchange of Ca2+-receptive protein complex (troponin) in the myofibrils of fast and slow skeletal muscles

In order to compare the role of the Ca²⁺-receptive protein (troponin), in the characteristic myofibrillar contractile response of chicken fast and slow skeletal muscles, the troponin in both kinds of myofibrils were partially exchanged, under slightly acidic conditions. The Ca²⁺- or Sr²⁺-activation of the ATPase of fast (or slow) skeletal myofibrils hybridized with slow (or fast) skeletal troponin profiles were also investigated. The results indicated that the Ca²⁺- or Sr²⁺-affinity of the myofibrillar ATPase activity were related to the species of troponin. This procedure for replacing troponin in myofibrils under physiological conditions in thus considered to be useful for the study of the Ca²⁺-regulatory mechanism in myofibrillar contraction.     

Regeneration of muscles after cardiotoxin injury. I. Cytological aspects

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

Differentiation of slow and fast fibers in tentacles of Sepia officinalis (Mollusca)

The tentacles of Sepia officinalis are cylindrical muscular structures that can be quickly everted and elongated to capture prey. The combination of both velocity and extensive elongation of the tentacles is due to the presence of both cross-striated and helical muscles. The complex organization and differentiation of different fibers has been studied in cuttlefish extracted from egg gel coats at different developmental stages, and in completely developed animals. Tentacle muscles start to differentiate centrifugally from the area close to the axial nervous system, where two types of myocytes can be recognized. These populations of myocytes, which may be distinguished morphologically and which express different myosin isoforms, give rise to fast and slow muscles. The presence in molluscs of slow and fast muscles arising from different populations of myocytes, as in vertebrate muscle development, could be considered as an example of evolutionary conservation.     

豚鼠眶外肌中快肌与慢肌纤维的小终板电流

豚鼠眶外肌中有两类不同电活动模式的肌纤维。一类纤维有动作电位,小终板电位时程较短并且较一致;另一类纤维不能产生动作电位,小终板电位时程较长而且多变。用电泳注射普胺天蓝分别标记这两类纤维各6条,并结合胆碱酯酶染色,结果表明,前一类肌纤维都是只有一个神经肌肉接头的快纤维;而另一类肌纤维都是有多个接头的慢纤维。根据双微电极电庄箱位实验结果,计算了上述决、慢肌纤维小终板电流下降相时间常数τ及其电压系数A。实验表明:(1)在静息电位附近,慢肌纤维小终板电流的τ值大于快肌纤维的;(2)慢肌纤维的A值比快肌纤维的小的多,即τ值较少随膜电位变化。本文对上述不同曾加以讨论。     

Trophic regulation of acetylcholinesterase isoenzymes in adult mammalian skeletal muscles

This work addresses the physiological regulation of skeletal muscle acetylcholinesterase (AChE) isoforms by examining endplate-enriched samples from adult rat gracilis muscles 48 h after: lowintensity treadmill exercise; obturator nerve transection; nerve impulse conduction blockade by tetrodotoxin; acetylcholine (ACh) receptor (AChR) inactivation by -bungarotoxin; and, addition of obturator nerve extracts to muscles in organ culture. Results document the important role(s) of functional AChRs and ACh-AChR interactions in the differential control of individual AChE isoenzymes. A theoretical model based on these and other findings considers that: AChR activation by spontaneously released ACh is the only neural factor required for the maintenance of G₁+G₂ AChE; the amount of A₁₂ AChE is determined by the combined effects of ACh and another neurogenic substance; although mechanisms intrinsic to myofibers control normal levels of G₄ AChE, enhanced production of this isoform is initiated through increasing the frequency of ACh-AChR interactions.Special issue dedicated to Dr. Frederick E. Samson     

Molecular forms and localization of acetylcholinesterase and nonspecific cholinesterase in regenerating skeletal muscles

Molecular forms and histochemical localization of acetylcholinesterase and nonspecific cholinesterase were analysed in muscle regenerates obtained from rat EDL and soleus muscles after ischaemic-toxic degeneration and irreversible inhibition of preexistent enzymes. Regenerating myotubes and myofibres produce the 16S AChE form in the absence of innervation. The 10S AChE form prevails over 4S form with maturation into striated fibres. Although the patterns of AChE molecular forms in normal EDL and soleus muscles differ significantly no such differences were observed in noninnervated regenerates from both muscles. Two types of focal accumulation of AChE appear on the sarcolemma of regenerating muscles: first, in places of former motor endplates and, second, in extrajunctional regions. The 4S form of nonspecific cholinesterase is prevailing in regenerating myotubes whereas its asymmetric forms or focal accumulations could not be identified reliably. The satellite cells which survive after muscle degeneration probably originate from some type of late myoblasts and transmit the information concerning the ability to synthesize the asymmetric AChE forms and to focally accumulate AChE to regenerating muscle cells. Synaptic basal lamina from former motor endplates may locally induce AChE accumulations in regenerating muscles.Special Issue Dedicated to Dr. Abel Lajtha.     

rAAV6-microdystrophin rescues aberrant Golgi complex organization in mdx skeletal muscles

Muscular dystrophies are a diverse group of severe degenerative muscle diseases. Recent interest in the role of the Golgi complex (GC) in muscle disease has been piqued by findings that several dystrophies result from mutations in putative Golgi-resident glycosyltransferases. Given this new role of the Golgi in sarcolemmal stability, we hypothesized that abnormal Golgi distribution, regulation and/or function may constitute part of the pathology of other dystrophies, where the primary defect is independent of Golgi function. Thus, we investigated GC organization in the dystrophin-deficient muscles of mdx mice, a mouse model for Duchenne muscular dystrophy. We report aberrant organization of the synaptic and extrasynaptic GC in skeletal muscles of mdx mice. The GC is mislocalized and improperly concentrated at the surface and core of mdx myofibers. Golgi complex localization is disrupted after the onset of necrosis and normal redistribution is impaired during regeneration of mdx muscle fibers. Disruption of the microtubule cytoskeleton may account in part for aberrant GC localization in mdx myofibers. Golgi complex distribution is restored to wild type and microtubule cytoskeleton organization is significantly improved by recombinant adeno-associated virus 6-mediated expression of DeltaR4-R23/DeltaCT microdystrophin showing a novel mode of microdystrophin functionality. In summary, GC distribution abnormalities are a novel component of mdx skeletal muscle pathology rescued by microdystrophin expression.     

Neural influence on the expression of acetylcholinesterase molecular forms in fast and slow rabbit skeletal muscles

With the aim of investigating the roles of motor innervation and activity on muscle characteristics, we studied the molecular forms of acetylcholinesterase (AChE) in fast-twitch (semimembranosus accessorius; SMa) and slow-twitch (semimembranosus proprius; SMp) muscles of the rabbit. We have shown that SMa and SMp express different patterns and tissue distribution of AChE forms and that the effect of long denervation varies with age. Three principal findings concerning expression of AChE molecular forms emerge from these studies. (1) The activity of AChE and the pattern of its molecular forms are particularly altered in adult denervated SMa and SMp muscles. AChE activity increases by 10-fold in both muscles, but asymmetric forms disappear in SMa and increase by 20-fold in SMp muscles. A similar alteration of AChE is found after tenotomy of these muscles, showing that the effect of denervation may be partly due to suppression of muscle activity. (2) The different changes occurring in the composition of AChE molecular forms in adult denervated SMa and SMp muscles are consistent with fluorescent staining with anti-AChE monoclonal antibodies and with DBA or VVA lectins, which bind to AChE asymmetric, collagen-tailed forms. These lectins poorly stain denervated SMa muscle surfaces but intensely stain neuromuscular junctions and extrasynaptic areas in denervated SMp muscle. (3) In contrast with the adult, denervation of 1-day-old muscles does not markedly modify the total amount of AChE or the proportions of its molecular forms, despite dramatic effects on muscle structure. These results are supported by studies of labeling with fluorescent DBA: the lectin only slightly stains the muscle fiber surface of denervated 15-day-old SMp muscle. Taken together, these data show that denervated muscles escape physiological regulation, producing increased levels of AChE with highly variable cellular distribution and patterns of molecular forms, depending on the age of operation and on the type of muscle.     

Effects of aging and life-long physical training on collagen in slow and fast skeletal muscle in rats

Summary Intramuscular collagen in a slow (m. soleus) and a fast (m. rectus femoris) skeletal muscle was studied by biochemical, morphometric, and immunohistochemical methods. Wistar white rats of 1, 4, 10, and 24 months were used as experimental animals. Our aim was to evaluate the effects of life-long physical training (treadmill running, 5 days a week for 1, 3, 9, and 23 months depending on the age attained). The biochemical concentration of collagen was higher in m. soleus than in m. rectus femoris and it increased in youth and in old age in m. soleus. The trained rats had higher concentrations of collagen than the untrained rats at 10 and 24 months. The morphometrically measured area-fractions of both the endomysium and perimysium were higher in m. soleus than in m. rectus femoris. The age-related increase in intramuscular connective tissue was of endomysial origin. The immunohistochemical staining of type-I, -III, and -IV collagens indicated the more collagenous nature of m. soleus as compared with m. rectus femoris for all major collagen types; this was most marked for type-IV collagen of basement membrane. The results indicate that both age and endurance-type physical training further distinguish the slow and fast muscles with respect to their connective tissue.     

Morphological differences between excitatory and inhibitory nerve terminals in cockroach coxal muscles

Two morphologically distinct types of neuromuscular junction on the coxal leg muscles of the cockroach, Periplaneta americana, which have been physiologically described as innervated by fast, slow and inhibitory nerve fibers, have been found. In one type of neuromuscular junction the axon terminal contains many round clear synaptic vesicles and contacts several sarcoplasmic extensions from the muscle fiber. The muscle processes adhere to the axon terminal for a short distance (short contact or SC type). The axon terminal of the other type of neuromuscular junction directly contacts the muscle fiber and no extensions of the muscle fiber are formed. The contact region is comparatively long (long contact or LC type). The nerve terminal contains many polymorphic synaptic vesicles. From a correlation of the present morphological findings and the previous physiological results, it may be suggested that the SC type of nerve terminal represents both fast and slow nerve terminals and the inhibitory terminal is of the LC type.     

Testosterone control of endplate and non-endplate acetylcholinesterase in the rat levator ani muscle

The time course of effects of castration (5–60 days) and testosterone treatment (15–60 days) of adult male rats were examined on the endplate (+EP) and non-endplate (–EP) acetylcholinesterase (AChE) of the androgen-dependent levator ani (LA) muscle. The thiocholine method was used to determine the enzyme activity. Castration caused LA muscle atrophy within 5 days but reduced the –EP and +EP AChE after 10 and 20 days, respectively. Following 30 days castration –EP and +EP AChE reached respectively 41% and 35% of control activity. Testosterone retrieval restored the control values of both muscle weight and total AChE after 15 and 60 days, respectively. Recovery of the +EP AChE preceded that of –EP AChE by 30 days. The results showed that in the rat LA muscle, +EP and –EP AChE depend on a continuous testosterone regulation that predominates at +EP region spreading thereafter to –EP region. Those data suggest a hormone regulation of AChE exerted indirectly through the synthesis and release of neurotrophic substances.     

Regulation of glucose uptake in rat slow and fast skeletal muscles

1. Regulation of glucose uptake was compared between extensor digitorum longus (EDL) and soleus (Sol) muscles in rats. 2. Insulin stimulated glucose uptake more in EDL than in Sol. 3. Under high concentrations of insulin, the glucose uptake was higher in EDL than Sol. 4. Inhibition of oxidative phosphorylation by anoxia or an uncoupler stimulated glucose uptake more in EDL than in Sol. 5. Anoxia abolished the effect of insulin on glucose uptake in both EDL and Sol. 6. The blocker to glucose transport system reduced glucose uptake more in Sol than in EDL.     

Influence of hindlimb unloading on the modulation of neurotransmitter secretion through the autoreceptor system

In experiments on neuromuscular junctions of fast (m. extensor digitorum longus, EDL) and slow (m. soleus) muscles of rats under hindlimb unloading of varied duration, we compared the intensity of spontaneous quantal secretion of neurotransmitter in response to potassium depolarization and activation of presynaptic cholinoreceptors with a nonhydrolyzable acetylcholine analog. Secretion was assessed by the mean frequency of miniature endplate potentials. In the controls, carbachol raised this index by 363% in EDL and by 62% in soleus. Secretion in the fast muscle was also more sensitive to [K⁺]. Hindlimb unloading abolished the sensitivity to carbachol in EDL while in soleus it did not change. Preservation of the sensitivity of the fast muscle to potassium depolarization suggested that unloading reduced the number of functional presynaptic receptors.     

Exercise-dependent regulation of glial cell line-derived neurotrophic factor (GDNF) expression in skeletal muscle and its importance for the neuromuscular system

The focus of this review is to highlight the importance of glial cell line-derived neurotrophic factor (GDNF) for the motor nervous system. GDNF is the most potent survival factor for motor neurons, where it enhances maintenance and survival of both developing and mature motor neurons in vivo and in vitro. GDNF aids in neuromuscular junction formation, maintenance, and plasticity, where skeletal muscle-derived GDNF may be responsible for this phenomenon. Increased levels of physical activity can increase GDNF protein levels in skeletal muscle, where alterations in acetylcholine and acetylcholine receptor activation may be involved in regulation of these changes observed. With inactivity and disuse, GDNF expression shows different patterns of regulation in the central and peripheral nervous systems. Due to its potent effects for motor neurons, GDNF is being extensively studied in neuromuscular diseases.     

Motor and sensory reinnervation of fast and slow mammalian muscles

Summary 1. The formation of endplates outside the original endplate region of a muscle fibre was studied in slow and fast rat muscles. It was found that such new endplates are readily formed on the soleus muscle, whereas hardly at all in the fast extensor digitorum longus. Most new endplates appear to be morphologically normal within 2 months after nerve implantation.2. The time course of recovery of slow and fast cat muscles was followed after crushing the sciatic nerve. It was found that the slow soleus muscle recovers more rapidly than the fast flexor hallucis longus muscle.3. The endplates of reinnervated cat muscles are more complicated than those of the control muscles, but have nevertheless fewer nerve terminals per endplate. Reinnervated muscles are more sensitive to curare and it is suggested that this is due to a decrease in transmitter release, for it was found that less acetylcholine is released from reinnervated rat hemidiaphragms than from control ones.4. Motor and sensory reinnervation of spindles and tendon organs was studied. At the time when motor reinnervation is almost completed the sensory endings from spindles and tendon organs are highly abnormal. Thus sensory reinnervation proceeds much more slowly than motor.     

Reciprocal neural regulation of extrajunctional acetylcholinesterase and collagen Q in rat muscles—The role of calcineurin signaling

There are two main differences regarding acetylcholinesterase (AChE) expression in the extrajunctional regions of fast and slow rat muscles: (1) the activity of AChE catalytic subunits (G1 form) is much higher in fast than in slow muscles, and (2) the activity of the asymmetric forms of AChE (A₈ and A₁₂) is quite high extrajunctionally in slow muscles but virtually absent in fast muscles. The latter is due to the absence of the expression of AChE-associated collagen Q (ColQ) in the extrajunctional regions of fast muscle fibers, in contrast to its ample expression in slow muscles. We showed that both differences are caused by different neural activation patterns of fast vs. slow muscle fibers, which determine the respective levels of mRNA of both proteins. Whereas the changes in AChE mRNA levels in fast and slow muscles, as well as the levels of ColQ mRNA levels in slow muscles, observed in response to exposing either slow or fast muscles to different muscle activation patterns, are completely reversible, the extrajunctional suppression of ColQ expression in fast muscle fibers seems to be irreversible. Calcineurin signaling pathway in muscles is activated by high-average sarcoplasmic calcium concentration resulting from tonic low-frequency muscle fiber activation pattern, typical for slow muscle fibers, but is inactive in fast muscle fibers, which are activated by infrequent high-frequency bursts of neural impulses. Application to rats of two inhibitors of calcineurin (tacrolimus-FK506 and cyclosporin A) demonstrated that the mRNA levels of both the AChE catalytic subunit and ColQ in the extrajunctional regions of the soleus muscle are regulated by the calcineurin signaling pathway, but in a reciprocal way. Under the conditions of low calcineurin activity, AChE expression is enhanced and that of ColQ is suppressed, and vice versa. Our results also indicated that different, calcineurin-independent regulatory pathways are responsible for the reduction of AChE expression during muscle denervation, and for maintaining high ColQ expression in the neuromuscular junctions of fast muscle fibers.