Fine structure,origin, and distribution density of the autonomic nerve endings in the tarsal muscle in the eyelid of the mouse

Summary The fine structure, origin, and distribution density of the autonomic nerve endings in the tarsal muscle of the mouse were studied by histochemistry and electron microscopy. With histochemical methods, the fine nerve plexus in the normal muscle shows both catecholamine-positive varicose fibers and acetylcholinesterase-active varicose fibers. The former are distributed more densely than the latter. After superior cervical ganglionectomy, the catecholamine-positive fibers disappear, while after pterygopalatine ganglionectomy, the acetylcholinesterase-active fibers vanish. In electron micrographs, the varicosities appear as expansions containing many synaptic vesicles. The axonal expansions partly lack a Schwann sheath and directly face the pinocytotic vesicle-rich zones of the smooth muscle cells. A relatively wide space, 0.1 to 1.0 m in width, lies between nerve expansion and muscle cell. The expansions can be classified into two types: Type I having small granular synaptic vesicles, and Type II having agranular vesicles instead of small granular synaptic vesicles. Type I undergoes degeneration after superior cervical ganglionectomy, while Type II degenerates after pterygopalatine ganglionectomy. This indicates that Type I corresponds to the synaptic ending of the adrenergic fiber originating from the superior cervical ganglion, and Type II to the synaptic ending of the cholinergic nerve fiber derived from the pterygopalatine ganglion. Type I is more frequent (88/10⁴ m² area of muscle) than Type II (17/10⁴ m²).     

Regulatory mechanism of contraction in the proboscis retractor muscle of a sipunculid worm,Phascolosoma scolops

Summary Regulatory mechanism of contraction in the proboscis retractor muscle of Phascolosoma scolops was studied by physiological measurements and cytochemical electron microscopy. The magnitude of K⁺-contracture was dependent on external Ca²⁺ concentration and the contracture disappeared in Ca²⁺-free solution. The K⁺-contracture was suppressed by application of procaine and Mn²⁺. Caffeine induced contracture even when external Ca²⁺ was absent. Ultrastructural observations of the retractor muscle cells showed the presence of a large number of vesicles (subsarcolemmal vesicles), corresponding to the sarcoplasmic reticulum in vertebrate skeletal muscle, underneath the plasma membrane. For the cytochemical electron microscopy, the muscle fibers were fixed with 1% OsO₄ solution containing 2% K-pyroantimonate. In the relaxed fibers, pyroantimonate precipitates were localized along the inner surface of plasma membrane and in the subsarcolemmal vesicles. In the contracting fibers, the precipitates were uniformly distributed in the myoplasm. The X-ray microanalysis revealed that the precipitates contained Ca. These results suggest that the contractile system is activated by the influx of extracellular Ca²⁺ as well as by the release of Ca²⁺ from the intracellular structures such as the inner surface of the plasma membrane and subsarcolemmal vesicles.     

The weak combined magnetic field,tuned to ion parametric resonance for Ca<Superscript>2+</Superscript>, stimulates dystrophin synthesis in the skeletal muscles of mdx mice subjected to cell therapy

The possibility of using bone marrow stem cells for treatment of Duchenne muscular dystrophy is intensely studied. Mdx mice are the most widely used laboratory model of Duchenne muscular dystrophy. One approach of cell therapy of muscular dystrophy is substitution of bone marrow in mdx mice after their X-ray irradiation. However, this method does not allow one to increase significantly dystrophin synthesis in muscular fibers of mdx mice. To improve the effect of transplanted cells on muscle regeneration, we additionally treated mdx mice subjected to transplantation of bone marrow cells with a weak combined magnetic field tuned to ion parametric resonance for Ca²⁺ (Ca²⁺-CMF). We found that, in irradiated chimeric 3 and 5 Gy mdx mice, additional treatment with Ca²⁺-CMF for 1 month resulted in significant increases in the portions of dystrophin-positive muscle fibers, by 15.8 and 18.3%, respectively, as compared to the control groups. Furthermore, the share of muscle fibers without centrally located nuclei also increased. We suggest that the magnetic field with these parameters may stimulate functioning of nuclei of donor cells, which were incorporated into muscle fibers.     

Laser Raman study of internally perfused muscle fibers effect of Mg2+, ATP and Ca2+

Raman spectra of an intact muscle fiber and of internally perfused fibers in capillary tubes have been obtained. The use of internal perfusion has insured a good control of the concentration of Ca²⁺, Mg²⁺ and ATP. The comparaison of the spectra obtained with the two types of fibers shows that the muscle structure is well preserved in capillary tubes. In addition, it appears that the sarcomere length has no significant effect on the Raman spectrum of muscle fibers. Our results on perfused fibers demonstrate that a fiber can be kept in the relaxed state for several hours, then displaying an intact fiber spectrum, when the concentration of ATP, Mg²⁺ and Ca²⁺ is maintained at 5, 2 and 0 mM, respectively. Therefore ATP and Mg²⁺ do not affect the Raman spectrum of muscle fibers. When one of these components is removed, or when Ca²⁺ is added, contraction occurs and causes major spectral changes. These results are interpreted as being due to strong electrostatic interactions between basic and acidic residues during contraction, and to a change of the α-helical content, or of the orientation, of some of the contractile proteins.     

Capillarization,mitochondrial densities,oxygen diffusion distances and innervation of red and white muscle of the lizard Dipsosaurus dorsalis

Summary The white and red regions of the iliofibularis muscle of the lizard Dipsosaurus dorsalis were analyzed using histologic and morphometric analysis. These regions are composed of fast glycolytic (FG) and both fast oxidative, glycolytic (FOG) and tonic fibers, respectively. Endplate morphology and number of endplates per fiber were estimated from fibers from both areas. Capillary volume densities of the red and white regions were quantified from transverse sections. Mitochondrial volume of fibers from the red and white regions were estimated from electron micrographs.All fibers from the white region of the iliofibularis possessed a single, well defined endplate, as did most red region fibers. The remaining red fibers (28±5%) possessed an average of 14.7±3 endplates each, distributed along the entire length of the fiber at intervals of approximately 1124 m.Red fibers possessed twice the mitochondrial volume of white fibers (7.6±0.4%, red; 3.8±0.3%, white). Mitochondria were distributed uniformly through the fibers from both regions. Capillary anisotropy was low ( = 1.018) in both regions. Capillary densities of the red region (629±35 mm^-2) were much greater than those of the corresponding White region (73±8 mm^-2).The data indicate that capillary densities, mitochondrial volumes and theoretical diffusion distances correlate well with the oxidative capacity of lizard muscle fibers. Tonic fibrs of this species appear oxidative and therefore metabolically capable of functioning during locomotion. The similar mitochondrial volumes and capillary densities of reptilian and mammalian muscles suggest that the greater oxidative capacity of mammalian muscle is due in part to possession of more oxidatively active mitochondria rather than to possession of more mitochondria per se.     

Perlecan deficiency causes muscle hypertrophy,a decrease in myostatin expression,and changes in muscle fiber composition

Perlecan is a component of the basement membrane that surrounds skeletal muscle. The aim of the present study is to identify the role of perlecan in skeletal muscle hypertrophy and myostatin signaling, with and without mechanical stress, using a mouse model (Hspg2^?/?-Tg) deficient in skeletal muscle perlecan. We found that myosin heavy chain (MHC) type IIb fibers in the tibialis anterior (TA) muscle of Hspg2^?/?-Tg mice had a significantly increased fiber cross-sectional area (CSA) compared to control (WT-Tg) mice. Hspg2^?/?-Tg mice also had an increased number of type IIx fibers in the TA muscle. Myostatin and its type I receptor (ALK4) expression was substantially decreased in the Hspg2^?/?-Tg TA muscle. Myostatin-induced Smad activation was also reduced in a culture of myotubes from the Hspg2^?/?-Tg muscle, suggesting that myostatin expression and its signaling were decreased in the Hspg2^?/?-Tg muscle. To examine the effects of mechanical overload or unload on fast and slow muscles in Hspg2^?/?-Tg mice, we performed tenotomy of the plantaris (fast) muscle and the soleus (slow) muscle. Mechanical overload on the plantaris muscle of Hspg2^?/?-Tg mice significantly increased wet weights compared to those of control mice, and unloaded plantaris muscles of Hspg2^?/?-Tg mice caused less decrease in wet weights compared to those of control mice. The decrease in myostatin expression was significantly profound in the overloaded plantaris muscle of Hspg2^?/?-Tg mice, compared with that of control mice. In contrast, overloading the soleus muscle caused no changes in either type of muscle. These results suggest that perlecan is critical for maintaining fast muscle mass and fiber composition, and for regulating myostatin signaling.     

Silencing SERCA1b in a few fibers stimulates growth in the entire regenerating soleus muscle

The neonatal isoform of the sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase 1 (SERCA1b) is a dominant Ca²⁺ pump in the young fibers of regenerating muscle. In vivo transfection of about 1% of the fibers with SERCA1b RNAi plasmid resulted in no apparent change in the transfected fibers, but enhanced the increase of fresh weight and fiber size in the whole regenerating rat soleus muscle, until the normal size was reached. Co-transfection of calcineurin inhibitor cain/cabin-1 with SERCA1b RNAi was sufficient to cut down the widespread growth stimulation, but the subsequent transfection of cain into the SERCA1b RNAi transfected muscle did not inhibit muscle growth. The SERCA1b RNAi preferably upregulated the expression of the NFAT reporter lacZ compared to controls when co-transfected into the fibers. Notably, perimuscular injection of interleukin-4 (IL-4) antibody but not that of an unrelevant antibody completely abolished the growth-promoting effect of SERCA1b RNAi. This indicates that silencing SERCA1b in a few fibers stimulates the calcineurin-NFAT-IL-4 pathway and fiber growth in the whole regenerating soleus. These results suggest the presence of an autocrine–paracrine coordination of growing muscle fibers, and put forward a new method to stimulate skeletal muscle regeneration.     

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca²⁺ signaling is a fundamental physiological process in living cells. Ca²⁺ sparks are the elementary units of Ca²⁺ signaling in the striated muscle fibers that appear as highly localized Ca²⁺ release events mediated by ryanodine receptor (RyR) Ca²⁺ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca²⁺ sparks could provide information on the intracellular Ca²⁺ handling properties of healthy and diseased striated muscles. Although Ca²⁺ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.Detailed here is an experimental protocol for measuring Ca²⁺ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca²⁺ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca²⁺ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca²⁺ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP₃R) and RyR contributes to Ca²⁺ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca²⁺ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).     

Sodium, Potassium, and Chloride Fluxes in Intercostal Muscle from Normal Goats and Goats with Hereditary Myotonia

In isolated bundles of external intercostal muscle from normal goats and goats with hereditary myotonia the following were determined: concentrations and unidirectional fluxes of Na⁺, K⁺, and Cl^-, extracellular volume, water content, fiber geometry, and core-conductor constants. No significant difference between the two groups of preparations was found with respect to distribution of fiber size, intracellular concentrations of Na⁺ or Cl^-, fiber water, resting membrane potential, or overshoot of action potential. The intracellular Cl^- concentration in both groups of preparations was 4 to 7 times that expected if Cl^- were distributed passively between intracellular and extracellular water. The membrane permeability to K (P_K) calculated from efflux data was (a) at 38°C, 0.365 x 10^-6 cm sec^-1 for normal and 0.492 x 10^-6 for myotonic muscle, and (b) at 25°C, 0.219 x 10^-6 for normal and 0.199 x 10^-6 for myotonic muscle. From Cl^- washout curves of normal muscle usually only three exponential functions could be extracted, but in every experiment with myotonic muscle there was an additional, intermediate component. From these data PP_cl could be calculated; it was 0.413 x 10^-6 cm sec^-1 for myotonic fibers and was 0.815 x 10^-6 cm sec^-1 for normal fibers. The resting membrane resistance of myotonic fibers was 4 to 6 times greater than that of normal fibers.     

Decrease in the electrogenic Contribution of Na,K-ATPase and the resting membrane potential as a possible mechanism of Ca<Superscript>2+</Superscript> accumulation in rat soleus muscle in a short-term gravity unloading

The resting membrane potential and electrogenic contribution of α1- and α2-isoforms of Na⁺/K⁺-ATPase in the rat soleus muscle at early stages of gravity unloading were analyzed. The role of L-type calcium channels in accumulation of calcium ions in the myoplasm under these conditions was estimated. After 3-day antiorthostatic suspension, the resting membrane potential of the muscle fibers decreased from ?71.0 ± 0.5 to ?66.8 ± 0.7 mV, the muscle excitability reduced, and a trend of muscle fatigue acceleration appeared. The electrogenic contribution of ouabain-sensitive α2-isoform of Na⁺/K⁺-ATPase, determined as the depolarization caused by 1μM ouabain, decreased after suspension from 6.2 ± 0.6 to 0.5 ± 0.8 mV. The contribution of ouabain-resistant α1-isoform of Na⁺/K⁺-ATPase, determined as an additional depolarization after addition of 500 μM ouabain, decreased from 4.6 ± 0.6 to 2.6 ± 0.6 mV. The intensity of Fluo-4AM fluorescence in individual muscle fibers increased after suspension more than fourfold, which suggests an elevated calcium concentration in the myoplasm. A local delivery of nifedipine, a blocker of the L-type calcium channels, to the muscle removed this effect. The existence of a selective mechanism suppressing the electrogenic contribution of Na⁺/K⁺-ATPase α2-isoform, which is the main cause of the muscle fiber membrane depolarization after 3-day suspension, is postulated. The depolarization can activate part of potential-sensitive L-type Ca²⁺ channels, causing the accumulation of calcium ions in the muscle fiber myoplasm.     

Electrophysiological analysis of the temperature-sensitive paralyticDrosophila mutant,para ts

Summary Paralysis of flight in the temperature-sensitiveDrosophila mutantpara ^ts was found to be dependent on the rate of heating (Fig. 2). The gradual nature of the onset of paralysis during the temperature elevation was revealed by recording the electrical responses of the thoracic flight muscle fibers, evoked by cervical stimulation (Figs. 3,4). A neural focus of the mutation was indicated by intracellular current injections into identified flight muscle fibers during paralysis (Fig. 5) and by electrical activity recorded from gynandromorph flies, mosaic forpara ^ts (Table 1). Recording from picrotoxin-treated flies excluded a previous explanation of paralysis by a temperature-induced augmentation of GABAergic inhibition (Fig. 6). Under the same treatment, evidence was presented for a heterogeneous increase of excitation threshold for spike generation in certain neurons.Abbreviations DLM dorsolongitudinal muscle - DVM dorsoventral muscle - ts temperature sensitive - GABA Gamma aminobutyric acid We wish to thank Dr. E. Lifschytz for providing the facilities for culturing flies in his laboratory. This research was supported by Grant No. 625 from the U.S.-Israel Binational Science Foundation to D.D.     

Role of calcium ions and spike activity in the neurotrophic control of membrane potential in rat muscle fibers

Blockade of calcium permeability produced an increase in postdenervation depolarization of rat diaphragm muscle fibers during in vitro experiments, while increased Ca²⁺ concentration in the sarcoplasm induced by caffeine led to hyperpolarization of the muscle membrane. Direct stimulation of the muscles or carbamylcholine application retarded the reduction of membrane potential in the muscle fibers. Verapamil and d-tubocurarine eliminated the hyperpolarizing effect of stimulation. The hyperpolarizing effects of carbamylcholine applied in conjunction with stimulation did not produce an accumulated action on the membrane. It is deduced that the factors governing membrane potential in the muscle fibers are acetylcholine and Ca²⁺ reaching the sarcoplasm mainly through the acetylcholine-sensitive ionic channels during the process of nerve impulse fluxes.S. V. Kurashov Medical Institute, Kazan', Ministry of Public Health of the RSFSR. Translated from Neirofiziologiya, Vol. 19, No. 4, pp. 449–456, July–August, 1987.     

Electron cytochemical study of the muscle cell surface

Summary The lectins wheat germ agglutinin and limulus polyphemus were used as cytochemical probes to study the ultrastructural localization of sialic acid at the cell surface of rat muscle fibers. In addition cytochemical studies employing strontium as an electron-dense marker were also carried out to investigate cation binding sites at the muscle cell surface. The results showed binding of the lectins to the glycocalyx, caveolae and the basal lamina of the muscle fibers. These binding sites matched the ones observed in the cytochemical studies using strontium as a marker. Based on these observations we suggest that the glycocalyx, caveolae and the basal lamina of the muscle fiber may be involved in the binding of Ca⁺⁺ and that significant amounts of Ca⁺⁺ may be normally present at the muscle cell surface.Supported by a grant from the Muscular Dystrophy Association and by Center Grant NS-1176 from the National Institute of Neurological and Communicative Disorders and Stroke     

Chronic stimulation of the spinal cord in developing chick embryo causes the differentiation of multiple clusters of acetylcholine receptor in the posterior latissimus dorsi muscle

Electrodes were implanted around the spinal cord of 7-day-old chick embryos and electric pulses delivered at 0.5-Hz frequency from the 10th to 15th day of incubation. At Day 15, the posterior latissimus dorsi (PLD) muscle, which, in control animals, is focally innervated, was dissected. The number and distribution of AChR clusters revealed by autoradiography after labeling with ¹²⁵I-α-bungarotoxin was quantitatively studied on isolated muscle fiber fragments and on serial sections of the whole muscle. After chronic stimulation, muscle fibers with multiple AChR clusters were observed. The distribution of the clusters appeared less regular than in the anterior latissimus dorsi muscle which, in control embryos, receives a multiple innervation. The total number of AChR clusters per PLD muscle increased about 1.8 times as a consequence of the stimulation without significant change of the total number of muscle fibers.     

Electrical and mechanical responses of chromatophore muscle fibers of the squid,Loligo opalescens,to nerve stimulation and drugs

Summary The muscle fibers of brown and red chromatophores in the skin of the squid, Loligo opalescens, respond to motor nerve stimulation with non-propagating excitatory postsynaptic potentials (e.p.s.p.'s) of fluctuating amplitude. Depending on the strength of stimulation several size classes of e.p.s.p.'s are found, indicating polyneuronal innervation. Facilitation and summation are minimal even though the reversal potential of the e.p.s.p.'s is close to zero.Acetylcholine (ACh) and 5-hydroxytryptamine (5-HT) have no effect on membrane characteristics of the muscle fiber, but ACh greatly augments the spontaneous quantal release of transmitter [increase in the frequency of miniature postsynaptic potentials (m.p.s.p.'s)] and thereby causes tonic contraction (miniature tetanus). 5-HT reduces the frequency of miniature potentials and abolishes tonic contraction. Inhibition of cholinesterase by eserine does not affect the amplitude or time course of e.p.s.p.'s and of m.p.s.p.'s. High concentrations of cholinergic blocking agents (atropine, banthine) reduce the postsynaptic effects of nerve stimulation in some cases. The natural transmitter substance of the motoneurones may not be ACh. The action of 5-HT appears to be intracellular.Neighboring muscle fibers are electrically coupled through low resistance pathways. These are most likely provided by the close junctions that form part of the myo-muscular junctions. The specific membrane resistance of the regular muscle fiber membrane was found to range from 1,056 to 1,320 Ohm×cm², that of the close junctions ranges from 12.8 to 22.6 Ohm×cm². The area occupied by close junctions is small, however, and only 10% of the current injected into one cell passes into the next. Some of the e.p.s.p.'s observed in a given muscle fiber most likely represent the electrotonic spread of the e.p.s.p.'s of the neighbor fibers. Of the six classes of e.p.s.p.'s observed in some muscle fibers, only two may originate in these fibers themselves.Chromatophores in aged preparations often exhibit pulsations. These are caused by spike potentials arising within muscle fibers whose membranes have become electrically excitable. Each spike is preceded by a generator depolarization. The electrical coupling of neighboring muscle cells permits conduction of the spike potentials throughout the set of muscle fibers of a pulsating chromatophore. Altered conditions within such preparations also lead to tonic contractions and contractures that are not necessarily accompanied by electrical activity. Several arguments are presented in support of the hypothesis that the tonic condition of nerve terminals (characterized by enhanced spontaneous transmitter release) and of muscle fibers (characterized by inability to relax) is due to an abnormal condition of intracellular calcium (lack of Ca-binding by sarcoplasmic reticulum or other storage sites).No evidence could be found for an inhibitory innervation of the chromatophore muscles. The nerve-induced relaxation of tonically contracted muscle fibers is caused by the action of motoneurones.Preliminary experiments on muscle fibers of the anterior byssus retractor muscle of Mytilus support the hypothesis that the tonic behavior (catch) of other molluscan muscles is due to mechanisms similar to those found in the chromatophore muscles.This investigation was supported by Public Health Service Grant No. NB 04145 from the National Institute of Neurological Diseases and Blindness. We are grateful to the director of the Friday Harbor Laboratories, Prof. R. L. Fernald for providing space and facilities for this investigation.Supported by a Training Grant GM 1194 from the National Institute of General Medical Sciences.     

Calcium dependent action potentials in skeletal muscle fibres of a beetle larva, Xylotrupes dichotomus

The ionic requirement for generating action potentials in ventral longitudinal muscle fibers dissected from beetle larvae was examined by conventional electrophysiological techniques. Muscle fibers that generated only graded responses in physiological saline were able to generate an all-or-none action potential when the potassium permeability of the membrane was inhibited by tetraethylammonium⁺ added to the saline. The peak of the action potential thus elicited was intimately related to the external Ca⁺⁺ concentration. The action potential was blocked by Co⁺⁺ which is known as a competitive inhibitor of Ca-spikes. Neither tetrodotoxin (3 μM) nor a Na-free condition effectively blocked the generation of the action potential. Mg⁺⁺ induced a shift in the peak of the action potential; this was, however, due to the stabilizing action of Mg⁺⁺ but not due to the penetration of Mg⁺⁺ through the muscle membrane. No action potential was elicited in the muscle fiber when immersed in a Ca-free, EGTA saline even when a high concentration of either Mg⁺⁺, Na⁺, or tetraethylammonium⁺ was present. The action potential of the larval muscle fiber was thus concluded to be a Ca-spike, through the channel of which Na⁺ or Mg⁺⁺ did not penetrate.     

Innervation regulates the metamorphic fates of larval abdominal muscles in the moth, Manduca sexta

 With the onset of metamorphosis, the abdominal muscles of the moth, Manduca sexta, follow one of three developmental fates: maintenance, respecification, or death. The maintained muscles retain their larval size and morphology throughout adult development. The respecified and dying muscles dedifferentiate, which involves regression, nuclear degeneration, and myofibril breakdown. Nuclei in both dying and respecified muscles also proliferate. The amount of nuclear degeneration is greater in the dying muscle fibers, and the amount of nuclear proliferation is greater in the respecified muscles. Four to ten days after pupation, the sizes of the respecified muscles stabilize while the dying muscles are lost. During regression, a subset of the respecified muscle fibers die. The surviving respecified muscle fibers grow and differentiate during the last half of adult development. In respecified muscles, denervation triggers an increased amount of nuclear degeneration and a decreased amount of nuclear proliferation. As a result, denervated respecified fibers experience increased muscle regression including an increased loss of muscle fibers and sometimes muscle death. Surviving respecified fibers still grow and differentiate yet are only 5 to 12% of the control size. Denervation triggers dedifferentiation in maintained muscles, resulting in fiber loss and occasionally muscle death. The percentage of fibers which dedifferentiate varies between different muscles. Denervation also triggers nuclear proliferation, with the amount of nuclear proliferation correlated with the extent of dedifferentiation of the individual muscle fibers. The dedifferentiated maintained fibers subsequently undergo differentiation in the absence of muscle growth. Received: 10 July 1997 / Accepted: 21 April 1998     

Ca2+-induced sarcoplasmic reticulum Ca2+ release in myotubularin-deficient muscle fibers

Skeletal muscle deficiency in the 3-phosphoinositide (PtdInsP) phosphatase myotubularin (MTM1) causes myotubular myopathy which is associated with severe depression of voltage-activated sarcoplasmic reticulum Ca²⁺ release through ryanodine receptors. In the present study we aimed at further understanding how Ca²⁺ release is altered in MTM1-deficient muscle fibers, at rest and during activation. While in wild-type muscle fibers, SR Ca²⁺ release exhibits fast stereotyped kinetics of activation and decay throughout the voltage range of activation, Ca²⁺ release in MTM1-deficient muscle fibers exhibits slow and unconventional kinetics at intermediate voltages, suggestive of partial loss of the normal control of ryanodine receptor Ca²⁺ channel activity. In addition, the diseased muscle fibers at rest exhibit spontaneous elementary Ca²⁺ release events at a frequency 30 times greater than that of control fibers. Eighty percent of the events have spatiotemporal properties of archetypal Ca²⁺ sparks while the rest take either the form of lower amplitude, longer duration Ca²⁺ release events or of a combination thereof. The events occur at preferred locations in the fibers, indicating spatially uneven distribution of the parameters determining spontaneous ryanodine receptor 1 opening. Spatially large Ca²⁺ release sources were obviously involved in some of these events, suggesting that opening of ryanodine receptors in one cluster can activate opening of ryanodine receptors in a neighboring one. Overall results demonstrate that opening of Ca²⁺-activated ryanodine receptors is promoted both at rest and during excitation-contraction coupling in MTM1-deficient muscle fibers. Because access to this activation mode is denied to ryanodine receptors in healthy skeletal muscle, this may play an important role in the associated disease situation.     

Reiji Natori, Setsuro Ebashi, and excitation-contraction coupling

The achievements of Natori and Ebashi, which greatly contributed to the progress in studies of excitation–contraction coupling, were reviewed. Natori succeeded in removing the cell membrane of an isolated fiber of skeletal muscle to prepare a skinned fiber, which still responded to an electrical stimulation with propagated contraction. Skinned fibers showed elastic extensibility beyond the elastic limit of intact muscle fibers. Based on this elasticity Natori predicted the presence of an elastic components, later found as connectin. Skinned fibers, an excellent experimental system, contributed greatly to the progress in subsequent studies. Ebashi showed that the essential principle of the relaxing factor was not the ATP-regenerating enzymes as generally thought, but a particulate fraction with MgATPase. Then he clearly showed that a minute amount of Ca²⁺ is necessary for the contractile reaction of actomyosin, and that the relaxing factor strongly accumulates Ca²⁺ in the presence of ATP and causes relaxation by the removal of Ca²⁺. He further discovered that the Ca²⁺-induced regulation of the contractile reaction of the myosin–actin system requires the presence of tropomyosin and a new protein, troponin. Troponin binds to a specific site on tropomyosin, which in turn binds to actin in the thin filament. Troponin is the Ca²⁺-receptive protein, and changes in troponin molecules upon Ca²⁺ binding is transmitted to actin through tropomyosin to regulate the actin–myosin interaction. Through these findings, the excitation was connected by Ca²⁺ with the contraction.