Diversity in expression of myosin heavy chain isoforms and M-band proteins in rat muscle spindles

Summary The composition of adult rat soleus muscle spindles, with respect to myosin heavy chain isoforms and M-band proteins, was studied by light-microscope immunohistochemistry. Serial sections were labelled with antibodies against slow tonic, slow twitch, fast twitch and neonatal myosin isoforms as well as against myomesin, M-protein and the MM form of creatine kinase. Intrafusal fiber types were distinguished according to the pattern of ATPase activity following acid and alkaline preincubations.Nuclear bag₁ fibers were always strongly stained throughout with anti-slow tonic myosin, were positive for anti-slow twitch myosin towards and in the C-region but were unstained with anti-fast twitch and anti-neonatal myosins. The staining of nuclear bag₂ fibers was in general highly variable. However, they were most often strongly stained by anti-slow tonic myosin in the A-region and gradually lost this reactivity towards the poles, whereas a positive reaction with anti-slow twitch myosins was found along the whole fiber. Regional staining variability with antineonatal and anti-fast myosins was apparent, often with decreasing intensity towards the polar regions. Nuclear chain fibers showed strong transient reactivity with anti-slow tonic myosin in the equatorial region, did not react with anti-slow twitch and were always evenly stained by anti-fast twitch and anti-neonatal myosins. All three intrafusal fiber types were stained with anti-myomesin. Nuclear bag₁ fibers lacked staining for M-protein, whereas bag₂ fibers displayed intermediate staining, with regional variability, often increasing in reactivity towards the polar regions. Chain fibers were always strongly stained by anti-M-protein. The MM form of creatine kinase was present in all three fiber types, but bag₁ fibers were less reactive and clear striations were not observed, in contrast to bag₂ and chain fibers. Out of 38 cross sectioned spindles two were found to have an atypical fiber composition, (lack of chain fibers) and a rather diverse staining pattern for the different antibodies tested.Taken together, the data show that in adult rat solcus, slow tonic and neonatal myosin heavy, chain isoforms are only expressed in the muscle spindle fibers and that each intrafusal fiber type has a unique, although variable, composition of myosin heavy chain isoforms and M-band proteins. We propose that both motor and sensory innervation might be the determining factors regulating the variable expression of myosin heavy chain isoforms and M-band proteins in intrafusal fibers of rat muscle spindles.     

Tissue inhibitor of metalloproteinase‐2 (TIMP‐2) regulates neuromuscular junction development via a β1 integrin‐mediated mechanism

Extracellular matrix (ECM) molecules play critical roles in muscle function by participating in neuromuscular junction (NMJ) development and the establishment of stable, cytoskeleton‐associated adhesions required for muscle contraction. Matrix metalloproteinases (MMPs) are neutral endopeptidases that degrade all ECM components. While the role of MMPs and their inhibitors, the tissue inhibitor of metalloproteinases (TIMPs), has been investigated in many tissues, little is known about their role in muscle development and mature function. TIMP‐2 ^?/? mice display signs of muscle weakness. Here, we report that TIMP‐2 is expressed at the NMJ and its expression is greater in fast‐twitch (extensor digitorum longus, EDL) than slow‐twitch (soleus) muscle. EDL muscle mass is reduced in TIMP‐2^?/? mice without a concomitant change in fiber diameter or number. The TIMP‐2^?/? phenotype is not likely due to increased ECM proteolysis because net MMP activity is actually reduced in TIMP‐2^?/? muscle. Most strikingly, TIMP‐2 colocalizes with β1 integrin at costameres in the wild‐type EDL and β1 integrin expression is significantly reduced in TIMP‐2^?/? EDL. We propose that reduced β1 integrin in fast‐twitch muscle may be associated with destabilized ECM‐cytoskeletal interactions required for muscle contraction in TIMP‐2^?/? muscle; thus, explaining the muscle weakness. Given that fast‐twitch fibers are lost in muscular dystrophies and age‐related sarcopenia, if TIMP‐2 regulates mechanotransduction in an MMP‐independent manner it opens new potential therapeutic avenues. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

Effect of chronic hypoxia on the capillarity of dog skeletal muscle

Capillarity and fiber composition were studied by the ATPase technique in frozen samples of sternothyroid muscle of dogs from sea level (SL) and high altitude (3,300–4,300 m) (HA). Capillary density (CD), capillary to fiber ratio (C:F) and fiber cross sectional area (FCSA) were measured. The mean CD was 791/mm² at SL and 743/mm² at HA. CD was linearly related to FCSA in the SL animals (CD=1112.8–0.10 FCSA; r=–0.63). In both SL and HA animals, C:F was linearly and positively correlated with FCSA. There was no significant difference between the two regression lines; therefore, only one line represents all the data (C:F=0.78+(5.19×10^–4) FCSA; r=0.77). Thus, at a given FCSA the C:F was the same for SL and HA dogs. Two types of fibers were identified: type I (slow twitch) (42%) and type II (fast twitch) (58%). No differences in fiber composition or FCSA were observed between the SL and HA dogs. These results indicate that moderate levels of hypoxia do not affect the capillarity of dog skeletal muscle.     

Opposite effects of cooling on twitch contractions of skeletal muscle isolated from tropical toads (Leptodactylidae) and northern frogs (Ranidae)

Cooling increases the twitch force of frog skeletal muscle (Rana temporaria; Rana pipiens), but decreases the twitch force of tropical toad muscle (Leptodactylus insularis). Action potentials and intramembranous charge movement in frog and toad fibers were slowed identically by cooling. Cooling increased the integral of twitch Ca²⁺ detected by aequorin in frog fibers (1.4-fold), while also decreasing the peak and slowing the rate of decay. Conversely, cooling decreased the integral (0.6-fold) and the peak of twitch Ca²⁺ in toad fibers, without affecting the rate of decay. The difference in entire Ca²⁺ transients may account for cold-induced twitch potentiation in frogs and twitch paralysis in toads. In sustained contractions of toad fibers, cooling markedly decreased maximum force caused by: (i) tetanic stimulation, (ii) two-microelectrode voltage clamp steps, (iii) high [K⁺], or (iv) caffeine. Maximum force in sustained contractions was decreased moderately by cooling frog fibers. Rapid rewarming and simultaneous removal of high [K⁺] or caffeine during a sustained contraction, caused toad muscle force to rise towards the value corresponding to the warm temperature. This did not occur after removing high [K⁺] or caffeine from toad fibers kept in the cold. Transmission electron micrographs showed no relevant structural differences. Parvalbumins are thought to promote relaxation of frog muscle in the cold. The unique parvalbumin isoforms in toad muscle apparently lack this property. Accepted: 27 August 1998     

Effect of breed of horse on muscle carnosine concentration

1. Muscle samples from the M. gluteus medius were obtained from six Quarter Horses (QH), six Thoroughbreds (TB), and five Standardbreds (SB) to determine carnosine values and fiber type percentages. 2. Muscle biopsies were for fiber type percentages and carnosine concentration. 3. QH had a lower percentage of slow twitch oxidative fibers and a higher percentage of past twitch glycolytic fibers than SB or TB. 4. Fast twitch oxidative-glycolytic fibers were lowest in the QH. 5. The QH had mean carnosine values significantly greater (P less than 0.01) than the mean values for SB and TB. 6. Across breeds muscle carnosine concentration was positively correlated (P less than 0.05; r = 0.53) with fast twitch glycolytic fiber percentage and negatively correlated (P less than 0.05, r = -0.51) with fast twitch oxidative fiber percentage. 7. Free intramuscular carnosine is believed to function as an intracellular buffer. Since carnosine was highest in the muscle of horses with the greatest percentage of fast twitch glycolytic fibers, these data are consistent with the proposed function of this dipeptide.     

去神经对快,慢肌纤维肌球蛋白ATPase影响的组织化学观察

本文用组织化学方法观察了豚鼠比目鱼肌(SOL)和腓骨第三肌(PT)在去神经后其快、慢纤维肌球蛋白ATPase特性的变化。在正常肌肉中Ⅰ型(慢)纤维和Ⅱ型(快)纤维分别具有酸和碱稳定ATPase活性。慢纤维在去神经后出现了碱稳定ATPase活性,而快纤维则无明显变化。结果表明,只有慢纤维的肌球蛋白ATPase特性才与神经支配有关。     

Increased effectiveness of a motorneuron after partial denervation of its target muscle in the cricket Teleogryllus oceanicus

Fibers of the metathoracic extensor tibia muscle of the cricket Teleogryllus oceanicus are innervated by a slow excitatory axon (slow fibers), a fast excitatory axon (fast fibers), or by both slow and fast axons (dual fibers). Sectioning metathoracic nerve 5 removes the fast axon input to the muscle but not that of the slow axon. Following such partial denervation, the mechanical responses initiated by the slow axon increase progressively for at least 30 days; twitch tensions reach 5–10 times those of control muscles and tetanic tensions 10–30 times control values. After sectioning nerve 5, resting membrane potentials decrease in those fibers which originally received fast axon input and the input resistance of all fiber types increases, including that of slow fibers which are not innervated through nerve 5. Excitatory junctional potentials (EJPs) initiated by the slow axon become larger following partial denervation, accounting in part for the larger contraction amplitudes. The increased input resistance is adequate to account for the larger EJPs in slow fibers but not for the proportionally greater increase in EJP amplitude in fibers which were formerly dually innervated. The change in EJP amplitude is abrupt in slow fibers and gradual in formerly dual fibers.     

THE SARCOPLASMIC RETICULUM, THE T SYSTEM, AND THE MOTOR TERMINALS OF SLOW AND TWITCH MUSCLE FIBERS IN THE GARTER SNAKE

Twitch and slow muscle fibers, identified morphologically in the garter snake, have been examined in the electron microscope. The transverse tubular system and the sarcoplasmic reticulum are separate entities distinct from each other. In twitch fibers, the tubular system and the dilated sacs of the sarcoplasmic reticulum form triads at the level of junction of A and I bands. In the slow fibers, the sarcoplasmic reticulum is severely depleted in amount and the transverse tubular system is completely absent. The junctional folds of the postsynaptic membrane of the muscle fiber under an "en grappe" ending of a slow fiber are not so frequent or regular in occurrence or so wide or so long as under the "en plaque" ending of a twitch fiber. Some physiological implications of these differences in fine structure of twitch and slow fibers are discussed. The absence of the transverse tubular system and reduction in amount of sarcoplasmic reticulum, along with the consequent disposition of the fibrils, the occurrence of multiple nerve terminals, and the degree of complexity of the post junctional folds of the sarcolemma appear to be the morphological basis for the physiological reaction of slow muscle fibers.     

Regulation of Slow and Fast Muscle Myofibrillogenesis by Wnt/β-Catenin and Myostatin Signaling

Deviation from proper muscle development or homeostasis results in various myopathic conditions. Employing genetic as well as chemical intervention, we provide evidence that a tight regulation of Wnt/β-catenin signaling is essential for muscle fiber growth and maintenance. In zebrafish embryos, gain-of-Wnt/β-catenin function results in unscheduled muscle progenitor proliferation, leading to slow and fast muscle hypertrophy accompanied by fast muscle degeneration. The effects of Wnt/β-catenin signaling on fast muscle hypertrophy were rescued by misexpression of Myostatin or p21^CIP/WAF, establishing an in vivo regulation of myofibrillogenesis by Wnt/β-catenin signaling and Myostatin. Epistatic analyses suggest a possible genetic interaction between Wnt/β-catenin and Myostatin in regulation of slow and fast twitch muscle myofibrillogenesis.     

In Vivo Studies on Fast and Slow Muscle Fibers in Cat Extraocular Muscles

In anesthetized in vivo preparations, responses of two types of extraocular muscle fibers have been studied. The small, multiply innervated slow fibers have been shown to be capable of producing propagated impulses, and thus have been labeled slow multi-innervated twitch fibers. Fast and slow multi-innervated twitch fibers are distinguished by impulse conduction velocities, by ranges of membrane potentials, by amplitudes and frequencies of the miniature end plate potentials, by responses to the intravenous administration of succinylcholine, by the frequency of stimulation required for fused tetanus, and by the velocities of conduction of the nerve fibers innervating each of the muscle fiber types.     

Double Sucrose-Gap Method Applied to Single Muscle Fiber of Xenopus laevis

Passive electrical properties (internal conductance, membrane conductance, low frequency capacity, and high frequency capacity obtained from the foot of the action potential) of normal and glycerol-treated muscle of Xenopus were determined with the intracellular microelectrode technique. The results show that the electrical properties of Xenopus muscle are essentially the same as those of frog muscle. Characteristics of the action potential of Xenopus muscle were also similar to those of frog muscle. Twitch tension of glycerol-treated muscle fibers of Xenopus recovered partially when left in normal Ringer for a long time (more than 6 h). Along with the twitch recovery, the membrane capacity increased. Single isolated muscle fibers of Xenopus were subjected to the double sucrose-gap technique. Action potentials under the sucrose gap were not very different from those obtained with the intracellular electrode, except for the sucrose-gap hyperpolarization and a slight tendency toward prolongation of the shape of action potential. Twitch contraction of the artificial node was recorded as a change of force from one end of the fiber under the sucrose gap. From the time-course of the recorded force and the sinusoidal stress-strain relationship at varying frequencies of the resting muscle fiber, the time-course of isotonic shortening of the node was recovered by using Fourier analysis. It was revealed that the recorded twitch force can approximately be regarded as isotonic shortening of the node.     

Expression of myosin heavy chain isoforms in developing rat muscle spindles

Summary The development of muscle spindles, with respect to the expression of myosin heavy chain isoforms was studied in rat hind limbs from 17 days of gestation up to seven days after birth. Serial cross-sections were labelled with antibodies against slow tonic, slow twitch and neonatal isomyosins, myomesin, laminin and neurofilament protein.At 17–18 days of gestation, a small population of primary myotubes expressing slow tonic myosin were identified as the earliest spindle primordia. These myotubes also expressed slow twitch and, to a lesser extent, neonatal myosin. At 19–20 days of gestation a second myotube became apparent; this staining strongly with anti-neonatal myosin. A day later this secondary myotube acquired reactivity to anti-slow tonic and anti-slow twitch myosins. By birth, a third myotube was present; this staining strongly with anti-neonatal myosin but otherwise unreactive with the other antibodies against myosin heavy chains. Three days after birth a fourth myotube, with identical reactivity to the third one, became apparent. Regional variation in the expression of isomyosins, which was present since birth in the two nuclear bag fibers was further enhanced: the nuclear bag₂ staining strongly with anti-slow tonic and antineonatal in the equatorial region and with decreasing intensity towards the poles, whilst with anti-slow twitch the stainability was low in the equatorial and high in the polar region. The nuclear bag₁ fiber showed a homogeneous staining: high with anti-slow tonic, moderate with anti-neonatal, and displayed stainability to antislow twitch myosin in the polar regions only. No regional variation was found along the chain fiber/myotube. At seven days after birth, the pattern of reactivity was similar to that found in the adult spindles, except for the bag₁ fiber which still expressed neonatal myosin.We show that slow tonic myosin is expressed from early development and it is a reliable marker of developing bag fibers. We suggest that muscle spindles are formed from special cell lineages of which the primary generation myotubes expressing slow tonic myosin represent the primordium of muscle spindles.     

Diversity in expression of myosin heavy chain isoforms and M-band proteins in rat muscle spindles

The composition of adult rat soleus muscle spindles, with respect to myosin heavy chain isoforms and M-band proteins, was studied by light-microscope immunohistochemistry. Serial sections were labelled with antibodies against slow tonic, slow twitch, fast twitch and neonatal myosin isoforms as well as against myomesin, M-protein and the MM form of creatine kinase. Intrafusal fiber types were distinguished according to the pattern of ATPase activity following acid and alkaline preincubations. Nuclear bag1 fibers were always strongly stained throughout with anti-slow tonic myosin, were positive for anti-slow twitch myosin towards and in the C-region but were unstained with anti-fast twitch and anti-neonatal myosins. The staining of nuclear bag2 fibers was in general highly variable. However, they were most often strongly stained by anti-slow tonic myosin in the A-region and gradually lost this reactivity towards the poles, whereas a positive reaction with anti-slow twitch myosins was found along the whole fiber. Regional staining variability with anti-neonatal and anti-fast myosins was apparent, often with decreasing intensity towards the polar regions. Nuclear chain fibers showed strong transient reactivity with anti-slow tonic myosin in the equatorial region, did not react with anti-slow twitch and were always evenly stained by anti-fast twitch and anti-neonatal myosins. All three intrafusal fiber types were stained with anti-myomesin. Nuclear bag1 fibers lacked staining for M-protein, whereas bag2 fibers displayed intermediate staining, with regional variability, often increasing in reactivity towards the polar regions. Chain fibers were always strongly stained by anti-M-protein. The MM form of creatine kinase was present in all three fiber types, but bag1 fibers were less reactive and clear striations were not observed, in contrast to bag2 and chain fibers. Out of 38 cross sectioned spindles two were found to have an atypical fiber composition (lack of chain fibers) and a rather diverse staining pattern for the different antibodies tested. Taken together, the data show that in adult rat soleus, slow tonic and neonatal myosin heavy chain isoforms are only expressed in the muscle spindle fibers and that each intrafusal fiber type has a unique, although variable, composition of myosin heavy chain isoforms and M-band proteins. We propose that both motor and sensory innervation might be the determining factors regulating the variable expression of myosin heavy chain isoforms and M-band proteins in intrafusal fibers of rat muscle spindles.     

The organization of muscle spindles in the tenuissimus muscle of the cat during late development

Intrafusal muscle fibers in the tenuissimus muscle of the cat develop as two separate groups; one being a single nuclear bag fiber while the other comprises a second nuclear bag fiber along with all the nuclear chain fibers. The groupings are very distinctive in the late fetus (55 days gestation) and remain so until 18 days of age. In the adult, the grouping is less distinctive but can often be recognized and followed for considerable distances within the capsular region of the spindle. Each group develops under its own basement membrane and is separated from the other by fibrocytes. ATPase histochemistry indicates the isolated single nuclear bag fiber is slow twitch while the fibers of the other group, the second bag and all the nuclear chains, are fast twitch. The organization of intrafusal fibers in late development into two groups of different fiber types is discussed in relation to their selective innervation by γ fibers.     

Zebrafish slow muscle cell migration induces a wave of fast muscle morphogenesis

The specification and morphogenesis of slow and fast twitch muscle fibers are crucial for muscle development. In zebrafish, Hedgehog is required for slow muscle fiber specification. However, less is known about signals that promote development of fast muscle fibers, which constitute the majority of somitic cells. We show that when Hedgehog signaling is blocked, fast muscle cell elongation is disrupted. Using genetic mosaics, we show that Hedgehog signal perception is required by slow muscle cells but not by fast muscle cells for fast muscle cell elongation. Furthermore, we show that slow muscle cells are sufficient to pattern the medial to lateral wave of fast muscle fiber morphogenesis even when fast muscle cells cannot perceive the Hedgehog signal. Thus, the medial to lateral migration of slow muscle fibers through the somite creates a morphogenetic signal that patterns fast muscle fiber elongation in its wake.     

AXIAL MUSCULATURE IN THE DOLPHIN (TURSIOPS TRUNCATUS): SOME ARCHITECTURAL AND HISTOCHEMICAL CHARACTERISTICS

In view of the supposition that a dolphin can swim faster than would be predicted based on its physical features and presumed muscle power potential, studies were initiated to reevaluate the assumptions made in reaching these conclusions. Several previous studies have shown that the architectural and histochemical properties of a skeletal muscle dictate its force, velocity and displacement properties. This study examined the muscle fiber lengths and tendon arrangements of the dorsal and ventral axial muscles in dolphins ( Tursiops truncatus ). Fiber type and fiber size distributions were determined to reflect the general biochemical characteristics of the musculature. The dorsal muscles had a higher mean fiber length (167 Vs. 90 mm) and the range within and across different dorsal muscles was less (141–199 vs. 37–185 mm) than in the ventral muscles. Both the dorsal and ventral muscles consisted of an overall mean of 50 percent slow twitch and 50 percent fast twitch fiber types. The fast twitch fibers were 67 percent larger (2,200 vs. 1,317 μ m ²) than the slow twitch fibers in the ventral and 38 percent larger (1,213 Vs. 879 μm²) in the dorsal muscles. In addition, the mean cross sectional area of the fibers in the ventral muscles was approximately 65 percent greater (1,750 vs. 1,072 μm²) than those in the dorsal muscles. The shorter, larger-diameter fibers of the ventral musculature give it a greater potential for force production for a given amount of muscle mass. In contrast, the dorsal muscles appear to be designed to optimize velocity and displacement, ( i.e. , longer fibers). These findings contribute to the information necessary for the determination of the power potential of the musculature of the dolphin.     

Structure and Function of the Transverse Tubular System in Crustacean Muscle Fibers

The structure and function of the transverse tubular system(TTS) in two types of crustacean muscle fibers are examined.Giant fibers from the barnacle,Balanus nubilus, which are gradedlycontracting, are compared with allor-none twitch fibers fromthe crab, Carcinus maenas. Both fiber types were found to havedeep sarcolemmal invaginations which serve both to increasethe fiber surface area and to kfeep the length of the tubulesshort enough for electrotonic propagation.The ultrastructureof the tubular system in both types of fiber is compared.Thesystem is better developed in Carcinus than in Balanus, butthe slow Balanus fibers do have a relatively well developedTTS and sarcoplasmic reticulum in contrast to slow vertebratefibers. The apparent high, membrane-capacitance values of crustaceanfibers are the result of investigators not taking into considerationthe large increase in surface area due to the sarcolemmal infoldings.Thetubular membranes in Carcinus fibers were found to be permselectiveto chloride ions, and could be made to swell (as confirmed byelectron microscopy) by establishing an outward gradient forchloride across them. The capacitance of the tubular membranerelative to the plasma membrane was found to increase when thetubuleswere swollen. The implication of a fiber having two spatiallyseparated, differentially permeable membranes on excitation-contractioncoupling is discussed.     

Contractile properties of the isolated rat soleus muscle and its single skinned soleus fibers at the early stage of gravitational unloading: Facts and hypotheses

The contractile properties of the postural soleus muscle were studied in rats at the early stage of gravitational unloading (three-day hindlimb suspension) with regard to different modes of muscle contraction (twitch and tetanic contraction of the isolated muscle and calcium-induced contraction of isolated skinned fibers). A significant (p < 0.01) enhancement of the peak twitch tension of the muscles of suspended rats without changes in time-dependent characteristics was observed, although the half-relaxation time tended to decrease. The fiber diameter did not change (42.37 ± 0.76 vs. 43.43 ± 1.15 μm in controls). The calcium-induced peak isometric tensions in control and unloaded soleus muscles were 37.6 ± 1.52 and 32.1 ± 1.05 mg, respectively (decrease significant at p < 0.05). No changes in threshold calcium concentration were recorded, but the pCa₅₀ value in unloaded muscles decreased from 6.05 ± 0.02 in controls to 5.97 ± 0.02 (p ≤ 0.05), indicating loss of myofibrillar calcium sensitivity. The cooperativity coefficient ηn in control animals was 3.46 ± 0.16, and in suspended ones it decreased to 3.08 ± 0.11 (p < 0.05). Analysis with the Fluo-4AM calcium probe demonstrated that the intracellular Ca²⁺ concentration increased significantly after hindlimb suspension, whereas the relative contents of titin or nebulin did not change.     

Aging alters contractile properties and fiber morphology in pigeon skeletal muscle

In this study, we tested the hypothesis that skeletal muscle from pigeons would display age-related alterations in isometric force and contractile parameters as well as a shift of the single muscle fiber cross-sectional area (CSA) distribution toward smaller fiber sizes. Maximal force output, twitch contraction durations and the force–frequency relationship were determined in tensor propatagialis pars biceps muscle from young 3-year-old pigeons, middle-aged 18-year-old pigeons, and aged 30-year-old pigeons. The fiber CSA distribution was determined by planimetry from muscle sections stained with hematoxylin and eosin. Maximal force output of twitch and tetanic contractions was greatest in muscles from young pigeons, while the time to peak force of twitch contractions was longest in muscles from aged pigeons. There were no changes in the force–frequency relationship between the age groups. Interestingly, the fiber CSA distribution in aged muscles revealed a greater number of larger sized muscle fibers, which was verified visually in histological images. Middle-aged and aged muscles also displayed a greater amount of slow myosin containing muscle fibers. These data demonstrate that muscles from middle-aged and aged pigeons are susceptible to alterations in contractile properties that are consistent with aging, including lower force production and longer contraction durations. These functional changes were supported by the appearance of slow myosin containing muscle fibers in muscles from middle-aged and aged pigeons. Therefore, the pigeon may represent an appropriate animal model for the study of aging-related alterations in skeletal muscle function and structure.