Rapid loss of sensitivity to tetrodotoxin by chick ventricular myocardial cells after separation from the heart

We reported previously that chick myocardial cells placed into monolayer cell culture lost tetrodotoxin (TTX) sensitivity when tested at 72 h. To further characterize the change, ventricular myocardial cells were dispersed from chick embryos 14–16 days old; these hearts are TTX-sensitive before dispersal. Intracellular microelectrode penetrations were made into spontaneously beating cells at 9–72 h after culturing. No TTX-sensitive cells were found. Spontaneous action potentials with concomitant contractions continued in the presence of TTX (8 μg/ml), and the maximum rate of rise of the action potentials (+ _max) (control of 2–20 V/sec) was not reduced. Since the cells did not adhere to the vessel before 9 h, suspensions of cells were studied 1–8 h after dispersal to determine the rapidity of the loss of TTX sensitivity; all cells which contracted spontaneously or responded to electrical stimulation continued to beat in TTX. Addition of cycloheximide or actinomycin D did not prevent the loss of TTX sensitivity. The loss is not due to the use of trypsin (0.01 %) because dispersal by collagenase also resulted in loss of TTX sensitivity. Furthermore, cells separated mechanically (from 8-day-old hearts) also lost TTX sensitivity. In addition, loss of TTX sensitivity did not occur in frog sartorius muscles organ cultured for several days in 0.01 % trypsin. The loss of TTX sensitivity occurred even in multilayered cell cultures. Chronic exposure to carbachol or isoproterenol did not prevent the loss. However, elevation of K⁺ in the medium (12–60 mM) prevented or reversed the loss of TTX sensitivity in some cells (˜50 %), although + _max remained low. Hence, the loss of TTX-sensitive fast Na⁺ channels upon cell dispersal (a) occurs very rapidly (less than 60 min), (b) is not due to the use of trypsin, (c) is independent of protein synthesis, (d) is not solely a function of cell association, (e) is not influenced by neurotransmitters, and (f) is prevented or reversed by culturing in elevated [K⁺]₀. The mechanism of the changes in characteristics of the cation channels remains to be elucidated.     

The mechanism of venom secretion from Duvernoy's gland of the snake Thamnophis sirtalis

The venom glands of snakes of the families Elapidae and Viperidae are thought to have evolved from Duvernoy's gland of colubrid ancestors. In highly venomous snakes elements of the external adductor musculature of the jaw insert fibers directly onto the capsule of the venom gland. These muscles, upon contraction, cause release of contents by increasing intraglandular pressure. In Thamnophis sirtalis, a colubrid, there is no direct connection between Duvernoy's gland and the adductor musculature. The anatomical arrangement of the gland, skull, adductor muscles, and the integument is such that contraction of the muscles may facilitate emptying of the gland. This hypothesis was tested by electrical stimulation of the muscles, which resulted in significantly greater release of secretion than elicited by controls. The results suggest a possible early step in the evolution of a more intimate association between venom glands and adductor musculature in highly venomous snakes.     

Slow-tonic MHC expression in paralyzed hindlimbs of fetal rats

Summary Whether nerve activity and active contraction of myotubes are essential for the assembly and initial differentiation of muscle spindles was investigated by paralyzing fetal rats with tetrodotoxin (TTX) from embryonic day 16 (E16) to E21, prior to and during the period when spindles typically form. TTX-treated soleus muscles were examined by light and electron microscopy for the presence of spindles and expression of myosin heavy chain (MHC) isoforms by the intrafusal fibers. Treatment with TTX did not inhibit the formation of a spindle capsule or the expression of a slow-tonic MHC isoform characteristic of intrafusal fibers, but did retard development of spindles. Spindles of TTX-treated E21 muscles usually consisted of one intrafusal fiber (bag₂) only rather than two fibers (bag₁ and bag₂) typically present in untreated (control) E21 spindles. Intrafusal fibers of TTX-treated spindles also had only one sensory region supplied by multiple afferents, and were devoid of motor innervation. These features are characteristic of spindles in normal E18–E19 muscles. Thus, nerve and/or muscle activity is not essential for the assembly of muscle spindles, formation of a spindle capsule, and transformation of undifferentiated myotubes into the intrafusal fibers containing spindle-specific myosin isoforms. However, activity may promote the maturation of intrafusal bundles, as well as the maturation of afferent and efferent nerve supplies to intrafusal fibers.     

ATP produced by myocardial sarcolemmal-bound creatine kinase is not preferentially used by the Na+ pump

It has been proposed (Grosse et al. (1980) Biochim. Biophys. Acta 603, 142–156) that membrane-bound creatine kinase and the ATP-dependent Na⁺ pump form a functional complex in cardiac sarcolemmal vesicles. In this model ADP produced at Na⁺ pump sites would be rephosphorylated by the creatine kinase for preferential delivery back to the Na⁺ pump. We have reexamined this hypothesis and find that under some conditions active Na⁺ transport can be stimulated by ATP produced by sarcolemmal creatine kinase. However, the characteristics of this stimulation are no different than stimulation produced by an added soluble ATP-regenerating system (phosphoenolpyruvate/pyruvate kinase). Thus, we are unable to detect coupling between the Na⁺ pump and sarcolemmal creatine kinase.     

Fiber-type specific expression of α-actinin isoforms in rat skeletal muscle

α-Actinins are actin-binding proteins, and two isoforms (α-actinin-2 and -3) are major structural components of the sarcomeric Z line in mammalian skeletal muscle. Based on human and knockout mice studies, α-actinin-3 is thought to be associated with muscle force output and high contraction velocities. However, fiber-type specific expression of α-actinin isoforms is not well understood and may vary among species. In this study, we investigated the expression of α-actinin isoforms and the difference between fiber types in rat skeletal muscle and compared it with those of humans and mice from previous reports. Soleus and plantaris muscles were analyzed immunohistochemically to identify muscle fiber types and α-actinin protein expression. α-Actinin-2 was stained in all muscle fibers in both the soleus and plantaris muscles; i.e., all α-actinin-3 co-expressed with α-actinin-2 in rat skeletal muscles. The proportions of α-actinin-3 expression, regardless of fiber type, were significantly higher in the plantaris (75.8 ± 0.6%) than the soleus (8.0 ± 1.7%). No α-actinin-3 expression was observed in type I fibers, whereas all type IIx+b fibers expressed α-actinin-3. α-Actinin-3 was also expressed in type IIa fibers; however, approximately 75% of type IIa fibers were not stained by α-actinin-3, and the proportion varied between muscles. The proportion of α-actinin-3 expression in type IIa fibers was significantly higher in the soleus muscle than the plantaris muscle. Our results showed that fiber-type specific expression of α-actinin isoforms in rats is more similar to that in humans compared to that of the mouse, whereas the proportion of α-actinin-3 protein varied between muscles.     

Postactivation potentiation, fiber type, and twitch contraction time in human knee extensor muscles.

In small mammals, muscles with shorter twitch contraction times and a predominance of fast-twitch, type II fibers exhibit greater posttetanic twitch force potentiation than muscles with longer twitch contraction times and a predominance of slow-twitch, type I fibers. In humans, the correlation between potentiation and fiber-type distribution has not been found consistently. In the present study, postactivation potentiation (PAP) was induced in the knee extensors of 20 young men by a 10-s maximum voluntary isometric contraction (MVC). Maximal twitch contractions of the knee extensors were evoked before and after the MVC. A negative correlation (r = -0. 73, P < 0.001) was found between PAP and pre-MVC twitch time to peak torque (TPT). The four men with the highest (HPAP, 104 +/- 11%) and lowest (LPAP, 43 +/- 7%) PAP values (P < 0.0001) underwent needle biopsies of vastus lateralis. HPAP had a greater percentage of type II fibers (72 +/- 9 vs. 39 +/- 7%, P < 0.001) and shorter pre-MVC twitch TPT (61 +/- 12 vs. 86 +/- 7 ms, P < 0.05) than LPAP. These data indicate that, similar to the muscles of small mammals, human muscles with shorter twitch contraction times and a higher percentage of type II fibers exhibit greater PAP.     

Induction of Oxidatively Modified Proteins in Skeletal Muscle by Electrical Stimulation and Its Suppression by Dietary Supplementation of (-)-Epigallocatechin Gallate

The oxidative stress produced by electrical stimulation-induced muscle contraction was examined in the skeletal muscle proteins of rats that had been fed on the dietary flavonoid, (-)-epigallocatechin gallate (EGCg). Electrical stimulation of the rat leg muscle every second day for a two-week period resulted in an increased (p<0.05) muscle weight and accumulation of oxidatively induced modified proteins. Similar stimulation conducted every day for only one week had no effect on the muscle weight or protein oxidation, although the rate of protein degradation increased. Rats fed on a 20% casein diet supplemented with 0.1% EGCg for 2 weeks responded to the electrical stimulation of muscle contraction by reducing the increased muscle protein carbonyl content when compared to their counterparts fed on a control diet. There was no change in activity of antioxidative enzymes in muscle tissue of the EGCg-fed rats receiving electrical stimulation. The results of this study show that the antioxidative property of EGCg was effective for suppressing oxidative modification of the skeletal muscle protein induced by electrical stimulation. This finding demonstrates that EGCg has a beneficial effect in vivo on the free radical-mediated oxidative damage to muscle proteins.     

Static stretch increases c-Jun NH2-terminal kinase activity and p38 phosphorylation in rat skeletal muscle

Physicalexercise and contraction increase c-Jun NH₂-terminal kinase(JNK) activity in rat and human skeletal muscle, and eccentriccontractions activate JNK to a greater extent than concentric contractions in human skeletal muscle. Because eccentric contractions include a lengthening or stretch component, we compared the effects ofisometric contraction and static stretch on JNK and p38, the stress-activated protein kinases. Soleus and extensor digitorum longus(EDL) muscles dissected from 50- to 90-g male Sprague-Dawley rats weresubjected to 10 min of electrical stimulation that produced contractions and/or to 10 min of stretch (0.24 N tension, 20-25% increase in length) in vitro. In the soleus muscle, contraction resulted in a small, but significant, increase in JNK activity (1.8-fold above basal) and p38 phosphorylation (4-fold). Static stretchhad a much more profound effect on the stress-activated proteinkinases, increasing JNK activity 19-fold and p38 phosphorylation 21-fold. Increases in JNK activation and p38 phosphorylation in response to static stretch were fiber-type dependent, with greater increases occurring in the soleus than in the EDL. Immunohistochemistry performed with a phosphospecific antibody revealed that activation ofJNK occurred within the muscle fibers. These studies suggest that thestretch component of a muscle contraction may be a major contributor tothe increases in JNK activity and p38 phosphorylation observed afterexercise in vivo.

     

Contractions of holothurian muscles

Summary In response to quick stretch, contraction is elicited in longitudinal retractor muscles of five tested species of holothurians, and in the pharyngeal retractor ofCucumaria. The effects of amplitude of stretch and rate of stretch are additive. Rates of contraction and repetitiveness of response, and spontaneous rhythmicity (especially in muscles ofLeptosynapta), correlate with mode of life.Contractile responses to stretch are abolished by anesthesia with procaine or magnesium. Responses are enhanced by physostigmine or prostigmine, blocked by d-tubocurarine. Responses to electric shocks persist after block of responses to stretch and after block of spontaneous activity by anesthesia, by cholinergic blockers or by Na replacement. Responses to both stretch and shock are abolished by reducing calcium or by agents which block Ca-conductance.It is postulated (1) that quick stretch stimulates the terminals of cholinergic nerves, (2) that conduction in these nerve fibers is by Na but is TTX resistant, (3) that the nerve endings activate conductance increase for Ca⁺⁺ in muscle fibers which initiate contractions.No muscle potentials were recorded by suction or pressure electrodes and no nexal junctions were observed between muscle fibers. The muscles were well innervated and synaptic endings and some neural somata were seen in the nerve bundles.Thanks are due to Dennis Willows, director and to staff, University of Washington Laboratories, Friday Harbor; to C.L. Singla of the University of Victoria for preparing and examining electron micrographs; to J.L.S. Cobb for commenting on electron micrographs; to Richard Meiss for designing and constructing ramp stretching device. C. Ladd Prosser was supported by NIH grant 5-R01 AM 12768-10 and George O. Mackie by grant no. A 1427, Nat. Sci. and Eng. Res. Council of Canada.     

Dihydropyridine Receptor-Ryanodine Receptor Uncoupling in Aged Skeletal Muscle

The mechanisms underlying skeletal muscle functional impairment and structural changes with advanced age are only partially understood. In the present study, we support and expand our theory about alterations in sarcolemmal excitation-sarcoplasmic reticulum Ca²⁺ release-contraction uncoupling as a primary skeletal muscle alteration and major determinant of weakness and fatigue in mammalian species including humans. To test the hypothesis that the number of RYR1 (ryanodine receptor) uncoupled to DHPR (dihydropyridine receptor) increases with age, we performed high-affinity ligand binding studies in soleus, extensor digitorum longus (EDL) and in a pool of several skeletal muscles consisting of a mixture of fast- and slow-twitch muscle fibers in middle-aged (14-month) and old (28-months) Fisher 344 Brown Norway F1 hybrids rats. The number of DHPR, RYR1, the coupling between both receptors expressed as the DHPR/RYR1 maximum binding capacity, and their dissociation constant for high-affinity ligands were measured. The DHPR/RYR1 ratio was significantly reduced in the three groups of muscles (pool: 1.03 ± 0.15 and 0.80 ± 0.11, soleus: 0.44 ± 0.12 and 0.26 ± 0.10, and EDL: 0.95 ± 0.14 and 0.68 ± 0.10, for middle-aged and old muscles, respectively). These data support the concept that DHPR-RYR1 uncoupling results in alterations in the voltage-gated sarcoplasmic reticulum Ca²⁺ release mechanism, decreases in myoplasmic Ca²⁺ elevation in response to sarcolemmal depolarization, reduced Ca²⁺ supply to contractile proteins and reduced contraction force with aging. Received: 26 August 1996/Revised: 30 December 1996     

Spatial fiber type distribution in normal human muscle Histochemical and tensiomyographical evaluation

The variability of fiber type distribution in nine limb muscles was examined with histochemical and tensiomyographical (TMG) methods in two groups of 15 men aged between 17 and 40 years. The aim of this study was to determine the extent to which the relative occurrence of different fiber types and subtypes varies within human limb muscles in function to depth and to predict fiber type proportions with a non-invasive TMG method.The distribution of different fiber types varied within the muscles, as a function of depth, with a predominance of type 2b fibers at the surface and type 1 fibers in deeper regions of the muscle. For all the analyzed muscles the contraction times measured at stimulus intensity 10% of supramaximal stimulus (10% MS) were significantly (p<0.05) shorter than the contraction times measured at 50% of supramaximal stimulus intensity (50% MS). The Pearson's correlation coefficient between percentage of type 1 muscle fibers measured at the surface of the muscle and contraction time at 10% MS, obtained by TMG was statistically significant (r=0.76,P<0.01). Also the Pearson's correlation coefficient between percentage of type 1 muscle fibers measured in the deep region of the muscle and contraction time at 50% MS obtained by TMG was also statistically significant (r=0.90,P<0.001).These findings suggest that the contraction time obtained by TMG may be useful for non-invasive examining of muscle fiber types spatial distribution in humans.     

Tetrodotoxin blocks mechanical response in mammalian muscle in the presence of tetrodotoxin-resistant action potentials

The effect of tetrodotoxin (TTX) (10(-5)-10(-6)M) on the mechanical activity and on the action potential of innervated and denervated muscle of the rat was studied. The twitch tension was reduced to 10 % of the control values within 20 min of TTX 10(-6) introduction. This effect was reversible. The mean twitch tension in the presence of 10(-6)M TTX expressed as a percentage of control was 9.3 +/- 2.4 (SEM) for innervated muscle and 10.9 +/- 2.5 for denervated muscle. The dose-effect twitch relation for denervated muscles was not significantly different from that observed in control innervated muscles in the 10(-3)-10(-6) TTX range. Action potentials of innervated muscles could not be elicited in 10(-6)M TTX. In the presence of this (TTX) fibers of chronically denervated muscles consistently responded to stimulation with action potentials which were slower and smaller but still with overshoot, contrasting with fibrillation potentials that had been described to be blocked by TTX.     

Interaction between the canine diaphragm and intercostal muscles in lung expansion.

Changes in intrathoracic pressure produced by the various inspiratory intercostals are essentially additive, but the interaction between these muscles and the diaphragm remains uncertain. In the present study, this interaction was assessed by measuring the changes in airway opening (DeltaPao) or transpulmonary pressure (DeltaPtp) in vagotomized, phrenicotomized dogs during spontaneous inspiration (isolated intercostal contraction), during isolated rectangular or ramp stimulation of the peripheral ends of the transected C(5) phrenic nerve roots (isolated diaphragm contraction), and during spontaneous inspiration with superimposed phrenic nerve stimulation (combined diaphragm-intercostal contraction). With the endotracheal tube occluded at functional residual capacity, DeltaPao during combined diaphragm-intercostal contraction was nearly equal to the sum of the DeltaPao produced by the two muscle groups contracting individually. However, when the endotracheal tube was kept open, DeltaPtp during combined contraction was 123% of the sum of the individual DeltaPtp (P < 0.001). The increase in lung volume during combined contraction was also 109% of the sum of the individual volume increases (P < 0.02). Abdominal pressure during combined contraction was invariably lower than during isolated diaphragm contraction. It is concluded, therefore, that the canine diaphragm and intercostal muscles act synergistically during lung expansion and that this synergism is primarily due to the fact that the intercostal muscles reduce shortening of the diaphragm. When the lung is maintained at functional residual capacity, however, the synergism is obscured because the greater stiffness of the rib cage during diaphragm contraction enhances the DeltaPao produced by the isolated diaphragm and reduces the DeltaPao produced by the intercostal muscles.     

Studies on the in Vitro Interaction of Electrical Stimulation and Ca++ Movement in Sarcoplasmic Reticulum

Sarcoplasmic reticulum fragments (S.R.F.) were isolated from skeletal and heart muscles. These fragments were found to take up Ca⁺⁺ very actively from media. When monophasic square waves were passed through the S.R.F. suspension, the Ca⁺⁺ uptake by S.R.F. was decreased. When the suspension was stimulated electrically after the Ca⁺⁺ was taken up by S.R.F., the initiation and the cessation of the stimulation were followed by the release and re-uptake of Ca⁺⁺ by S.R.F., respectively. The degree of inhibition of the Ca⁺⁺ uptake as well as of the Ca⁺⁺ release by electrical stimulation was dependent on the voltage and the frequency of stimulation. The presence of inorganic phosphate or oxalate modified the influence of electrical stimulation on the release and the uptake of Ca⁺⁺ by S.R.F. Attempts were made to observe the release of Ca⁺⁺ by electrical stimulation from unfractionated sarcoplasmic reticulum remaining in myofibers, and the interaction of the released Ca⁺⁺ with myofibrils in vitro. For this purpose, the glycerol-extracted fiber was selected as a muscle model, since it contains both sarcoplasmic reticulum and myofibrils. It was found that electrical stimulation of skeletal and heart glycerol-extracted fibers resulted in the contraction of fibers. It appeared that the contraction of glycerol fibers by electrical stimulation was caused by the Ca⁺⁺ release from sarcoplasmic reticulum by stimulation.     

Ultrastructural study of two kinds of muscle in sea anemones: The existence of fast and slow muscles

The sea anemones studied have two morphological types of muscle fiber. Types A and B are distinguishable on the basis of myofilament patterns, size of fibers, responses to fixation, and staining with methylene blue. Observation of the muscle in both resting and contracted states has shown that the two types do not result from differences in contraction state of the muscle. The fine structural characteristics distinguishing A and B fibers are similar to those which distinguish fast and slow muscle fibers in higher animals. The distribution of A and B fibers in Stomphia and Aiptasia is consistent with the distribution of fast and slow muscles in these two species. It is proposed that the A and B fibers represent two morphologically distinct kinds of smooth muscle, and that the capacity for fast and slow contraction in the muscles of Stomphia and Aiptasia, and possibly in all actinians, is due to morphological differentiation in the muscle system.     

Selective inhibition of ATPase activity during contraction alters the activation of p38 MAP kinase isoforms in skeletal muscle

Muscle contractions strongly activate p38 MAP kinases, but the precise contraction‐associated sarcoplasmic event(s) (e.g., force production, energetic demands, and/or calcium cycling) that activate these kinases are still unclear. We tested the hypothesis that during contraction the phosphorylation of p38 isoforms is sensitive to the increase in ATP demand relative to ATP supply. Energetic demands were inhibited using N‐benzyl‐p‐toluene sulphonamide (BTS, type II actomyosin) and cyclopiazonic acid (CPA, SERCA). Extensor digitorum longus muscles from Swiss Webster mice were incubated in Ringer's solution (37°C) with or without inhibitors and then stimulated at 10 Hz for 15 min. Muscles were immediately freeze‐clamped for metabolite and Western blot analysis. BTS and BTS + CPA treatment decreased force production by 85%, as measured by the tension time integral, while CPA alone potentiated force by 310%. In control muscles, contractions resulted in a 73% loss of ATP content and a concomitant sevenfold increase in IMP content, a measure of sustained energetic imbalance. BTS or CPA treatment lessened the loss of ATP, but BTS + CPA treatment completely eliminated the energetic imbalance since ATP and IMP levels were nearly equal to those of non‐stimulated muscles. The independent inhibition of cytosolic ATPase activities had no effect on contraction‐induced p38 MAPK phosphorylation, but combined treatment prevented the increase in phosphorylation of the γ isoform while the α/β isoforms unaffected. These observations suggest that an energetic signal may trigger phosphorylation of the p38γ isoform and also may explain how contractions differentially activate signaling pathways. J. Cell. Biochem. 114: 1445–1455, 2013. © 2013 Wiley Periodicals, Inc.     

M-wave modulation at relative levels of maximal voluntary contraction

Frequency (mean and median power frequency, f and f _m) and amplitude (average rectified and root mean square values, ARV and rms), parameters of the M-wave, and the dorsiflexor force parameters of the anterior tibial muscles were measured in seven healthy human subjects. Intermittent, voluntary contractions at relative intensities (40%, 60%, and 80%) of maximal voluntary contraction (MVC) were performed in conjunction with electrical stimulation. The M-wave parameter changes were measured over the course of the isometric contractions. At higher force levels, M-wave potentiation was observed as increases in both ARV and rms. The ARV augmentation attained levels as high as 206.1 (SD 7.4)% of resting values after both initial and final contractions of 80% MVC, reaching statistical significance (P < 0.01). The f and f _m failed to show a significant difference at any level of contraction. It was surmised that potentiation of the M-wave was the result of an increased contribution of muscle fibre type IIb recruited during higher contraction levels, reflecting the change to larger, deeper innervating motoneurons as the intensity of contraction, as a percentage of MVC, rose. Recruitment of type IIb fibres, which have been reported to have a higher energy potential and frequency content, were thought to reflect changes in the local, excitability threshold of some motor units as the force intensity increased during the intermittent voluntary contractions. It is suggested that the M-wave elicited after contractions has the potential to reflect, to some extent, motor unit recruitment changes resulting from the preceding contractions, and that through comparisons of M-wave amplitude parameters, contributions of varying fibre types over the course of a contraction may be indicated.     

Adaptations of diaphragm and medial gastrocnemius muscles to inactivity.

The effects of 2 wk of inactivity on in vitro contractile properties of diaphragm and medial gastrocnemius (MG) muscles were examined in adult hamsters. In addition, inactivity effects on fiber-type proportions and cross-sectional areas were studied. Inactivity of the right hemidiaphragm or MG muscle was induced by either tetrodotoxin (TTX) blockade of nerve impulses or denervation (DNV). Inactivity effects on diaphragm or MG were compared with corresponding sham (saline-treated or untreated control) muscles. After both TTX- and DNV-induced inactivity, isometric twitch contraction and half-relaxation times were prolonged, maximum tetanic force decreased, and fatigue resistance improved. Proportions of type I and II fibers in both diaphragm and MG were unaffected by TTX- and DNV-induced inactivity. However, in both muscles, type I fibers hypertrophied, whereas type II fibers atrophied. In diaphragm, contractile and morphometric adaptations after DNV were generally more pronounced than those induced by TTX. In addition, compared with corresponding untreated or saline-treated control groups, inactivity effects (both TTX and DNV) on MG were generally greater than those induced in diaphragm, with the exception of hypertrophy of type I fibers. We conclude that inactivity exerts differential effects on type I and II fibers in both diaphragm and MG. Yet, these morphometric adaptations cannot completely account for the adaptations in muscle contractile and fatigue properties after inactivity.     

Flight initiations in Drosophila melanogaster are mediated by several distinct motor patterns

We have monitored the patterns of activation of five muscles during flight initiation of Drosophila melanogaster: the tergotrochanteral muscle (a mesothoracic leg extensor), dorsal longitudinal muscles #3, #4 and #6 (wing depressors), and dorsal ventral muscle #Ic (a wing elevator). Stimulation of a pair of large descending interneurons, the giant fibers, activates these muscles in a stereotypic pattern and is thought to evoke escape flight initiation. To investigate the role of the giant fibers in coordinating flight initiation, we have compared the patterns of muscle activation evoked by giant fiber stimulation with those during flight initiations executed voluntarily and evoked by visual and olfactory stimuli. Visually elicited flight initiations exhibit patterns of muscle activation indistinguishable from those evoked by giant fiber stimulation. Olfactory-induced flight initiations exhibit patterns of muscle activation similar to those during voluntary flight initiations. Yet only some benzaldehyde-induced and voluntary flight initiations exhibit patterns of muscle activation similar to those evoked by giant fiber stimulation. These results indicate that visually elicited flight initiations are coordinated by the giant fiber circuit. By contrast, the giant fiber circuit alone cannot account for the patterns of muscle activation observed during the majority of olfactory-induced and voluntary flight initiations.Abbreviations DLM/DLMn dorsal longitudinal muscle/motor neuron - DVM/DVMn dorsal ventral muscle/motor neuron - GF(s) giant fiber interneuron (s) - PSI peripherally synapsing interneuron - TTM/TTMn tergotrochanteral muscle/motor neuron     

Cultured slow vs. fast skeletal muscle cells differ in physiology and responsiveness to stimulation

In vitro studies have used protein markers to distinguish between myogenic cells isolated from fast and slow skeletal muscles. The protein markers provide some support for the hypothesis that satellite cells from fast and slow muscles are different, but the data are equivocal. To test this hypothesis directly, three-dimensional skeletal muscle constructs were engineered from myogenic cells isolated from fast tibialis anterior (TA) and slow soleus (SOL) muscles of rats and functionality was tested. Time to peak twitch tension (TPT) and half relaxation time (RT_1/2) were 30% slower in constructs from the SOL. The slower contraction and relaxation times for the SOL constructs resulted in left shift of the force-frequency curve compared with those from the TA. Western blot analysis showed a 60% greater quantity of fast myosin heavy chain in the TA constructs. 14 days of chronic low-frequency electrical stimulation resulted in a 15% slower TPT and a 14% slower RT_1/2, but no change in absolute force production in the TA constructs. In SOL constructs, slow electrical stimulation resulted in an 80% increase in absolute force production with no change in TPT or RT_1/2. The addition of cyclosporine A did not prevent the increase in force in SOL constructs after chronic low-frequency electrical stimulation, suggesting that calcineurin is not responsible for the increase in force. We conclude that myogenic cells associated with a slow muscle are imprinted to produce muscle that contracts and relaxes slowly and that calcineurin activity cannot explain the response to a slow pattern of electrical stimulation. tissue engineering; calcineurin; electrical stimulation; engineered muscle; bioreactors