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     

Differential changes in myoelectric characteristics of slow and fast fatigable frog muscle fibres during long-lasting activity.

The electrical activity of different muscle fibre types during fatigue at varying stimulation frequency and fibre stretch was studied. Extracellular action potentials (ECAPs) were recorded from isolated frog muscle fibres at initial length and stretched by 15%, 25% and 35% and stimulated for 180 s by suprathreshold pulses with frequencies of 5, 6.7 and 10Hz. The changes in ECAP negative phase duration (T(0)), propagation velocity of excitation (PV), potential power spectrum and its median frequency (MDF) were analysed for the period of uninterrupted activity (endurance time, ET). Slow (SMF) and fast (FMF) fatigable muscle fibre types were distinguished by the rate of PV decrease during ET. With the increase of stimulation frequency and fibre stretch, the rate of ECAP parameter changes increased and was larger in FMF, but this proportion was reversed with stretching over 25% and 10Hz stimulation. In both fibre types the power spectrum shift to lower frequencies during continuous activity was more pronounced with higher stimulation frequency. In FMFs the rates of MDF changes were positively and more strongly correlated with the rates of PV changes, whilst in SMFs the inverse correlation between the rates of changes of MDF and T(0) was stronger. The results indicate specific adaptation of slow and fast fatigable muscle fibres to stretch and activation frequency due to the differences in their membrane processes.     

Time course adaptations in rat skeletal muscle isomyosins during compensatory growth and regression

The purpose of this study was to ascertain the time course of change during both compensatory growth (hypertrophy) and subsequent growth regression on myosin isoform expression in rodent fast-twitch plantaris muscle in response to functional overload (induced by removal of synergists). Peak hypertrophy of the plantaris muscle (92%) occurred after 9 wk of overload. After 7 wk of overload regression (induced by a model of hindlimb unweighting), muscle weight returned to within 30% of control values. Myofibril protein content (mg/g muscle) remained relatively constant throughout the overload period but became significantly depressed relative to control values after 7 wk of regression. However, when expressed on a per muscle basis (mg/muscle) no differences existed at this time point (t = 7 wk regression). The distribution of native myosin isoforms in the myofibril protein pool of the overloaded plantaris muscle reflected a progressive increase (23% at t = 9 wk; P less than 0.001) in the relative proportion of slow myosin (Sm). This change was also accompanied by increases in intermediate myosin (Im) as well as the repression of the fast myosin one (Fm1) isoform (P less than 0.001). These shifts in Sm and Fm1 isoform expression were gradually reversed during the regression period, whereas Im remained elevated relative to control values. These adaptive changes in myosin isoform expression during both hypertrophy and regression were further supported by concomitant shifts in both myosin adenosinetriphosphatase (ATPase) activity (decreased during overload) and slow myosin light chain (SLC) expression. However, during regression the changes in myosin isoform expression and myosin ATPase were not as synchronous as they were during overload. Estimation of the mixed myosin heavy chain (MHC) half-life (t 1/2), using a linear model that assumes zero-order synthesis and first-order degradation kinetics, revealed t 1/2 values of approximately 19 and 10 days for the overload and regression periods, respectively. Collectively these data suggest that 1) skeletal muscle myosin isoforms and corresponding ATPase activity are in a dynamic state of change, although not completely synchronous, in response to altered muscle stress, and 2) the kinetics of change in the mixed MHC protein pool are slower during compensatory growth compared with regression of growth.     

Regionalized adaptations and muscle fiber proliferation in stretch-induced enlargement

The relative contribution of increases in fiber area to stretch-induced muscle enlargement was evaluated in the slow tonic fibers of the anterior latissimus dorsi of adult Japanese quails. A weight corresponding to 10% of the bird's body mass was attached to one wing. Thirty days of stretch in 34 birds averaged 171.8 +/- 13.5% increase in muscle mass and 23.5 +/- 0.8% increase in muscle fiber length. The volume density of noncontractile tissue increased in middle and distal regions of stretch-enlarged muscles. Mean fiber cross-sectional area increased 56.7 +/- 12.3% in the midregion of stretched muscles. Further analysis indicated slow beta-fiber hypertrophy occurred in proximal, middle, and distal regions; however, fast alpha-type fiber hypertrophy was limited to middle regions of stretched muscles. Stretched muscles had a significant increase in the frequency of slow beta-fibers that were less than 500 microns 2 in all regions and fast alpha-type fibers in middle and distal regions. Total fiber number was determined after nitric acid digestion of connective tissue in 10 birds. Fiber number increased 51.8 +/- 19.4% in stretched muscle. These results are the first to clearly show that muscle fiber proliferation contributes substantially to adult skeletal muscle stretch-induced enlargement, although we do not know whether the responses of the slow tonic anterior latissimus dorsi might be similar or different from mammalian twitch muscle.     

Heat, mechanics, and myosin ATPase in normal and hypertrophied heart muscle

In this paper we review our previous work on the myothermic economy of isometric force production in compensated cardiac hypertrophy secondary to pulmonary artery constriction (pressure overload) and/or thyrotoxicosis (volume overload). Hypertrophy-induced changes in isotonic and isometric twitch mechanics are correlated with accompanying changes in actin-activated myosin ATPase and heat liberation. Heat measurements were made with rapid, high-sensitivity thermopiles on right ventricular papillary muscles from normal and hypertrophied rabbit hearts. Total activity-related heat was separated into initial and recovery heat. Initial heat was separated into a tension-dependent component (TDH) relating to cross-bridge activity, and a tension-independent component (TIH) relating to excitation-contraction coupling. There were oppositely directed changes in most parameters studied in pressure overload hypertrophy (P) as compared with thyrotoxic hypertrophy (T). Thus, in P there was depression (30-50% in the rate of isometric force production, mechanical Vmax, TDH and TDH rate, myosin ATPase, TIH, and prolongation in time-to-peak twitch tension, whereas in T all parameters were oppositely changed except for no change in TIH. Thyrotoxicosis following pressure overload reversed the P-induced changes in all parameters. There was a direct, linear relation between in vitro actin-activated myosin ATPase and in vivo TDH. However, TDH per unit twitch tension or tension-time integral varied inversely with ATPase, making force production more economical than normal in P muscles and less economical than normal in T muscles. These cellular changes beneficially equip P hearts for slow, high-pressure, economical pumping the T hearts for fast, high-volume, uneconomical pumping. The differences are similar to those between slow and fast skeletal muscle and between neonatal and adult skeletal muscle. The mechanism of these changes is discussed in terms of an enzyme kinetic scheme of chemomechanical coupling in actomyosin interaction.     

Response to denervation of rabbit soleus and gastrocnemius muscles. Time-course study of postnatal changes in myosin isoforms,fiber types,and contractile properties

Summary— In contrast to general belief, the response of rabbit muscles to denervation is maturation to slow-like type muscles [7]. We report now an investigation by biochemical, morphological, and mechanical studies of the time course effects of muscle denervation on the slow-type soleus and fast-type gastrocnemius to help clucidate the mechanism of maturation of rabbit denervated muscles to slow-like muscles. In both muscles, denervation induced selective progressive atrophy of most fast fibers and hypertrophy of many slow fibers which displayed wide Z-lines; this was accompanied by the appearance of hybrid LC1F- and LC1E-associated slow myosins. The percentage of slow myosins increased with age similarly in the contralateral and denervated soleus. On the other hand, the percentage of slow myosins remained low in the contralateral gastrocnemius, whereas it increased to 95% in the denervated gastrocnemius; in the denervated gastrocnemius, the percentage of slow myosins reached 50% at about 35 days postnatal. At this age, the maximal shortening velocity of the denervated gastrocnemius and its twitch contraction time were already those of a slow-type muscle. This suggests that in addition to myosin, other proteins contributed to the mechanical properties of the denervated gastrocnemius. Transformation of rabbit denervated muscles to slow-like type muscles, which are associated with a lower energy requirement and higher muscle endurance than fast-type muscles, may constitute an adequate model for human neuromuscular pathology.     

The response of fast and slow nuclear bag fibres and nuclear chain fibres in isolated cat muscle spindles to fusimotor stimulation, and the effect of intrafusal contraction on the sensory endings.

1. The mechanical behaviour of intrafusal muscle fibres during fusimotor stimulation and passive stretch was observed directly in muscle spindles isolated from the cat tenuissimus muscle. 2. Mammalian intrafusal muscle fibres are of three functional types. Most spindles contain one slow nuclear bag fibre, one fast nuclear bag fibre, and four or five nuclear chain fibres. 3. Contraction in slow nuclear bag fibres is characterized by a long latency and very slow initial velocity, whereas the latency for the other intrafusal fibres is short and the inital velocity rapid. The mean time for maximum contraction (at 75 Hz to 100 Hz) and relaxation is significantly longer for slow nuclear bag fibres (0-8s) than for other intrafusal fibres (0-5 s). The contraction time of fast nuclear bag fibres is sometimes longer than that of nuclear chain fibres but the mean values are not significantly different; a difference in the time to attain 90% contraction is more obvious. 4. At low stimulation frequencies (10 Hz) contraction in slow nuclear bag fibres and in most fast nuclear bag fibres is smooth whereas nuclear chain fibres exhibit marked oscillations. Single stimuli elicit small local twitches in nuclear chain fibres and occasionally in fast nuclear bag fibres but produce no visible effect in slow nuclear bag fibres. 5. Maximum contraction of slow and fast nuclear bag fibres at body temperature is attained at a stimulation frequency of 75 Hz to 100 Hz, whereas a frequency of 150 Hz or more is required for maximum contraction of nuclear chain fibres. At 50 Hz at body temperature contraction in nuclear bag fibres is at least half the maximum, whereas in many spindles nuclear chain fibres show only a very small contraction at this frequency. 6. Contraction in slow nuclear bag fibres occurs at one or two discrete foci, most of which lie in the intracapsular region beyond the end of the fluid space. Weak contraction extends the primary sensory spiral by a small amount (2%-8%) at a low velocity (5%-10%s-1). When the fibre is passively stretched the spiral opens and then creeps back to about 75% of the extension at the end of the stretch due to yielding in the poles of fibre; creep is complete in 0-5s to 2-5s. 7. Contraction in fast nuclear bag fibres also occurs at one or two discrete foci, most of which lie in the intracapsular region beyond the end of the fluid space. Shortening of sarcomeres at the foci and extension of the sensory spiral are, however, up to eight times greater (up to 25%) than in slow nuclear bag fibres, and the velocity of stretch of the spiral is three to eight times greater (25%-40%s-1). Fast nuclear bag fibres exhibit little or no creep following passive stretch. 8. Contraction in the nuclear chain fibre bundle is localized to the intracapsular region, centered on a point in the intracapsular region between 0-9 mm and 1-6 mm from the spindle equator. Maximal contraction stretches primary and secondary sensory endings by 15% to 20%, at 30% to 40% s-1...     

Work overload induced changes in fast and slow skeletal muscle myosin heavy chain gene expression

Work induced hypertrophy of the slow postural soleus and the fast phasic plantaris muscles was produced by tenotomy of the synergistic gastrocnemius muscle. Increases in weight of both muscles were associated with proportionately even larger increases in total RNA and mRNA levels. Alterations in levels of specific myosin heavy chain (MHC) isoform mRNAs were measured using the slot blot procedure with radioactively labelled oligonucleotides as probes. Type 1 MHC gene expression was unaffected in both muscles by work overload, whereas type 2a was deinduced in the soleus and type 2b was deinduced in the plantaris. The neonatal MHC gene was transiently reinduced in the plantaris.     

Disuse and passive stretch cause rapid alterations in expression of developmental and adult contractile protein genes in skeletal muscle

Contractile proteins exist as a number of isoforms that show a developmental and tissue-specific pattern of expression. Using gene-specific cDNA probes, the expression of the sarcomeric myosin heavy chain (MHC) multi-gene family and of cardiac (foetal) alpha-actin was analysed in three different rat hindlimb muscles immobilised for 5 days in either the shortened or lengthened positions. For each of the MHC genes normally expressed in adult muscle (slow, IIA and IIB), the effect of disuse alone (immobilisation in the shortened position) upon expression was markedly different to that of passive stretch (immobilisation in the lengthened position) in each of the three muscles. However, the same adult sarcomeric myosin heavy chain gene can be affected in a different, or even opposite, manner by either disuse or passive stretch depending on the muscle in which it is being expressed. The fast IIB MHC gene, for example, exhibits a rapid induction in the slow postural soleus muscle, in response to disuse but no such induction occurs in the faster plantaris and gastrocnemius muscles. Furthermore, the induction of this gene in the soleus was prevented by passive stretch. The MHC gene, normally only expressed in embryonic skeletal muscle, showed a similar response in all three muscles and was reinduced in adult muscle in response to passive stretch but not by disuse alone. In contrast, the isoform of alpha-actin which is normally only present in significant quantities in embryonic skeletal muscle and which is reduced postnatally, is not reinduced by passive stretch but is reduced still further by immobilisation in the shortened position.     

Stimulation of slow skeletal muscle fiber gene expression by calcineurin in vivo

Adult skeletal muscle fibers can be categorized into fast and slow twitch subtypes based on specialized contractile and metabolic properties and on distinctive patterns of muscle gene expression. Muscle fiber-type characteristics are dependent on the frequency of motor nerve stimulation and are thought to be controlled by calcium-dependent signaling. The calcium, calmodulin-dependent protein phosphatase, calcineurin, stimulates slow fiber-specific gene promoters in cultured skeletal muscle cells, and the calcineurin inhibitor, cyclosporin A, inhibits slow fiber gene expression in vivo, suggesting a key role of calcineurin in activation of the slow muscle fiber phenotype. Calcineurin has also been shown to induce hypertrophy of cardiac muscle and to mediate the hypertrophic effects of insulin-like growth factor-1 on skeletal myocytes in vitro. To determine whether activated calcineurin was sufficient to induce slow fiber gene expression and hypertrophy in adult skeletal muscle in vivo, we created transgenic mice that expressed activated calcineurin under control of the muscle creatine kinase enhancer. These mice exhibited an increase in slow muscle fibers, but no evidence for skeletal muscle hypertrophy. These results demonstrate that calcineurin activation is sufficient to induce the slow fiber gene regulatory program in vivo and suggest that additional signals are required for skeletal muscle hypertrophy.     

Cyclical stretch induces structural changes in atrial myocytes

Atrial fibrillation (AF) often occurs in the presence of an underlying disease. These underlying diseases cause atrial remodelling, which make the atria more susceptible to AF. Stretch is an important mediator in the remodelling process. The aim of this study was to develop an atrial cell culture model mimicking remodelling due to atrial pressure overload. Neonatal rat atrial cardiomyocytes (NRAM) were cultured and subjected to cyclical stretch on elastic membranes. Stretching with 1 Hz and 15% elongation for 30 min. resulted in increased expression of immediate early genes and phosphorylation of Erk and p38. A 24‐hr stretch period resulted in hypertrophy‐related changes including increased cell diameter, reinduction of the foetal gene program and cell death. No evidence of apoptosis was observed. Expression of atrial natriuretic peptide, brain natriuretic peptide and growth differentiation factor‐15 was increased, and calcineurin signalling was activated. Expression of several potassium channels was decreased, suggesting electrical remodelling. Atrial stretch‐induced change in skeletal α‐actin expression was inhibited by pravastatin, but not by eplerenone or losartan. Stretch of NRAM results in elevation of stress markers, changes related to hypertrophy and dedifferentiation, electrical remodelling and cell death. This model can contribute to investigating the mechanisms involved in the remodelling process caused by stretch and to the testing of pharmaceutical agents.     

Extracellular signal-regulated kinase pathway is differentially involved in beta-agonist-induced hypertrophy in slow and fast muscles

The molecular mechanisms controlling -adrenergic receptor agonist (BA)-induced skeletal muscle hypertrophy are not well known. We presently report that BA exerts a distinct muscle- and muscle fiber type-specific hypertrophy. Moreover, we have shown that pharmacologically or genetically attenuating extracellular signal-regulated kinase (ERK) signaling in muscle fibers resulted in decreases (P < 0.05) in fast but not slow fiber type-specific reporter gene expressions in response to BA exposure in vitro and in vivo. Consistent with these data, forced expression of MAPK phosphatase 1, a nuclear protein that dephosphorylates ERK1/2, in fast-twitch skeletal muscle ablated (P < 0.05) the hypertrophic effects of BA feeding (clenbuterol, 20 parts per million in water) in vivo. Further analysis has shown that BA-induced phosphorylation and activation of ERK occurred to a greater (P < 0.05) extent in fast myofibers than in slow myofibers. Analysis of the basal level of ERK activity in slow and fast muscles revealed that ERK1/2 is activated to a greater extent in fast- than in slow-twitch muscles. These data indicate that ERK signaling is differentially involved in BA-induced hypertrophy in slow and fast skeletal muscles, suggesting that the increased abundance of phospho-ERK1/2 and ERK activity found in fast-twitch myofibers, compared with their slow-twitch counterparts, may account, at least in part, for the fiber type-specific hypertrophy induced by BA stimulation. These data suggest that fast myofibers are pivotal in the adaptation of muscle to environmental cues and that the mechanism underlying this change is partially mediated by the MAPK signaling cascade. muscle fiber type; mitogen-activated protein kinase signaling pathways; mechanism     

Mechanisms underlying the asynchronous replacement of myosin light chain isoforms during stimulation-induced fibre-type transformation of skeletal muscle

During the fibre-type transformation induced by chronic electrical stimulation of rabbit fast-twitch muscle, replacement of the fast forms of the two classes of myosin light chain by their slow isoforms occurs asynchronously. Studies of total cellular myosin light chains and of the slow-to-fast transition now justify the conclusion that the asynchrony is due to switching between the expression of fast and slow genes for the two light chain classes at sequential stages of the transformation process.     

Effects of Electrical Stimulation on Molecular Forms of Butyrylcholinesterase in Denervated Fast and Slow Latissimus Dorsi Muscles of Newly Hatched Chicken

The effects of denervation and direct electrical stimulation upon the activity and the molecular form distribution of butyrylcholinesterase (BuChE) were studied in fast-twitch posterior latissimus dorsi (PLD) and in slow-tonic anterior latissimus dorsi (ALD) muscles of newly hatched chicken. In PLD muscle, denervation performed at day 2 substantially reduced the rate of rapid decrease of BuChE specific activity which takes place during normal development, whereas in the case of ALD muscle little change was observed. Moreover, the asymmetric forms which were dramatically reduced in denervated PLD muscle were virtually absent in denervated ALD muscle at day 14. Denervated PLD and ALD muscles were stimulated from day 4 to day 14 of age. Two patterns of stimulation were applied, either 5-Hz frequency (slow rhythm) or 40-Hz frequency (fast rhythm). Both patterns of stimulation provided the same number of impulses per day (about 61,000). In PLD muscle, electrical stimulation almost totally prevented the postdenervation loss in asymmetric forms and led to a decrease in BuChE specific activity. In ALD muscle, electrical stimulation partially prevented the asymmetric form loss which occurs after denervation. This study emphasizes the role of evoked muscle activity in the regulation of BuChE asymmetric forms in the fast PLD muscle and the differential response of denervated slow and fast muscles to electrical stimulation.