Mammalian Skeletal Muscle Fibers Research Articles

Molecular regulation of stretch activation.

Stretch activation is defined as a delayed increase in force after rapid stretches. Although there is considerable evidence for stretch activation in isolated cardiac myofibrillar preparations, few studies have measured mechanisms of stretch activation in mammalian skeletal muscle fibers. We measured stretch activation following rapid step stretches [∼1%-4% sarcomere length (SL)] during submaximal Ca2+ activations of rat permeabilized slow-twitch skeletal muscle fibers before and after protein kinase A (PKA), which phosphorylates slow myosin binding protein-C. PKA significantly increased stretch activation during low (∼25%) Ca2+ activation and accelerated rates of delayed force development (kef) during both low and half-maximal Ca2+ activation. Following the step stretches and subsequent force development, fibers were rapidly shortened to original sarcomere length, which often elicited a shortening-induced transient force overshoot. After PKA, step shortening-induced transient force overshoot increased ∼10-fold following an ∼4% SL shortening during low Ca2+ activation levels. kdf following step shortening also increased after PKA during low and half-maximal Ca2+ activations. We next investigated thin filament regulation of stretch activation. We tested the interplay between cardiac troponin I (cTnI) phosphorylation at the canonical PKA and novel tyrosine kinase sites on stretch activation. Native slow-skeletal Tn complexes were exchanged with recombinant human cTn complex with different human cTnI N-terminal pseudo-phosphorylation molecules: 1) nonphosphorylated wild type (WT), 2) the canonical S22/23D PKA sites, 3) the tyrosine kinase Y26E site, and 4) the combinatorial S22/23D + Y26E cTnI. All three pseudo-phosphorylated cTnIs elicited greater stretch activation than WT. Following stretch activation, a new, elevated stretch-induced steady-state force was reached with pseudo-phosphorylated cTnI. Combinatorial S22/23D + Y26E pseudo-phosphorylated cTnI increased kdf. These results suggest that slow-skeletal myosin binding protein-C (sMyBP-C) phosphorylation modulates stretch activation by a combination of cross-bridge recruitment and faster cycling kinetics, whereas cTnI phosphorylation regulates stretch activation by both redundant and synergistic mechanisms; and, taken together, these sarcomere phosphoproteins offer precision targets for enhanced contractility.

Differential regulation of Actn2 and Actn3 expression during unfolded protein response in C2C12 myotubes.

ACTN2 and ACTN3 encode sarcomeric α-actinin-2 and α-actinin-3 proteins, respectively, that constitute the Z-line in mammalian skeletal muscle fibers. In human ACTN3, a nonsense mutation at codon 577 that encodes arginine (R) produces the R577X polymorphism. Individuals having a homozygous 577XX genotype do not produce α-actinin-3 protein. The 577XX genotype reportedly occurs in sprint and power athletes in frequency lower than in the normal population, suggesting that α-actinin-3 deficiency diminishes fast-type muscle function. Among humans who carry 577R alleles, varying ACTN3 expression levels under certain conditions can have diverse effects on atheletic and muscle performance. However, the factors that regulate ACTN3 expression are unclear. Here we investigated whether the unfolded protein response (UPR) under endoplasmic reticulum (ER) stress regulates expression of Actn3 and its isoform Actn2 in mouse C2C12 myotubes. Among UPR-related transcription factors, XBP1 upregulated Actn2, whereas XBP1, ATF4 and ATF6 downregulated Actn3 promoter activity. Chemical induction of ER stress increased Actn2 mRNA levels, but decreased those for Actn3. ER stress also decreased α-actinin-3 protein levels, whereas levels of α-actinin-2 were unchanged. The intracellular composition of muscle contraction-related proteins was altered under ER stress, in that expression of parvalbumin (a fast-twitch muscle-specific protein) and troponin I type 1 (skeletal, slow) was suppressed. siRNA-induced suppression of Actn3 mimicked the inhibitory effect of ER stress on parvalbumin levels. Thus, endogenous expression levels of α-actinin-3 can be altered by ER stress, which may modulate muscle performance and athletic aptitudes, particularly in humans who carry ACTN3 577R alleles.

Phosphorylated ERK1/2 protein levels are closely associated with the fast fiber phenotypes in rat hindlimb skeletal muscles.

The relationship between the extracellular signal-regulated kinase 1 and 2 (ERK1/2), one of the mitogen-activated protein kinases (MAPKs), and mammalian skeletal muscle fiber phenotype is unclear. We looked at this relationship in three in vivo conditions in male Wistar rats. First, the levels of phosphorylated (active) ERK1/2 protein were closely associated with the fiber type composition of sedentary rat hindlimb muscles: highest in the superficial portion of the gastrocnemius (100% fast fibers), lower in the plantaris (~ 80% fast fibers), and lowest in the soleus (~ 15% fast fibers). Second, during growth, there was a gradual decrease in the percentage of fast fibers from 40% at 3weeks to 1.5% at 65weeks and a concomitant gradual decrease in the levels of phosphorylated ERK1/2 in the soleus muscle. Third, sciatic nerve denervation induced a significant decrease in the weight of both the soleus and plantaris, but a slow-to-fast fiber type shift and increase in phosphorylated ERK1/2 protein were observed only in the soleus. Although only a few fast and fast + slow hybrid fibers of the denervated soleus muscle reacted positively to the anti-phosphorylated ERK1/2 antibody by immuno-histochemical analysis, our results suggest that the phosphorylated form of ERK1/2 seems to be closely related to the fast fiber phenotype program. Further evidence for this relationship was provided by the observation that several slow fiber phenotype-specific proteins, i.e., Hsp72, Hsp60, and PGC-1, changed in the opposite direction of the levels of phosphorylated ERK1/2 protein.

Muscle Fiber‐type Differences in Mice Selected for Wheel Running Behavior

Mammalian skeletal muscle fibers contain varying amounts of myosin isoforms for slow and fast myosin heavy‐chains, (Types 1, 2A, 2X, 2B,). Muscle fibers containing different isoform profiles typically correlate with variable force, speed, and fatigue resistance. We investigated the isoform profiles of various hindlimb muscles in two lines of mice, one of which has been undergoing laboratory selection for high wheel‐running behavior for over 80 generations. We hypothesized that the selected line would typically have the most type 1 or type 2a myosin. This isoform is most often expressed in muscle with higher oxidative capacity and therefore greater fatigue resistance. We collected lower leg muscles including the quadriceps, soleus, medial and lateral gastrocnemius, and plantaris from the breeding pairs of generation 80 from replicated selected and control lines.Some data were previously reported from generations collected over a decade ago, but many muscles were not included, and we wished to see if further changes to the isoform profiles have arisen. SDS PAGE and densitometry were used to quantify the myosin isoforms.Preliminary analysis using densitometry shows that the leg muscles of high‐running mice had a higher concentration of type 1 myosin, or in other cases, a shift towards slower type 2 fibers (i.e. 2x to 2a). The findings were highly muscle‐specific, and indicate shifts in muscle protein expression has resulted from selection.Our new catalog of isoform profiles will serve as a baseline for comparison of two future studies – one following the development and subsequent senescence of muscle function with aging, and the second determining the effect of exercise activity on both selected and control lines.With the aging study we hope to identify whether activity during the lifetime is as important and genetic factors in preventing the decline of muscle performance with age. For the exercise study, we hope to determine the influence of genetics on shaping exercise performance for individuals which descend from sedentary or active lines. All of these data address long‐standing nature vs nurture debates, but also bear on human clinical muscle disease, aging, and activity.Support or Funding InformationThis research was supported by California State University, Long Beach and the National Institute of General Medical Sciences of the National Institutes of Health under Award Numbers; 8UL1GM118979‐02; 8TL4GM118980‐02; 8RL5GM118978‐02. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. We would also like to thank Dr. Theodore Garland from University of California, Riverside and Dr. Angela Horner from California State University, San Bernardino.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mechanical isolation, and measurement of force and myoplasmic free [Ca2+] in fully intact single skeletal muscle fibers.

Mechanical dissection of single intact mammalian skeletal muscle fibers permits real-time measurement of intracellular properties and contractile function of living fibers. A major advantage of mechanical over enzymatic fiber dissociation is that single fibers can be isolated with their tendons remaining attached, which allows contractile forces (in the normal expected range of 300-450 kN/m2) to be measured during electrical stimulation. Furthermore, the sarcolemma of single fibers remains fully intact after mechanical dissection, and hence the living fibers can be studied with intact intracellular milieu and normal function and metabolic properties, as well as ionic control. Given that Ca2+ is the principal regulator of the contractile force, measurements of myoplasmic free [Ca2+] ([Ca2+]i) can be used to further delineate the intrinsic mechanisms underlying changes in skeletal muscle function. [Ca2+]i measurements are most commonly performed in intact single fibers using ratiometric fluorescent indicators such as indo-1 or fura-2. These Ca2+ indicators are introduced into the fiber by pressure injection or by using the membrane-permeable indo-1 AM, and [Ca2+]i is measured by calculating a ratio of the fluorescence at specific wavelengths emitted for the Ca2+-free and Ca2+-bound forms of the dye. We describe here the procedures for mechanical dissection, and for force and [Ca2+]i measurement in intact single fibers from mouse flexor digitorum brevis (FDB) muscle, which is the most commonly used muscle in studies using intact single fibers. This technique can also be used to isolate intact single fibers from various muscles and from various species. As an alternative to Ca2+ indicators, single fibers can also be loaded with fluorescent indicators to measure, for instance, reactive oxygen species, pH, and [Mg2+], or they can be injected with proteins to change functional properties. The entire protocol, from dissection to the start of an experiment on a single fiber, takes ∼3 h.

Characterization of Different Localized Ca2+ Signals in Skeletal Muscle Fibers

Discrete localized Ca2+ signals (LCS) occur in many cell types. In skeletal muscle fibers they have been shown e.g. to be elicited as a result of an osmotic shock or mechanical skinning of isolated muscle fibers. Indicating their possible pathophysiological importance, spontaneous LCS have been observed in the mdx mouse model of human Duchenne muscular dystrophy and in wild type mouse muscle fibers after severe mechanical stress. In addition, there is a variety of different types of intricate subcellular Ca2+ signaling in micro-domains of mammalian skeletal muscle fibers.

Mammalian Skeletal Muscle Fibres Promote Non-Muscle Stem Cells and Non-Stem Cells to Adopt Myogenic Characteristics

Skeletal muscle fibres are unique cells in large animals, often composed of thousands of post-mitotic nuclei. Following skeletal muscle damage, resident stem cells, called satellite cells, commit to myogenic differentiation and migrate to carry out repair. Satellite stem cells migrate on muscle fibres through amoeboid movement, which relies on dynamic cell membrane extension and retraction (blebbing). It is not known whether blebbing is due to the intrinsic properties of satellite cells, or induced by features of the myofibre surface. Here, we determined the influence of the muscle fibre matrix on two important features of muscle regeneration: the ability to migrate and to differentiate down a myogenic lineage. We show that the muscle fibre is able to induce amoeboid movement in non-muscle stem cells and non-stem cells. Secondly, we show that prolonged co-culture on myofibres caused amniotic fluid stem cells and breast cancer cells to express MyoD, a key myogenic determinant. Finally, we show that amniotic fluid stem cells co-cultured on myofibres are able to fuse and make myotubes that express Myosin Heavy Chain.

Neuromuscular NMDA Receptors Modulate Developmental Synapse Elimination.

At birth, each mammalian skeletal muscle fiber is innervated by multiple motor neurons, but in a few weeks, all but one of those axons retracts (Redfern, 1970) and differential activity between inputs controls this phenomenon (Personius and Balice-Gordon, 2001; Sanes and Lichtman, 2001; Personius et al., 2007; Favero et al., 2012). Acetylcholine, the primary neuromuscular transmitter, has long been presumed to mediate this activity-dependent process (O'Brien et al., 1978), but glutamatergic transmission also occurs at the neuromuscular junction (Berger et al., 1995; Grozdanovic and Gossrau, 1998; Mays et al., 2009). To test the role of neuromuscular NMDA receptors, we assessed their contribution to muscle calcium fluxes in mice and tested whether they influence removal of excess innervation at the end plate. Developmental synapse pruning was slowed by reduction of NMDA receptor activation or expression and by reduction of glutamate production. Conversely, pruning is accelerated by application of exogenous NMDA. We also found that NMDA induced increased muscle calcium only during the first 2 postnatal weeks. Therefore, neuromuscular NMDA receptors play previously unsuspected roles in neuromuscular activity and synaptic pruning during development. In normal adult muscle, each muscle fiber is innervated by a single axon, but at birth, fibers are multiply innervated. Elimination of excess connections requires neural activity; because the neuromuscular junction (NMJ) is a cholinergic synapse, acetylcholine has been assumed to be the critical mediator of activity. However, glutamate receptors are also expressed at the NMJ. We found that axon removal in mice is slowed by pharmacological and molecular manipulations that decrease signaling through neuromuscular NMDA receptors, whereas application of exogenous NMDA at the NMJ accelerates synapse elimination and increases muscle calcium levels during the first 2 postnatal weeks. Therefore, neuromuscular NMDA receptors play previously unsuspected roles in neuromuscular activity and elimination of excess synaptic input during development.

Membrane glucocorticoid receptors are localised in the extracellular matrix and signal through the MAPK pathway in mammalian skeletal muscle fibres.

Many studies have previously suggested the existence of stress hormone receptors on the cell membrane of many cell types, including skeletal muscle fibres; however, the exact localisation of these receptors and how they signal to the rest of the cell is poorly understood. In this study, we investigated the localisation and the mechanism(s) underlying the physiological functions of these receptors in mouse skeletal muscle cells. We found that the receptors were present throughout muscle development and that, in adult muscle fibres, they were localised in the extracellular matrix, satellite cells (muscle stem cells) and close to mitochondria. We also found that they signalled to the rest of the cell by activating enzymes called mitogen-activated protein kinases. From these results we suggest that, at physiological concentrations, stress hormones may be important in skeletal muscle differentiation, repair and regeneration. A number of studies have previously proposed the existence of glucocorticoid receptors on the plasma membrane of many cell types, including skeletal muscle fibres. However, their exact localisation and the cellular signalling pathway(s) they utilise to communicate with the rest of the cell are still poorly understood. In this study, we investigated the localisation and the mechanism(s) underlying the non-genomic physiological functions of these receptors in mouse skeletal muscle cells. The results show that the receptors were localised in the cytoplasm in myoblasts, in the nucleus in myotubes, in the extracellular matrix, in satellite cells and in the proximity of mitochondria in adult muscle fibres. Also, they bound laminin in a glucocorticoid-dependent manner. Treating small skeletal muscle fibre bundles with the synthetic glucocorticoid beclomethasone dipropionate increased the phosphorylation (=activation) of extracellular signal-regulated kinases 1 and 2, c-Jun N-terminal kinase and p38 mitogen-activated protein kinase. This occurred within 5min and depended on the fibre type and the duration of the treatment. It was also abolished by the glucocorticoid receptor inhibitor, mifepristone, and a monoclonal antibody against the receptor. From these results we conclude that the non-genomic/non-canonical physiological functions of glucocorticoids, in adult skeletal muscle fibres, are mediated by a glucocorticoid receptor localised in the extracellular matrix, in satellite cells and close to mitochondria, and involve activation of the mitogen-activated protein kinase pathway.

Cholesterol removal from adult skeletal muscle impairs excitation-contraction coupling and aging reduces caveolin-3 and alters the expression of other triadic proteins.

Cholesterol and caveolin are integral membrane components that modulate the function/location of many cellular proteins. Skeletal muscle fibers, which have unusually high cholesterol levels in transverse tubules, express the caveolin-3 isoform but its association with transverse tubules remains contentious. Cholesterol removal impairs excitation–contraction (E–C) coupling in amphibian and mammalian fetal skeletal muscle fibers. Here, we show that treating single muscle fibers from adult mice with the cholesterol removing agent methyl-β-cyclodextrin decreased fiber cholesterol by 26%, altered the location pattern of caveolin-3 and of the voltage dependent calcium channel Cav1.1, and suppressed or reduced electrically evoked Ca2+ transients without affecting membrane integrity or causing sarcoplasmic reticulum (SR) calcium depletion. We found that transverse tubules from adult muscle and triad fractions that contain ~10% attached transverse tubules, but not SR membranes, contained caveolin-3 and Cav1.1; both proteins partitioned into detergent-resistant membrane fractions highly enriched in cholesterol. Aging entails significant deterioration of skeletal muscle function. We found that triad fractions from aged rats had similar cholesterol and RyR1 protein levels compared to triads from young rats, but had lower caveolin-3 and glyceraldehyde 3-phosphate dehydrogenase and increased Na+/K+-ATPase protein levels. Both triad fractions had comparable NADPH oxidase (NOX) activity and protein content of NOX2 subunits (p47phox and gp91phox), implying that NOX activity does not increase during aging. These findings show that partial cholesterol removal impairs E–C coupling and alters caveolin-3 and Cav1.1 location pattern, and that aging reduces caveolin-3 protein content and modifies the expression of other triadic proteins. We discuss the possible implications of these findings for skeletal muscle function in young and aged animals.

Prox1 transcription factor regulates satellite cell and skeletal muscle fiber phenotype and function

Prospero related homeobox 1 (Prox1) transcription factor is important for the development of several organs, including the heart, liver and lymphatic vessels. Prox1 gene polymorphisms have recently been related to insulin resistance. We show here that Prox1 is expressed in adult slow skeletal muscle fibers and satellite cells, both in rodents and in humans. Prox1 expression is increased during mouse C2C12 cell differentiation and in primary human myoblast differentiation into myotubes, and its silencing leads to failure in differentiation. Mechanistically, Prox1 activates calcineurin signaling both in vivo in skeletal muscles and in C2C12 cells. Overexpression of Prox1 in fast skeletal muscle induced the expression of slow muscle fiber-related myofibrillar and calcium-signaling genes, while deletion of Prox1 lead to the upregulation of the fast myosin heavy chain isoforms in slow soleus muscle. With lineage tracing, we showed that Prox1 expressing satellite cells proliferate and contribute to muscle regeneration after cardiotoxin-induced muscle injury. After acute running exercise, the expression of Prox1 was downregulated in mouse gastrocnemius muscle. We have identified Prox1 as an important transcription factor regulating mammalian skeletal muscle fiber type and satellite cell differentiation and phenotype. This provides novel information how skeletal muscle function is regulated, and is important in terms of understanding normal skeletal muscle physiology and muscle diseases.

Potassium Regulation of the NaK-ATPase Pump Currents in Mammalian Skeletal Muscle Fibers

Adult skeletal muscles express two Na,K-ATPase isoforms, α1 and α2. α2, the sole isoform expressed in the transverse tubules, comprises up to 90% of total Na,K-ATPase. α2 in resting muscle operates well below its maximum turnover rate; but is rapidly stimulated, by yet unknown mechanisms, during muscle contraction, helping maintaining excitation and resist fatigue. We tested the hypothesis that α2 activity may be regulated by extracellular K over the physiological range of K concentrations that occur in the t-tubules. Na,K-ATPase driven current (Ipump) was measured in enzymatically isolated FDB fibers voltage clamped at −90 mV using a two microlectrode amplifier. Ipump was identified as a ouabain- and temperature-sensitive outward current activated by Ko in fibers rendered electrically passive. Intracellular solutions (filling both electrodes) contained (mM): 1EGTA, 0.5CaCl2, 5MgCl2, 20MOPS, 5gluthathion-H2, 90K-asparte, and 5ATP-Na2, 5-phosphocreatine-Na2, and 30Na-aspartate to promote forward pump cycling. External solutions contained (mM): 2CaCl2, 1MgCl2, 10glucose, 10MOPS; and inversely varying amounts of KCl (0-40) and NaCL (154-114) to maintain osmolarity. TTX (400nM), nifedipine (20 μM), Ba (1mM) and 9-anthracene-carboxilic-acid (200μM) were added to block main ionic currents. All solutions had pH=7.4 and 300mOmal/kgH2O. Ipump was activated in a concentration-dependent manner by raising [K]o from 0 to 40mM, and eliminated by washing out K. Ipump was prevented by pretreatment with 10μM ouabain or inhibited by addition of ouabain, and was steeply temperature-dependent. Maximal Ipump vs. [K]o was fitted to obtain the Km for pump activation by K. Results demonstrate that α2 in skeletal muscle is regulated by changes in extracellular K over tens of mM, suggesting that this isoform is adapted to respond to expected demands of Na/K transport in the t-tubules during sustained activity. (Supported by the National institutes of Health, USA)

Calcium Sparklets in Intact Mammalian Skeletal Muscle Fibers Expressing the Embryonic CaV1.1 Splice Variant

The embryonic splice variant of the voltage-gated L-type calcium channel (CaV1.1e) displays an altered voltage-dependence and gating kinetics as compared to that expressed in adult skeletal muscle. Because the adult CaV1.1a only opens slowly at strong depolarizations, its contribution as a source of calcium influx during action potentials is negligible. In contrast, calcium influx through the embryonic CaV1.1e substantially contributes to depolarization-induced calcium transients in fetal muscles and in cultured myotubes. In a genetically modified mouse (CaV1.1αE29), which exclusively expresses the embryonic CaV1.1e variant also in adult muscle the calcium influx component is maintained throughout life. Utilizing this mouse model, calcium release events - calcium sparklets - were recorded in enzymatically isolated, intact adult skeletal muscle fibers from the m. flexor digitorum longus using the fluorescent calcium probe fluo-8 and the fast confocal scanner (ZeissLive) in the x-y mode. While control animals did not display such events, CaV1.1αE29 mice spontaneously generated sparklets with a frequency of 9.2∗10-4±6∗10-4 Hz/μm2 (19 fibers; mean±SEM). The role of external calcium as the trigger was tested by either removing calcium from the external solution or by the application of 5μM nizoldipine to block the calcium current through CaV1.1e. Both interventions resulted in a complete loss of the events. Identified sparklets (n=311) were characterized by an average amplitude (αF/F0) of 0.287±0.005, a full-width at half-maximum of 3.05±0.05 µm, and duration of 235±4 ms, clearly different from the properties of calcium sparks on saponin-permeabilized adult mammalian skeletal muscle fibers. These findings indicate that the sustained expression of the CaV1.1e splice variant gives rise to spontaneous calcium entry events (sparklets) in adult muscle fibers and that their properties are distinct from calcium sparks arising from ryanodine receptors. Support: OTKA-NN107765, FWF P23479, W1101, TAMOP-4.2.4.A./2-11-1-2012-0001.

Usage of a localised microflow device to show that mitochondrial networks are not extensive in skeletal muscle fibres.

In cells, such as neurones and immune cells, mitochondria can form dynamic and extensive networks that change over the minute timescale. In contrast, mitochondria in adult mammalian skeletal muscle fibres show little motility over several hours. Here, we use a novel three channelled microflow device, the multifunctional pipette, to test whether mitochondria in mouse skeletal muscle connect to each other. The central channel in the pipette delivers compounds to a restricted region of the sarcolemma, typically 30 µm in diameter. Two channels on either side of the central channel use suction to create a hydrodynamically confined flow zone and remove compounds completely from the bulk solution to internal waste compartments. Compounds were delivered locally to the end or side of single adult mouse skeletal muscle fibres to test whether changes in mitochondrial membrane potential were transmitted to more distant located mitochondria. Mitochondrial membrane potential was monitored with tetramethylrhodamine ethyl ester (TMRE). Cytosolic free [Ca2+] was monitored with fluo-3. A pulse of carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP, 100 µM) applied to a small area of the muscle fibre (30 µm in diameter) produced a rapid decrease in the mitochondrial TMRE signal (indicative of depolarization) to 38% of its initial value. After washout of FCCP, the TMRE signal partially recovered. At distances greater than 50 µm away from the site of FCCP application, the mitochondrial TMRE signal was unchanged. Similar results were observed when two sites along the fibre were pulsed sequentially with FCCP. After a pulse of FCCP, cytosolic [Ca2+] was unchanged and fibres contracted in response to electrical stimulation. In conclusion, our results indicate that extensive networks of interconnected mitochondria do not exist in skeletal muscle. Furthermore, the limited and reversible effects of targeted FCCP application with the multifunctional pipette highlight its advantages over bulk application of compounds to isolated cells.

A membrane glucocorticoid receptor mediates the rapid/non-genomic actions of glucocorticoids in mammalian skeletal muscle fibres.

Glucocorticoids (GCs) are steroid hormones released from the adrenal gland in response to stress. They are also some of the most potent anti-inflammatory and immunosuppressive drugs currently in clinical use. They exert most of their physiological and pharmacological actions through the classical/genomic pathway. However, they also have rapid/non-genomic actions whose physiological and pharmacological functions are still poorly understood. Therefore, the primary aim of this study was to investigate the rapid/non-genomic effects of two widely prescribed glucocorticoids, beclomethasone dipropionate (BDP) and prednisolone acetate (PDNA), on force production in isolated, intact, mouse skeletal muscle fibre bundles. The results show that the effects of both GCs on maximum isometric force (Po) were fibre-type dependent. Thus, they increased Po in the slow-twitch fibre bundles without significantly affecting that of the fast-twitch fibre bundles. The increase in Po occurred within 10 min and was insensitive to the transcriptional inhibitor actinomycin D. Also, it was maximal at ∼250 nM and was blocked by the glucocorticoid receptor (GCR) inhibitor RU486 and a monoclonal anti-GCR, suggesting that it was mediated by a membrane (m) GCR. Both muscle fibre types expressed a cytosolic GCR. However, a mGCR was present only in the slow-twitch fibres. The receptor was more abundant in oxidative than in glycolytic fibres and was confined mainly to the periphery of the fibres where it co-localised with laminin. From these findings we conclude that the rapid/non-genomic actions of GCs are mediated by a mGCR and that they are physiologically/therapeutically beneficial, especially in slow-twitch muscle fibres.

Examination of the Subsarcolemmal Tubular System of Mammalian Skeletal Muscle Fibers

A subsarcolemmal tubular system network (SSTN) has been detected in skeletal muscle fibers by confocal imaging after the removal of the sarcolemma. Here we confirm the existence and resolve the fine architecture and the localization of the SSTN at an unprecedented level of detail by examining extracellularly applied tubular system markers in skeletal muscle fiber preparations with a combination of three imaging modalities: confocal fluorescence microscopy, direct stochastic optical reconstruction microscopy, and tomographic electron microscopy. Three-dimensional reconstructions showed that the SSTN was a dense two-dimensional network within the subsarcolemmal space around the fiber, running ∼500–600 nm underneath and parallel to the sarcolemma. The SSTN is composed of tubules ∼95 nm in width with ∼60% of the tubules directed transversely and >30% directed longitudinally. The deeper regular transverse tubules located at each A-I boundary of the sarcomeres branched from the SSTN, indicating individual transverse tubules that form triads are continuous with, but do not directly contact the sarcolemma. This suggests that the SSTN plays an important role in affecting the exchange of deeper tubule lumina with the extracellular space.

The Contribution of CLC-1 and Kir Channels to the Resting Electrical Properties of Skeletal Muscle Fibers from Normal and Myotonic Mice

The membrane resistance (Rm) of mammalian skeletal muscle fibers at rest has been typically assumed to be dominated by the chloride conductance (gCl). We have demonstrated, in fibers internally equilibrated with 70 mM Cl, bathed in TEA-Cl (145mM), and voltage-clamped at −20mV, that the majority of the Cl currents (ICl; ∼1mA/cm2) are contributed by 9-ACA-sensitive chloride (ClC-1) channels expressed in the transverse tubular system (TTS) membranes. Likewise, in fibers internally equilibrated with 150mM K, bathed with 150mM KMeSO4, and clamped at 0mV, the majority of the K-currents arise from inward rectifier K (KIR; ∼1mA/cm2) channels in the TTS. Here we address the question: what are the relative contributions of gCl and gKIR to Rm in intact fibers bathed in Tyrode (4mM K and 155mM Cl; RP∼-90mV)? Short fibers isolated from flexor digitorum brevis (FDB) muscles were impaled with 2 microelectrodes and stimulated with step current pulses (ΔI<30nA; 400ms) eliciting small voltage responses (ΔV<15mV). Rm and Cm were calculated by fitting ΔV traces to the solution of equations for a short cylindrical cable (Hodgkin & Nakajima, JPhysiol. 221:105-120.1972). For fibers of adult control mice, Rm in Tyrode is 721±52 cm2 (n=7); this value increased to 1,672±116 cm2 (n=5) by blocking gKIR with 10mM Rb (or 1mM Ba), and to 1,804±190 cm2 (n=5) by blocking gCl with 1mM 9-ACA. The results demonstrate that, unlike commonly assumed, gCl and gKIR represent approximately 58% and 42% (respectively) of the blockable resting conductances in fibers of normal mice. In contrast, similar analysis in fibers of young HSALR mice, which have ∼80% reduction in ClC-1 expression, gCl and gKIR contributions represent approximately 23% and 77%, respectively. Supported by NIH grants AR047664, AR041802, and AR054816.

The delayed rectifier potassium conductance in the sarcolemma and the transverse tubular system membranes of mammalian skeletal muscle fibers

A two-microelectrode voltage clamp and optical measurements of membrane potential changes at the transverse tubular system (TTS) were used to characterize delayed rectifier K currents (IKV) in murine muscle fibers stained with the potentiometric dye di-8-ANEPPS. In intact fibers, IKV displays the canonical hallmarks of KV channels: voltage-dependent delayed activation and decay in time. The voltage dependence of the peak conductance (gKV) was only accounted for by double Boltzmann fits, suggesting at least two channel contributions to IKV. Osmotically treated fibers showed significant disconnection of the TTS and displayed smaller IKV, but with similar voltage dependence and time decays to intact fibers. This suggests that inactivation may be responsible for most of the decay in IKV records. A two-channel model that faithfully simulates IKV records in osmotically treated fibers comprises a low threshold and steeply voltage-dependent channel (channel A), which contributes ∼31% of gKV, and a more abundant high threshold channel (channel B), with shallower voltage dependence. Significant expression of the IKV1.4 and IKV3.4 channels was demonstrated by immunoblotting. Rectangular depolarizing pulses elicited step-like di-8-ANEPPS transients in intact fibers rendered electrically passive. In contrast, activation of IKV resulted in time- and voltage-dependent attenuations in optical transients that coincided in time with the peaks of IKV records. Normalized peak attenuations showed the same voltage dependence as peak IKV plots. A radial cable model including channels A and B and K diffusion in the TTS was used to simulate IKV and average TTS voltage changes. Model predictions and experimental data were compared to determine what fraction of gKV in the TTS accounted simultaneously for the electrical and optical data. Best predictions suggest that KV channels are approximately equally distributed in the sarcolemma and TTS membranes; under these conditions, >70% of IKV arises from the TTS.

Twitchin can regulate the ATPase cycle of actomyosin in a phosphorylation-dependent manner in skinned mammalian skeletal muscle fibres

The effect of twitchin, a thick filament protein of molluscan muscles, on the actin–myosin interaction at several mimicked sequential steps of the ATPase cycle was investigated using the polarized fluorescence of 1.5-IAEDANS bound to myosin heads, FITC-phalloidin attached to actin and acrylodan bound to twitchin in the glycerol-skinned skeletal muscle fibres of mammalian. The phosphorylation-dependent multi-step changes in mobility and spatial arrangement of myosin SH1 helix, actin subunit and twitchin during the ATPase cycle have been revealed. It was shown that nonphosphorylated twitchin inhibited the movements of SH1 helix of the myosin heads and actin subunits and decreased the affinity of myosin to actin by freezing the position and mobility of twitchin in the muscle fibres. The phosphorylation of twitchin reverses this effect by changing the spatial arrangement and mobility of the actin-binding portions of twitchin. In this case, enhanced movements of SH1 helix of the myosin heads and actin subunits are observed. The data imply a novel property of twitchin incorporated into organized contractile system: its ability to regulate the ATPase cycle in a phosphorylation-dependent fashion by changing the affinity and spatial arrangement of the actin-binding portions of twitchin.

Longitudinal and transversal propagation of excitation along the tubular system of rat fast‐twitch muscle fibres studied by high speed confocal microscopy

Mammalian skeletal muscle fibres possess a tubular (t-) system that consists of regularly spaced transverse elements which are also connected in the longitudinal direction. This tubular network provides a pathway for the propagation of action potentials (APs) both radially and longitudinally within the fibre, but little is known about the actual radial and longitudinal AP conduction velocities along the tubular network in mammalian skeletal muscle fibres. The aim of this study was to track AP propagation within the t-system network of fast-twitch rat muscle fibres with high spatio-temporal resolution when the t-system was isolated from the surface membrane. For this we used high speed confocal imaging of AP-induced Ca(2+) release in contraction-suppressed mechanically skinned fast-twitch fibres where the t-system can be electrically excited in the absence of the surface membrane. Supramaximal field pulses normally elicited a synchronous AP-induced release of Ca(2+) along one side of the fibre axis which propagated uniformly across the fibre. In some cases up to 80 or more adjacent transverse tubules failed to be excited by the field pulse, while adjacent areas responded with normal Ca(2+) release. In these cases a continuous front of Ca(2+) release with an angle to the scanning line was observed due to APs propagating longitudinally. From these observations the radial/transversal and longitudinal AP conduction velocities along the tubular network deeper in the fibre under our conditions (19 ± 1°C) ranged between 8 and 11 μm ms(-1) and 5 to 9 μm ms(-1), respectively, using different methods of estimation. The longitudinal propagation of APs appeared to be markedly faster closer to the edge of the fibre, in agreement with the presence of dense longitudinal connections immediately below the surface of the fibre and more sparse connections at deeper planes within the fibre. During long trains of closely spaced field pulses the AP-elicited Ca(2+) releases became non-synchronous along the fibre axis. This is most likely caused by local tubular K(+) accumulation that produces local depolarization and local slowing of AP propagation. Longitudinally propagating APs may reduce such inhomogeneities by exciting areas of delayed AP onset. Clearly, the longitudinal tubular pathways within the fibre for excitation are used as a safety mechanism in situations where a local depolarization obstructs immediate excitation from the sarcolemma. Results obtained from this study also provide an explanation for the pattern of contractures observed in rippling muscle disease.