Adult Skeletal Muscle Fibers Research Articles

Rapid actin-cytoskeleton-dependent recruitment of plasma membrane-derived dysferlin at wounds is critical for muscle membrane repair.

Deficits in membrane repair may contribute to disease progression in dysferlin-deficient muscular dystrophy. Dysferlin, a type-II transmembrane phospholipid-binding protein, is hypothesized to regulate fusion of repair vesicles with the sarcolemma to facilitate membrane repair, but the dysferlin-containing compartments involved in membrane repair and the mechanism by which these compartments contribute to resealing are unclear. A dysferlin-pHluorin [dysf-pH-sensitive green fluorescent protein (pHGFP)] muscle-specific transgenic mouse was developed to examine the dynamic behavior and subcellular localization of dysferlin during membrane repair in adult skeletal muscle fibers. Live-cell confocal microscopy of uninjured adult dysf-pHGFP muscle fibers revealed that dysferlin is highly enriched in the sarcolemma and transverse tubules. Laser-wounding induced rapid recruitment of ∼30 μm of local dysferlin-containing sarcolemma, leading to formation of stable dysferlin accumulations surrounding lesions, endocytosis of dysferlin, and formation of large cytoplasmic vesicles from distal regions of the fiber. Disruption of the actin cytoskeleton decreased recruitment of sarcolemma-derived dysferlin to lesions in dysf-pHGFP fibers without affecting endocytosis and impaired membrane resealing in wild-type fibers, similar to findings in dysferlin deficiency (a 2-fold increase in FM1-43 uptake). Our data support a new mechanism whereby recruitment of sarcolemma-derived dysferlin creates an active zone of high lipid-binding activity at wounds to interact with repair vesicles and facilitate membrane resealing in skeletal muscle.

β1a490–508, a 19-Residue Peptide from C-Terminal Tail of Cav1.1 β1a Subunit, Potentiates Voltage-Dependent Calcium Release in Adult Skeletal Muscle Fibers

The α1 and β1a subunits of the skeletal muscle calcium channel, Cav1.1, as well as the Ca2+ release channel, ryanodine receptor (RyR1), are essential for excitation-contraction coupling. RyR1 channel activity is modulated by the β1a subunit and this effect can be mimicked by a peptide (β1a490–524) corresponding to the 35-residue C-terminal tail of the β1a subunit. Protein-protein interaction assays confirmed a high-affinity interaction between the C-terminal tail of the β1a and RyR1. Based on previous results using overlapping peptides tested on isolated RyR1, we hypothesized that a 19-amino-acid residue peptide (β1a490–508) is sufficient to reproduce activating effects of β1a490–524. Here we examined the effects of β1a490–508 on Ca2+ release and Ca2+ currents in adult skeletal muscle fibers subjected to voltage-clamp and on RyR1 channel activity after incorporating sarcoplasmic reticulum vesicles into lipid bilayers. β1a490–508 (25 nM) increased the peak Ca2+ release flux by 49% in muscle fibers. Considerably fewer activating effects were observed using 6.25, 100, and 400 nM of β1a490–508 in fibers. β1a490–508 also increased RyR1 channel activity in bilayers and Cav1.1 currents in fibers. A scrambled form of β1a490–508 peptide was used as negative control and produced negligible effects on Ca2+ release flux and RyR1 activity. Our results show that the β1a490–508 peptide contains molecular components sufficient to modulate excitation-contraction coupling in adult muscle fibers.

Telomeres Shorten in Response to Oxidative Stress in Mouse Skeletal Muscle Fibers

Aging phenotypes are dictated by myriad cellular changes including telomere shortening. In most tissues, telomere shortening is accelerated during replication if unrepaired oxidative damage to telomere sequences is present. However, the effect of reactive oxygen species exposure on skeletal muscle telomeres is unknown. We sought to determine if oxidative stress shortens telomeres in isolated adult rodent skeletal muscle fibers. Flexor digitorum brevis muscles were dissected from male mice (C57BL/6, long telomere and CAST/Ei, wild-derived, short telomere) and dissociated into single fibers. Fibers were cultured at an oxygen tension of 2%-5% for 5 days in control, hydrogen peroxide (oxidant), or a combination of N-acetylcysteine (antioxidant) and oxidant containing media. Telomere length, telomerase enzyme activity, and protein content of TRF1 and TRF2 were subsequently measured. In both strains, oxidative stress resulted in significant telomere shortening in isolated skeletal muscle fibers, likely by different mechanisms. Telomerase activity was not altered by oxidative stress treatment but was significantly different between strains, with greater telomerase activity in long-telomere-bearing C57BL/6 mice. These results provide important insights into mechanisms by which oxidative stress could shorten skeletal muscle telomeres.

Whole-cell voltage clamp on skeletal muscle fibers with the silicone-clamp technique.

Control of membrane voltage and membrane current measurements are of critical importance for the study of numerous aspects of skeletal muscle physiology and pathophysiology. The silicone-clamp technique makes use of a conventional patch-clamp apparatus to achieve whole-cell voltage clamp of a restricted portion of a fully differentiated adult skeletal muscle fiber. The major part of an isolated muscle fiber is insulated from the extracellular medium with silicone grease and the tip of a single microelectrode connected to the amplifier is then inserted within the fiber through the silicone layer. The method is extremely easy to implement. It represents an alternative to the traditional vaseline-gap isolation and two or three microelectrodes voltage-clamp techniques. The present chapter reviews the benefits of the silicone-clamp technique and provides updated detailed insights into its practical implementation.

Optogenetic Control of Skeletal Muscle Excitability

We assessed the feasibility of using light-activated channels and pumps to modulate the excitability of adult skeletal muscle fibers. Fibers were enzymatically isolated from FDB muscles of the mouse and studied under current- or voltage-clamp conditions using a two-microelectrode amplifier. Fibers were kept in Tyrode at a resting (or holding) potential of −90mV. Contraction was arrested by adding 40mM EGTA to the pipettes' solution. Plasmids coding channelrhodopsin-2-EYFP [CHR2; Addgene] and archaerhodopsin-3-ECFP [ARCH; subcloned from pFCK-ARCH-CFP (Addgene) into pECFPN1], were transfected into FDB fibers via in vivo electroporation. Expression was evaluated 7-10 days later. CHR2 and ARCH were excited with the 488nm and 514nm laser lines from an argon laser. Light power was controlled with an acousto-optic modulator, and focused onto selected areas (10-20μm) of the fibers via optic fibers. TPLSM imaging showed that CHR2 and ARCH were targeted to t-tubules and surface membranes. In fibers expressing CHR2, 1-3ms light pulses of increasing power elicited graded transient depolarizations until an action potential (AP) was triggered. The amplitude and FDHM of light-triggered APs were similar to those in response to current pulses. At all powers used, illuminating non-transfected fibers had no effect. Repetitive (<10Hz) pulsed illuminations elicited trains of APs with no failures. Failures at higher frequencies could be reduced by increasing the muscle fiber's input resistance (e.g. by blocking the chloride conductance with 9-AC), and/or increasing the illumination power. In voltage-clamped fibers, 40ms light pulses elicited slowly decaying inward currents with amplitudes graded with the applied illumination power. When fibers expressing ARCH were illuminated (at 514nm) otherwise supra-threshold current pulses became ineffective to trigger APs. Our results demonstrate that optogenetic tools can be used to excite or depress the excitability of adult skeletal muscle fibers.Supported by NIH/NIAMS grants AR047664, AR041802, and AR054816.

AICAR inhibits oxygen consumption by intact skeletal muscle cells in culture

Activation of 5' adenosine monophosphate-activated protein kinase (AMPK) with aminoimidazole carboxamide ribonucleotide (AICAR) increases skeletal muscle glucose uptake and fatty acid oxidation. The purpose of these experiments was to utilize AICAR to enhance palmitate consumption by mitochondria in cultured skeletal muscle cells. In these experiments, we treated C2C12 myotubes or adult single skeletal muscle fibers with varying concentrations of AICAR for different lengths of time. Surprisingly, acute AICAR exposure at most concentrations (0.25-1.5 mM), but not all (0.1 mM), modestly inhibited oxygen consumption even though AICAR increased AMPK phosphorylation. The data suggest that AICAR inhibited oxygen consumption by the cultured muscle in a non-specific manner. The results of these experiments are expected to provide valuable information to investigators interested in using AICAR in cell culture studies.

Elevated nuclear Foxo1 suppresses excitability of skeletal muscle fibers

Forkhead box O 1 (Foxo1) controls the expression of proteins that carry out processes leading to skeletal muscle atrophy, making Foxo1 of therapeutic interest in conditions of muscle wasting. The transcription of Foxo1-regulated proteins is dependent on the translocation of Foxo1 to the nucleus, which can be repressed by insulin-like growth factor-1 (IGF-1) treatment. The role of Foxo1 in muscle atrophy has been explored at length, but whether Foxo1 nuclear activity affects skeletal muscle excitation-contraction (EC) coupling has not yet been examined. Here, we use cultured adult mouse skeletal muscle fibers to investigate the effects of Foxo1 overexpression on EC coupling. Fibers expressing Foxo1-green fluorescent protein (GFP) exhibit an inability to contract, impaired propagation of action potentials, and ablation of calcium transients in response to electrical stimulation compared with fibers expressing GFP alone. Evaluation of the transverse (T)-tubule system morphology, the membranous system involved in the radial propagation of the action potential, revealed an intact T-tubule network in fibers overexpressing Foxo1-GFP. Interestingly, long-term IGF-1 treatment of Foxo1-GFP fibers, which maintains Foxo1-GFP outside the nucleus, prevented the loss of normal calcium transients, indicating that Foxo1 translocation and the atrogenes it regulates affect the expression of proteins involved in the generation and/or propagation of action potentials. A reduction in the sodium channel Nav1.4 expression in fibers overexpressing Foxo1-GFP was also observed in the absence of IGF-1. We conclude that increased nuclear activity of Foxo1 prevents the normal muscle responses to electrical stimulation and that this indicates a novel capability of Foxo1 to disable the functional activity of skeletal muscle.

Using a novel coculture model to dissect the role of intramuscular lipid load on skeletal muscle insulin responsiveness under reduced estrogen conditions

Reductions in estrogen function lead to adiposity and peripheral insulin resistance. Significant metabolic changes have been found in adipocytes and skeletal muscle with disruptions in the estrogen-signaling axis; however, it is unclear if intercellular communication exists between these tissues. The purpose of this study was to examine the impact of isolated adipocytes cocultured with single adult skeletal muscle fibers (SMF) collected from control female (SHAM) and ovariectomized female (OVX) mice. In addition, a second purpose was to compare differential effects of primary adipocytes from omental and inguinal adipose depots on SMF from these same groups. OVX SMF displayed greater lipid content, impaired insulin signaling, and lower insulin-induced glucose uptake compared with SHAM SMF without coculture. In the SHAM group, regardless of the adipose depot of origin, coculture induced greater intracellular lipid content compared with control SHAM SMF. The increased lipid in the SMF was associated with impaired insulin-induced glucose uptake when adipocytes were of omental, but not inguinal, origin. Coculture of OVX SMF with omental or inguinal adipocytes resulted in higher lipid content but no further reduction in insulin-induced glucose uptake compared with control OVX SMF. The data indicate that, in the OVX condition, there is a threshold for lipid accumulation in skeletal muscle beyond which there is no further impairment in insulin responsiveness. These results also demonstrate depot-specific effects of adipocyte exposure on skeletal muscle glucose uptake and further implicate a role for increased intracellular lipid storage in the pathogenesis of insulin resistance when estrogen levels are reduced.

Type 1 Inositol (1,4,5)-Trisphosphate Receptor Activates Ryanodine Receptor 1 to Mediate Calcium Spark Signaling in Adult Mammalian Skeletal Muscle

Functional coupling between inositol (1,4,5)-trisphosphate receptor (IP(3)R) and ryanodine receptor (RyR) represents a critical component of intracellular Ca(2+) signaling in many excitable cells; however, the role of this mechanism in skeletal muscle remains elusive. In skeletal muscle, RyR-mediated Ca(2+) sparks are suppressed in resting conditions, whereas application of transient osmotic stress can trigger activation of Ca(2+) sparks that are restricted to the periphery of the fiber. Here we show that onset of these spatially confined Ca(2+) sparks involves interaction between activation of IP(3)R and RyR near the sarcolemmal membrane. Pharmacological prevention of IP(3) production or inhibition of IP(3)R channel activity abolishes stress-induced Ca(2+) sparks in skeletal muscle. Although genetic ablation of the type 2 IP(3)R does not appear to affect Ca(2+) sparks in skeletal muscle, specific silencing of the type 1 IP(3)R leads to ablation of stress-induced Ca(2+) sparks. Our data indicate that membrane-delimited signaling involving cross-talk between IP(3)R1 and RyR1 contributes to Ca(2+) spark activation in skeletal muscle.

Elevated extracellular glucose and uncontrolled type 1 diabetes enhance NFAT5 signaling and disrupt the transverse tubular network in mouse skeletal muscle

The transcription factor nuclear factor of activated T-cells 5 (NFAT5) is a key protector from hypertonic stress in the kidney, but its role in skeletal muscle is unexamined. Here, we evaluate the effects of glucose hypertonicity and hyperglycemia on endogenous NFAT5 activity, transverse tubular system morphology and Ca(2+) signaling in adult murine skeletal muscle fibers. We found that exposure to elevated glucose (25-50 mmol/L) increased NFAT5 expression and nuclear translocation, and NFAT-driven transcriptional activity. These effects were insensitive to the inhibition of calcineurin A, but sensitive to both p38α mitogen-activated protein kinases and phosphoinositide 3-kinase-related kinase inhibition. Fibers exposed to elevated glucose exhibited disrupted transverse tubular morphology, characterized by swollen transverse tubules and an increase in longitudinal connections between adjacent transverse tubules. Ca(2+) transients elicited by a single, brief electric field stimuli were increased in amplitude in fibers challenged by elevated glucose. Muscle fibers from type 1 diabetic mice exhibited increased NFAT5 expression and transverse tubule disruptions, but no differences in electrically evoked Ca(2+) transients. Our results suggest the hypothesis that these changes in skeletal muscle could play a role in the pathophysiology of acute and severe hyperglycemic episodes commonly observed in uncontrolled diabetes.

Kinetics of nuclear-cytoplasmic translocation of Foxo1 and Foxo3A in adult skeletal muscle fibers

In skeletal muscle, the transcription factors Foxo1 and Foxo3A control expression of proteins that mediate muscle atrophy, making the nuclear concentration and nuclear-cytoplasmic movements of Foxo1 and Foxo3A of therapeutic interest in conditions of muscle wasting. Here, we use Foxo-GFP fusion proteins adenovirally expressed in cultured adult mouse skeletal muscle fibers to characterize the time course of nuclear efflux of Foxo1-GFP in response to activation of the insulin-like growth factor-1 (IGF-1)/phosphatidylinositol-3-kinase (PI3K)/Akt pathway to determine the time course of nuclear influx of Foxo1-GFP during inhibition of this pathway and to show that Akt mediates the efflux of nuclear Foxo1-GFP induced by IGF-1. Localization of endogenous Foxo1 in muscle fibers, as determined via immunocytochemistry, is consistent with that of Foxo1-GFP. Inhibition of the nuclear export carrier chromosome region maintenance 1 by leptomycin B (LMB) traps Foxo1 in the nucleus and results in a relatively rapid rate of Foxo1 nuclear accumulation, consistent with a high rate of nuclear-cytoplasmic shuttling of Foxo1 under control conditions before LMB application, with near balance of unidirectional influx and efflux. Expressed Foxo3A-GFP shuttles ∼20-fold more slowly than Foxo1-GFP. Our approach allows quantitative kinetic characterization of Foxo1 and Foxo3A nuclear-cytoplasmic movements in living muscle fibers under various experimental conditions.

Decrease of MMP-9 Activity Improves Soleus Muscle Regeneration

The regeneration of skeletal muscles relies on the function of satellite cells that are quiescent myogenic precursors associated with adult skeletal muscle fibers. Upon injury, the satellite cells are activated, divide extensively, and differentiate into new myofibers. These events are accompanied by the remodeling of the surrounding extracellular matrix, which is mediated by variety of factors, including matrix metalloproteinases (MMPs). Regeneration of certain type of muscles, such as Soleus slow twitch muscle, is often inefficient and hindered by the development of fibrosis. Here, we studied the effect of inhibition of MMP-9 and MMP-2 activity on the Soleus muscle regeneration in vivo and on the in vitro differentiation of myoblasts derived from this muscle. Using in situ and in-gel zymography, we tested the activity of these two MMPs in vivo, during regeneration of the muscle, and in vitro, during differentiation of the myoblasts. We also analyzed the histology of regenerating muscles and morphology of differentiating myoblasts. All these analyses showed that treatment with doxycycline and anti-MMP-9, but not MMP-2 antibody, significantly improved Soleus muscle regeneration and ameliorated development of excessive fibrosis, as well as delayed myoblast proliferation and differentiation in vitro.

HMGB1 mediates muscle fatigue via TLR4 - a possible mechanism for muscle fatigue in patients with inflammatory myopathies

Backgroundand objectivesIdiopathic inflammatory myopathies (IIMs) are chronic inflammatory diseases characterised by muscle weakness and the mechanisms are still unclear. High mobility group box protein 1 (HMGB1) is often found together...

Biophysical Properties of a Novel Cationic Channel in the Outer and Inner Membranes of Nuclei from Adult Skeletal Muscle Fibers

In spite of some limited progress, the physiological role of cationic ion channels within the nuclear envelope remains largely unknown. Since biophysically distinct channels have been reported within the nuclear envelope of different cells, it is likely that nuclear membrane ion homeostasis is cell-type specific. Here we provide the first identification and characterization of the biophysical properties of the most prominently observed ion channel expressed within the inner and outer membrane of nuclei from adult skeletal muscle fibers. Excised inside-out single channel recordings were obtained from individual nuclei acutely isolated from Flexor Digitorum Brevis (FDB) fibers of wild-type mice. The outer membrane of nuclei was readily accessible following isolation. For measurements of channels from the inner membrane, nuclei were treated with 1 % (w/v) sodium citrate in order to remove the outer membrane. We found that the predominant ion channel expressed in both the inner and outer nuclear membrane was a cationic channel that conducts monovalent ions with slight preference for potassium over sodium ions (a PK/PNa ∼1.22). A 10,000-fold difference in the concentration of free Ca2+ between the pipette and bath solutions did not affect the channel reversal potential in symmetric KCl (∼0 mV), indicating that Ca2+ ions permeation is negligible. The maximum conductance of the channel in the outward direction was ∼162 pS. The mean open probability (PO) was ∼0.7 and voltage-independent between −50 mV to +50 mV. We suggest that this novel monovalent cationic channel within the inner and outer membrane of skeletal muscle nuclei provides a counter-current mechanism that minimizes voltage change across the nuclear membrane. This research was supported by NIH K01 award AR060831(to V.Y.) and NIH R01 grant AR44657 (to R.T.D).

Localization and Dynamics of Phosphatidylinositol 4,5-Bisphosphate (PIP2) in Adult Skeletal Muscle Fibers

PIP2 is a precursor of important second messengers, and by itself is a direct modulator of the activity of ion channels and transporters. We investigated the localization and dynamic changes in PIP2 levels in live adult muscle fibers by expressing two PIP2 sensors: an EGFP construct of the pleckstrin homology (PH) domain of the phospholipase delta1 subunit (PH-EGFP), and a construct of the PIP2 binding domain of the tubby protein (EGFP-tubby). EGFP-tubby has higher affinity and specificity for PIP2 than PH-EGFP, and has been used to sequester PIP2 in the plasma membrane. Their respective plasmids (∗) were transfected by in vivo electroporation of FDB muscles. Two-photon laser scanning microscopy (TPLSM) shows that both EGFP-tubby and PH-EGFP are efficiently expressed in muscle fibers and that they are distributed in a double-banded pattern indicating localization at the transverse tubular system (TTS) membranes (in addition to the sarcolemma). Peak/baseline ratio analysis of TPLSM images suggests that there is a larger proportion of EGFP-tubby than PH-EGFP associated with the TTS membranes. Although the expression of PH-EGFP does not change neither the voltage-dependence nor the amplitude of Ca2+ release signals detected with Rhod-5N, the expression of EGFP-tubby apparently left-shifts their voltage-dependence by >10 mV. To further investigate the localization and dynamics of PIP2 in the TTS, we performed fluorescence resonance energy transfer (FRET) studies. The voltage-dependence of FRET signals, based on the translocation of the lipophilic anion dipicrylamine (DPA), shows that the EGFP tags of both PH-EGFP and EGFP-tubby are anchored within ∼6-9 nm of the TTS membrane. (∗)The plasmids were kindly provided to us by Dr. Tamas Balla, NICHD, NIH. This work was supported by NIH grants AR047664, AR041802, and AR054816.

Localization and Regulation of the N Terminal Splice Variant of PGC-1αin Adult Skeletal Muscle Fibers

The transcriptional coactivator peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) regulates expression of genes for metabolism and muscle fiber type. Recently, a novel splice variant of PGC-1α (NT-PGC-1α, amino acids 1–270) was cloned and found to be expressed in muscle. Here we use Flag-tagged NT-PGC-1α to examine the subcellular localization and regulation of NT-PGC-1α in skeletal muscle fibers. Flag-NT-PGC-1α is located predominantly in the myoplasm. Nuclear NT-PGC-1α can be increased by activation of protein kinase A. Activation of p38 MAPK by muscle activity or of AMPK had no effect on the subcellular distribution of NT-PGC-1α. Inhibition of CRM1-mediated export only caused relatively slow nuclear accumulation of NT-PGC-1α, indicating that nuclear export of NT-PGC-1α may be mediated by both CRM1-dependent and -independent pathways. Together these results suggest that the regulation of NT-PGC-1α in muscle fibers may be very different from that of the full-length PGC-1α, which is exclusively nuclear.

Dysferlin Forms a Dimer Mediated by the C2 Domains and the Transmembrane Domain In Vitro and in Living Cells

Dysferlin was previously identified as a key player in muscle membrane repair and its deficiency leads to the development of muscular dystrophy and cardiomyopathy. However, little is known about the oligomerization of this protein in the plasma membrane. Here we report for the first time that dysferlin forms a dimer in vitro and in living adult skeletal muscle fibers isolated from mice. Endogenous dysferlin from rabbit skeletal muscle exists primarily as a ∼460 kDa species in detergent-solubilized muscle homogenate, as shown by sucrose gradient fractionation, gel filtration and cross-linking assays. Fluorescent protein (YFP) labeled human dysferlin forms a dimer in vitro, as demonstrated by fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analyses. Dysferlin also dimerizes in living cells, as probed by fluorescence resonance energy transfer (FRET). Domain mapping FRET experiments showed that dysferlin dimerization is mediated by its transmembrane domain and by multiple C2 domains. However, C2A did not significantly contribute to dimerization; notably, this is the only C2 domain in dysferlin known to engage in a Ca-dependent interaction with cell membranes. Taken together, the data suggest that Ca-insensitive C2 domains mediate high affinity self-association of dysferlin in a parallel homodimer, leaving the Ca-sensitive C2A domain free to interact with membranes.

Fluvastatin-induced alterations of skeletal muscle function in hypercholesterolaemic rats

Although statins, the most widely used drugs in the treatment of hyperlipidaemia, are generally accepted as efficient and safe drugs their side-effects on skeletal muscle have been reported with increasing frequency. The lack of an animal model in which these side effects would consistently be observed is one of the important drawbacks in studying statin associated myopathy. To overcome this and enable the studying of the effects of fluvastatin on skeletal muscles an animal model with high blood cholesterol levels was developed. In these animals cholesterol levels rose more than seven fold (from 1.5 ± 0.1 to 10.7 ± 2.0 mmol/l; n = 15 and 16) with a dramatic increase in low density lipoprotein/high density lipoprotein ratio (from 0.29 ± 0.02 to 1.56 ± 0.17). While the latter was reversed by statin treatment, an elevation in blood creatine kinase (CK) level indicated the presence of muscle wasting. Fibers from m. extensor digitorum longus (EDL) showed significant reduction in cross sectional area in the statin treated groups. Statin treatment also decreased the proliferation and fusion of skeletal myotubes in culture. In line with this, resting intracellular calcium concentration ([Ca(2+)](i)) was reduced in statin treated satellite cells and myotubes. On the other hand, in adult skeletal muscle fibers statin treatment increased resting [Ca(2+)](i) (116 ± 4 nM vs. 151 ± 5 nM; n = 33 and 34) and decreased both twitch and tetanic force both in EDL and m. soleus. In addition, in m. soleus the duration of twitch and tetanic force was shortened. These results clearly indicate that statin administration in these animals results in a myopathy characterized by decreased muscle force and elevated plasma CK level.

New insights on sarcoplasmic reticulum calcium regulation in muscle fatigue

a consistent observation with fatigue in skeletal muscle is a decline in the amplitude of the myoplasmic Ca2+ transient, which is thought to result primarily from a reduced Ca2+ flux through the ryanodine receptor (RyR1) of the sarcoplasmic reticulum (SR) ([Fig. 1][1]). This in turn is thought to

Culture methods and initial characterization of intercostal skeletal fibers isolated from the adult mouse

Primary cultures of single adult skeletal muscle fibers dissociated from locomotor muscles adhered to glass coverslips are routine models in muscle biology. Such models allow the functional and biological properties of live muscle fibers to be probed under carefully controlled conditions. Single fiber models also make muscle fibers highly accessible to various procedures such as the expression of exogenous proteins, electric field stimulation to drive contraction and pharmacological treatment. Such isolated fiber cultures have not been established for the intercostal muscles, despite the critical function of these muscles and the role of respiration in the mortality of many muscular diseases. We have developed an adherent culture system based on single intercostal muscle fibers isolated from the adult mouse for use in physiological and biochemical studies. We also provide initial characterization and demonstrate several common techniques in this new model system with comparisons to the established Flexor Digitorum Brevis muscle primary culture model.