Isolated Flexor Digitorum Brevis Research Articles

\u03b2-arrestin 1 regulates \u03b22-adrenergic receptor-mediated skeletal muscle hypertrophy and contractility

Backgroundβ2-adrenergic receptors (β2ARs) are the target of catecholamines and play fundamental roles in cardiovascular, pulmonary, and skeletal muscle physiology. An important action of β2AR stimulation on skeletal muscle is anabolic growth, which has led to the use of agonists such as clenbuterol by athletes to enhance muscle performance. While previous work has demonstrated that β2ARs can engage distinct signaling and functional cascades mediated by either G proteins or the multifunctional adaptor protein, β-arrestin, the precise role of β-arrestin in skeletal muscle physiology is not known. Here, we tested the hypothesis that agonist activation of the β2AR by clenbuterol would engage β-arrestin as a key transducer of anabolic skeletal muscle growth.MethodsThe contractile force of isolated extensor digitorum longus muscle (EDL) and calcium signaling in isolated flexor digitorum brevis (FDB) fibers were examined from the wild-type (WT) and β-arrestin 1 knockout mice (βarr1KO) followed by chronic administration of clenbuterol (1 mg/kg/d). Hypertrophic responses including fiber composition and fiber size were examined by immunohistochemical imaging. We performed a targeted phosphoproteomic analysis on clenbuterol stimulated primary cultured myoblasts from WT and βarr1KO mice. Statistical significance was determined by using a two-way analysis with Sidak’s or Tukey’s multiple comparison test and the Student’s t test.ResultsChronic administration of clenbuterol to WT mice enhanced the contractile force of EDL muscle and calcium signaling in isolated FDB fibers. In contrast, when administered to βarr1KO mice, the effect of clenbuterol on contractile force and calcium influx was blunted. While clenbuterol-induced hypertrophic responses were observed in WT mice, this response was abrogated in mice lacking β-arrestin 1. In primary cultured myoblasts, clenbuterol-stimulated phosphorylation of multiple pro-hypertrophy proteins required the presence of β-arrestin 1.ConclusionsWe have identified a previously unappreciated role for β-arrestin 1 in mediating β2AR-stimulated skeletal muscle growth and strength. We propose these findings could have important implications in the design of future pharmacologic agents aimed at reversing pathological conditions associated with skeletal muscle wasting.

Sarcolipin overexpression improves muscle energetics and reduces fatigue.

Sarcolipin (SLN) is a regulator of sarcoendoplasmic reticulum calcium ATPase in skeletal muscle. Recent studies using SLN-null mice have identified SLN as a key player in muscle thermogenesis and metabolism. In this study, we exploited a SLN overexpression (Sln(OE)) mouse model to determine whether increased SLN level affected muscle contractile properties, exercise capacity/fatigue, and metabolic rate in whole animals and isolated muscle. We found that Sln(OE) mice are more resistant to fatigue and can run significantly longer distances than wild-type (WT). Studies with isolated extensor digitorum longus (EDL) muscles showed that Sln(OE) EDL produced higher twitch force than WT. The force-frequency curves were not different between WT and Sln(OE) EDLs, but at lower frequencies the pyruvate-induced potentiation of force was significantly higher in Sln(OE) EDL. SLN overexpression did not alter the twitch and force-frequency curve in isolated soleus muscle. However, during a 10-min fatigue protocol, both EDL and soleus from Sln(OE) mice fatigued significantly less than WT muscles. Interestingly, Sln(OE) muscles showed higher carnitine palmitoyl transferase-1 protein expression, which could enhance fatty acid metabolism. In addition, lactate dehydrogenase expression was higher in Sln(OE) EDL, suggesting increased glycolytic capacity. We also found an increase in store-operated calcium entry (SOCE) in isolated flexor digitorum brevis fibers of Sln(OE) compared with WT mice. These data allow us to conclude that increased SLN expression improves skeletal muscle performance during prolonged muscle activity by increasing SOCE and muscle energetics.

Mice with RyR1 mutation (Y524S) undergo hypermetabolic response to simvastatin.

BackgroundStatins are widely used drugs for the treatment of hyperlipidemia. Though relatively safe, some individuals taking statins experience rhabdymyolysis, muscle pain, and cramping, a condition termed statin-induced myopathy (SIM). To determine if mutations in the skeletal muscle calcium (Ca2+) release channel, ryanodine receptor type 1 (RyR1), enhance the sensitivity to SIM we tested the effects of simvastatin, the statin that produces the highest incidence of SIM in humans, in mice with a mutation (Y524S, ‘YS’) in RyR1. This mutation is associated with malignant hyperthermia in humans. Exposure of mice with the YS mutation to mild elevations in environmental temperature produces a life-threatening hypermetabolic response (HMR) that is characterized by increased oxygen consumption (VO2), sustained muscle contractures, rhabdymyolysis, and elevated core body temperature.MethodsWe assessed the ability of simvastatin to induce a hypermetabolic response in the YS mice using indirect calorimetry and to alter Ca2+ release via RyR1 in isolated flexor digitorum brevis (FDB) fibers from WT and YS mice using fluorescent Ca2+ indicators. We also tested the ability of 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) to protect against the simvastatin effects.ResultsAn acute dose of simvastatin triggers a hypermetabolic response in YS mice. In isolated YS muscle fibers, simvastatin triggers an increase in cytosolic Ca2+ levels by increasing Ca2+ leak from the sarcoplasmic reticulum (SR). With higher simvastatin doses, a similar cytosolic Ca2+ increase occurs in wild type (WT) muscle fibers. Pre-treatment of YS and WT mice with AICAR prevents the response to simvastatin.ConclusionsA mutation in RyR1 associated with malignant hyperthermia increases susceptibility to an adverse response to simvastatin due to enhanced Ca2+ release from the sarcoplasmic reticulum, suggesting that RyR1 mutations may underlie enhanced susceptibility to statin-induced myopathies. Our data suggest that AICAR may be useful for treating statin myopathies.

Atypical NFATc1 Regulation in Cultured Murine Intercostal Muscles

The transcription factor NFATc1 controls the transformation of skeletal muscle fibers from fast to slow type in response to muscle activity. In the canonical pathway of NFATc1 activation, muscle activity triggers dephosphorylation of NFATc1 via the calcium dependant phosphatase calcineurin, activating NFATc1 by exposing nuclear localization sequences. Reversal of this process during periods of muscle inactivity is accomplished by constitutive kinase activity, with phosphorylation of NFAT leading to nuclear export. Using a virally expressed fluorescent NFATc1‐GFP construct, these activities can be observed as a relative accumulation or egress of NFATc1 in the nuclei of live cultured muscle fibers during electrical stimulation. Under stimulation paradigms that elicit strong nuclear influx of NFATc1 in isolated flexor digitorum brevis muscle fibers and significant influx in fibers isolated from the soleus, we have observed significant nuclear egress of NFATc1 in fibers isolated from the intercostal respiratory muscles. This suggests a possible role for activity‐inducible kinases in the intercostal muscles outside the canonical NFATc1 regulatory scheme.

No evidence for inositol 1,4,5-trisphosphate–dependent Ca2+ release in isolated fibers of adult mouse skeletal muscle

The presence and role of functional inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) in adult skeletal muscle are controversial. The current consensus is that, in adult striated muscle, the relative amount of IP3Rs is too low and the kinetics of Ca2+ release from IP3R is too slow compared with ryanodine receptors to contribute to the Ca2+ transient during excitation–contraction coupling. However, it has been suggested that IP3-dependent Ca2+ release may be involved in signaling cascades leading to regulation of muscle gene expression. We have reinvestigated IP3-dependent Ca2+ release in isolated flexor digitorum brevis (FDB) muscle fibers from adult mice. Although Ca2+ transients were readily induced in cultured C2C12 muscle cells by (a) UTP stimulation, (b) direct injection of IP3, or (c) photolysis of membrane-permeant caged IP3, no statistically significant change in calcium signal was detected in adult FDB fibers. We conclude that the IP3–IP3R system does not appear to affect global calcium levels in adult mouse skeletal muscle.

Quantal Analysis of Endplate Potentials in Mouse Flexor Digitorum Brevis Muscle.

The isolated flexor digitorum brevis (FDB) muscle from mice is extremely well suited to rapid acquisition of data and analysis of neurotransmitter release and action at neuromuscular junctions, because the muscle and its tibial nerve supply are simple to dissect and its constituent muscle fibers are short (<1 mm) and isopotential along their length. Methods are described here for dissection of FDB, stimulation of the tibial nerve, microelectrode recording from individual muscle fibers, and quantal analysis of endplate potentials (EPPs) and miniature endplate potentials (MEPPs). Curr. Protoc. Mouse Biol. 1:429-444 © 2011 by John Wiley & Sons, Inc.

Modulation of sarcoplasmic reticulum Ca2+ release in skeletal muscle expressing ryanodine receptor impaired in regulation by calmodulin and S100A1.

In vitro, calmodulin (CaM) and S100A1 activate the skeletal muscle ryanodine receptor ion channel (RyR1) at submicromolar Ca(2+) concentrations, whereas at micromolar Ca(2+) concentrations, CaM inhibits RyR1. One amino acid substitution (RyR1-L3625D) has previously been demonstrated to impair CaM binding and regulation of RyR1. Here we show that the RyR1-L3625D substitution also abolishes S100A1 binding. To determine the physiological relevance of these findings, mutant mice were generated with the RyR1-L3625D substitution in exon 74, which encodes the CaM and S100A1 binding domain of RyR1. Homozygous mutant mice (Ryr1(D/D)) were viable and appeared normal. However, single RyR1 channel recordings from Ryr1(D/D) mice exhibited impaired activation by CaM and S100A1 and impaired CaCaM inhibition. Isolated flexor digitorum brevis muscle fibers from Ryr1(D/D) mice had depressed Ca(2+) transients when stimulated by a single action potential. However, during repetitive stimulation, the mutant fibers demonstrated greater relative summation of the Ca(2+) transients. Consistently, in vivo stimulation of tibialis anterior muscles in Ryr1(D/D) mice demonstrated reduced twitch force in response to a single action potential, but greater summation of force during high-frequency stimulation. During repetitive stimulation, Ryr1(D/D) fibers exhibited slowed inactivation of sarcoplasmic reticulum Ca(2+) release flux, consistent with increased summation of the Ca(2+) transient and contractile force. Peak Ca(2+) release flux was suppressed at all voltages in voltage-clamped Ryr1(D/D) fibers. The results suggest that the RyR1-L3625D mutation removes both an early activating effect of S100A1 and CaM and delayed suppressing effect of CaCaM on RyR1 Ca(2+) release, providing new insights into CaM and S100A1 regulation of skeletal muscle excitation-contraction coupling.

The Role of Ryanodine Receptor Phosphorylation in Skeletal Muscle Excitation-Contraction Coupling

Activation of the type 1 ryanodine receptor (RyR1) during skeletal muscle excitation-contraction (EC) coupling allows the release of stored Ca2+ required for muscle contraction. While RyR1 is a well-known substrate of protein kinase A (PKA), the physiological significance of this phosphorylation event is poorly defined. PKA is known to increase the open probability of the channel in lipid bilayers by inducing phosphorylation of a specific residue (S2844). Knock-in mice harboring phosphorylation site deficient (RyR1-S2844A) and phosphomimetic (RyR1-S2844D) mutations were generated in order to probe the consequence of this phosphorylation event in skeletal muscle EC coupling. Specific force measurements were performed on extensor digitorum longus (EDL) muscles and intracellular Ca2+ release was examined in isolated flexor digitorum brevis (FDB) muscle fibers using confocal microscopy. While isoproterenol (ISO) enhanced both Ca2+ release and muscle force in wild type (WT) mice, the positive effects of ISO were abrogated in samples from RyR1-S2844A mice. This demonstrates the enhancing effects of RyR1 phosphorylation during adrenergic stimulation in skeletal muscle. While transient phosphorylation of RyR1 exerts positive effects, chronic phosphorylation, as occurs in animal heart failure models, is thought to be detrimental to muscle function. We tested this hypothesis by measuring muscle force production and intracellular Ca2+ in RyR1-S2844D mice. At 3-months of age, muscle function and Ca2+ release were similar to WT controls. However, muscle force production and intracellular Ca2+ release were both blunted by 6-months of age suggesting that a pathological Ca2+ leak induced by the S2844D mutation impairs muscle function.

DHPR α1S subunit controls skeletal muscle mass and morphogenesis

The alpha1S subunit has a dual function in skeletal muscle: it forms the L-type Ca(2+) channel in T-tubules and is the voltage sensor of excitation-contraction coupling at the level of triads. It has been proposed that L-type Ca(2+) channels might also be voltage-gated sensors linked to transcriptional activity controlling differentiation. By using the U7-exon skipping strategy, we have achieved long-lasting downregulation of alpha1S in adult skeletal muscle. Treated muscles underwent massive atrophy while still displaying significant amounts of alpha1S in the tubular system and being not paralysed. This atrophy implicated the autophagy pathway, which was triggered by neuronal nitric oxide synthase redistribution, activation of FoxO3A, upregulation of autophagy-related genes and autophagosome formation. Subcellular investigations showed that this atrophy was correlated with the disappearance of a minor fraction of alpha1S located throughout the sarcolemma. Our results reveal for the first time that this sarcolemmal fraction could have a role in a signalling pathway determining muscle anabolic or catabolic state and might act as a molecular sensor of muscle activity.

The Qγ component of intra‐membrane charge movement is present in mammalian muscle fibres, but suppressed in the absence of S100A1

S100A1 is a Ca(2+) binding protein that modulates excitation-contraction (EC) coupling in skeletal and cardiac muscle. S100A1 competes with calmodulin for binding to the skeletal muscle SR Ca(2+) release channel (the ryanodine receptor type 1, RyR1) at a site that also interacts with the C-terminal tail of the voltage sensor of EC coupling, the dihydropyridine receptor. Ablation of S100A1 leads to delayed and decreased action potential evoked Ca(2+) transients, possibly linked to altered voltage sensor activation. Here we investigate the effects of S100A1 on voltage sensor activation in skeletal muscle utilizing whole-cell patch clamp electrophysiology to record intra-membrane charge movement currents in isolated flexor digitorum brevis (FDB) muscle fibres from wild-type and S100A1 knock-out (KO) mice. In contrast to recent reports, we found that FDB fibres exhibit two distinct components of intra-membrane charge movement, an initial rapid component (Q(beta)), and a delayed, steeply voltage dependent 'hump' component (Q(gamma)) previously recorded primarily in amphibian but not mammalian fibres. Surprisingly, we found that Q(gamma) was selectively suppressed in S100A1 KO fibres, while the Q(beta) component of charge movement was unaffected. This result was specific to S100A1 and not a compensatory result of genetic manipulation, as transient intracellular application of S100A1 restored Q(gamma). Furthermore, we found that exposure to the RyR1 inhibitor dantrolene suppressed a similar component of charge movement in FDB fibres. These results shed light on voltage sensor activation in mammalian muscle, and support S100A1 as a positive regulator of the voltage sensor and Ca(2+) release channel in skeletal muscle EC coupling.

Calcium entry in skeletal muscle

Calcium entry in skeletal muscle

Decreased Skeletal Muscle Intramembrane Charge Movement And Contractile Activation In S100A1 Knock Out Mice

S100A1, a calcium binding protein expressed in skeletal and cardiac muscle, modulates Ca2+ signaling. We have previously shown that isolated flexor digitorum brevis (FDB) skeletal muscle fibers from S100A1 knock out (ko) mice exhibit reduced Ca2+ transients with delayed onset in response to single action potentials compared to wild type (wt) FDB fibers (Prosser et al, 2008). Utilizing the potentiometric dye di-8-ANEPPS, we now show no differences in action potential properties between wt and ko fibers. Using whole cell voltage clamp of single isolated fibers we examined surface and transverse tubule membrane electrical properties in wt and ko fibers. Preliminary non-linear capacitive current measurements indicate that maximum charge moved (Qmax) is less in ko than wt fibers (wt Qmax = 43.6 ± 3.4 nC/μF, ko Qmax = 32.0 ± 3.5 nC/μF; p < .05). The temporally delayed, steeply voltage dependent component of intramembrane charge movement at intermediate depolarizations (Qγ) is less pronounced in ko than in wt fibers, consistent with a steeper voltage dependence of charge moved (wt k = 7.2 ± .5, ko k = 10.8 ± 1.2, p < .05). The midpoint voltage of charge moved was not different (wt Vhalf = -26.1 ± 2.8 mV, ko Vhalf = -28.5 ± 1.5 mV). Both the amplitude and the voltage dependence of Cav1 Ca2+ currents were similar in wt and ko fibers. In vivo maximal specific force (force normalized to muscle mass) was significantly decreased in ko compared with wt muscles (wt Po = 1.52 ± .09 g/mg, ko Po = 1.11 ± .09 g/mg; p < .05). These results are consistent with muscles lacking S100A1 having decreased charge moved during membrane depolarization, causing depressed SR Ca2+ release, and ultimately decreased force generation.

Restricted Distribution of mRNAs Encoding a Sarcoplasmic Reticulum or Transverse Tubule Protein in Skeletal Myofibers

Calsequestrin (CSQ) and dihydropyridine receptor (DHPR) are muscle cell proteins that are directed into the endoplasmic reticulum (ER) during translation. The former is subsequently found in the sarcoplasmic reticulum (SR) and the latter in the transverse tubule membrane. To elucidate the potential role of mRNA targeting within muscle cells, we have analyzed the localization of CSQ and DHPR proteins and mRNAs in primary cultured rat myotubes, in skeletal muscle cryosections, and in isolated flexor digitorum brevis muscle fibers. In the myotube stage of differentiation, the mRNAs distributed throughout the cell, mimicking the distribution of the endogenous ER marker proteins. In the adult skeletal myofibers, however, both CSQ and DHPRalpha1 transcripts located perinuclearly and in cross-striations flanking Z lines beneath the sarcolemma, a distribution pattern that sharply contrasted the interfibrillar distribution of typical ER proteins. Interestingly, all nuclei of the myofibers were transcriptionally active. In summary, the mRNAs encoding either a resident SR protein or a transverse tubule protein were located beneath the sarcolemma, implying that translocation of the respective proteins to the lumen of ER takes place at this location.

Direct block of native and cloned (Kir2.1) inward rectifier K+ channels by chloroethylclonidine.

1. We have investigated the inhibition of inwardly rectifying potassium channels by the alpha-adrenergic agonist/antagonist chloroethylclonidine (CEC). We used two preparations; two-electrode voltage-clamp of rat isolated flexor digitorum brevis muscle and whole-cell patch-clamp of cell lines transfected with Kir2.1 (IRK1). 2. In skeletal muscle and at a membrane potential of -50 mV, chloroethylclonidine (CEC), an agonist at alpha2-adrenergic receptors and an antagonist at alpha1x-receptors, was found to inhibit the inward rectifier current with a Ki of 30 microM. 3. The inhibition of skeletal muscle inward rectifier current by CEC was not mimicked by clonidine, adrenaline or noradrenaline and was not sensitive to high concentrations of alpha1-(prazosin) or alpha2-(rauwolscine) antagonists. 4. The degree of current inhibition by CEC was found to vary with the membrane potential (approximately 70% block at -50 mV c.f. approximately 10% block at -190 mV). The kinetics of this voltage dependence were further investigated using recombinant inward rectifier K+ channels (Kir2.1) expressed in the MEL cell line. Using a two pulse protocol, we calculated the time constant for block to be approximately 8 s at 0 mV, and the rate of unblock was described by the relationship tau=exp((Vm+149)/22) s. 5. This block was effective when CEC was applied to either the inside or the outside of patch clamped cells, but ineffective when a polyamine binding site (aspartate 172) was mutated to asparagine. 6. The data suggest that the clonidine-like imidazoline compound, CEC, inhibits inward rectifier K+ channels independently of alpha-receptors by directly blocking the channel pore, possibly at an intracellular polyamine binding site.

Mechanosensitive ion channels in skeletal muscle from normal and dystrophic mice.

1. We examined the activity of single mechanosensitive ion channels in recordings from cell-attached patches on myoblasts, differentiated myotubes and acutely isolated skeletal muscle fibres from wild-type and mdx and dy mutant mice. The experiments were concerned with the role of these channels in the pathophysiology of muscular dystrophy. 2. The predominant form of channel activity recorded with physiological saline in the patch electrode arose from an approximately 25 pS mechanosensitive ion channel. Channel activity was similar in undifferentiated myoblasts isolated from all three strains of mice. By contrast, channel activity in mdx myotubes was approximately 3-4 times greater than in either wild-type or dy myotubes and arose from a novel mode of mechanosensitive gating. 3. Single mechanosensitive channels in acutely isolated flexor digitorum brevis fibres had properties indistinguishable from those of muscle cells grown in tissue culture. The channel open probability in mdx fibres was approximately 2 times greater than the activity recorded from wild-type fibres. The overall level of activity in fibres, however, was roughly an order of magnitude smaller than in myoblasts or myotubes. 4. Histological examination of the flexor digitorum brevis fibres from mdx mice showed no evidence of myonecrosis or regenerating fibres, suggesting that the elevated channel activity in dystrophin-deficient muscle precedes the onset of fibre degeneration. 5. An early step in the dystrophic process of the mdx mouse, which leads to pathophysiological Ca2+ entry, may be an alteration in the mechanisms that regulate mechanosensitive ion channel activity.

Critical evaluation of cytosolic calcium determination in resting muscle fibres from normal and dystrophic (mdx) mice

The fluorescent probe Fura-2 AM was used to determine cytosolic free calcium concentration in soleus muscle and in isolated flexor digitorum brevis fibres. This required a precise calibration; therefore, each calibration parameter was studied in situ. The influence of the dye concentration on calcium measurements was also examined. This precise calibration technique was used to compare absolute free calcium concentration in resting preparations from dystrophic (mdx) and control (C57) mice. We showed that the behavior of the dye was not similar in C57 and in mdx muscles. For this reason, we did not confirm the previous results that cytosolic free calcium concentration is increased in mdx muscles.

Effect of electrical stimulation on intracellular triacylglycerol in isolated skeletal muscle

The contribution of intracellular triacylglycerol (TG) as a substrate for skeletal muscle during electrical stimulation is equivocal. Therefore, the purpose of this study was to investigate the effect of electrical stimulation on the TG content in the isolated intact rat flexor digitorum brevis skeletal muscle preparation by use of two different stimulation protocols. Muscles were electrically stimulated for 1 h either continuously at 1 Hz or intermittently (30 s on, 60 s off) at 5 Hz while incubated in 21 degrees C Krebs bicarbonate buffer (pH 7.4) that contained 11 mM glucose. Control muscles were either frozen immediately after excision or incubated for 1 h. TG content was significantly decreased (P less than 0.05) compared with control concentrations in both stimulated muscle groups, with the greatest reduction (60%) occurring after 5-Hz intermittent stimulation. These data indicate that intramuscular TG is hydrolyzed in response to electrical stimulation in the isolated flexor digitorum brevis muscle preparation. In addition, the type of stimulation (higher frequency intermittent vs. lower frequency continuous) employed influences the amount of intracellular TG hydrolyzed.

Effect of adrenergic agonists on phosphoinositide breakdown in rat skeletal muscle preparations

Adrenergic regulation of phosphoinositide breakdown in rat skeletal muscle was investigated in 30-min incubations with 10 mM LiCl. In rat hemidiaphragms, prelabelled with D- myo-[2- 3H]inositol, addition of α-agonists (epinephrine, norepinephrine, phenylephrine) induced a 5-8-fold increase of [ 3H]inositol monophosphate accumulation. This could be prevented by inclusion of α-antagonists (phentolamine, prazosin). β-Agonists and/or β-antagonists had no effect. Similar experiments with isolated flexor digitorum brevis muscle fibers yielded confirmatory results. Functional integrity of β-receptor mediated processes was suggested by the β-agonist-induced increase of glucose 6-phosphate in hemidiaphragms and cAMP in fiber preparations. The results indicate that phosphoinositide breakdown in differentiated rat skeletal muscle is, at least in part, under α-adrenergic control.