Adult Skeletal Muscle Fibers Research Articles

Functional expression of transgenic α1sDHPR channels in adult mammalian skeletal muscle fibres

We investigated the effects of the overexpression of two enhanced green fluorescent protein (EGFP)-tagged α1sDHPR variants on Ca2+ currents (ICa), charge movements (Q) and SR Ca2+ release of muscle fibres isolated from adult mice. Flexor digitorum brevis (FDB)muscles were transfected by in vivo electroporation with plasmids encoding for EGFP-α1sDHPR-wt and EGFP-α1sDHPR-T935Y (an isradipine-insensitive mutant). Two-photon laser scanning microscopy (TPLSM) was used to study the subcellular localization of transgenic proteins, while ICa, Q and Ca2+ release were studied electrophysiologically and optically under voltage-clamp conditions. TPLSM images demonstrated that most of the transgenic α1sDHPR was correctly targeted to the transverse tubular system (TTS). Immunoblotting analysis of crude extracts of transfected fibres demonstrated the synthesis of bona fide transgenic EGFP-α1sDHPR-wt in quantities comparable to that of native α1sDHPR. Though expression of both transgenic variants of the alpha subunit of the dihydropyridine receptor (α1sDHPR) resulted in ∼50% increase in Q, they surprisingly had no effect on the maximal Ca2+ conductance (gCa) nor the SR Ca2+ release. Nonetheless, fibres expressing EGFP-α1sDHPR-T935Y exhibited up to 70% isradipine-insensitive ICa (ICa-ins) with a right-shifted voltage dependence compared to that in control fibres. Interestingly, Qand SRCa2+ release also displayed right-shifted voltage dependence in fibres expressing EGFP-α1sDHPR-T935Y. In contrast, the midpoints of the voltage dependence of gCa, Q and Ca2+ release were not different from those in control fibres and in fibres expressing EGFP-α1sDHPR-wt. Overall, our results suggest that transgenic α1sDHPRs are correctly trafficked and inserted in the TTS membrane, and that a substantial fraction of the mworks as conductive Ca2+ channels capable of physiologically controlling the release of Ca2+ from the SR. A plausible corollary of this work is that the expression of transgenic variants of the α1sDHPR leads to the replacement of native channels interacting with the ryanodine receptor 1 (RyR1), thus demonstrating the feasibility of molecular remodelling of the triads in adult skeletal muscle fibres.

On the localization of ClC-1 in skeletal muscle fibers

On the localization of ClC-1 in skeletal muscle fibers

Visualization of Dysferlin Self-Interaction Using Fluorescence Resonance Energy Transfer and Fluctuation Correlation Spectroscopy

Mutations in the dysferlin gene cause progressive muscle weakness and muscle wasting. Previously dysferlin was found to play a key role in the Ca2+-activated membrane repair process of striated myocytes. However, it remains to be explored as to how dysferlin is involved in muscle membrane repair. Here we report for the first time that dysferlin forms a homodimer in living cells as well as in detergent-solubilized cell lysates. Sucrose gradient fractionation experiments showed that endogenous dysferlin from digitonin-solubilized skeletal muscle membranes migrated into heavy fractions. A self-associated fluorescent protein labeled dysferlin (ECFP-dysferlin and EYFP-dysferlin) complex in living AAV293 cells and adult skeletal muscle fibers was demonstrated by fluorescence resonance energy transfer (FRET) assay. Importantly, fluorescent protein tag at the N terminus of dysferlin did not impair the function of dysferlin in restoring the membrane repair efficiency in dysferlin-deficient skeletal muscle fibers. FRET between ECFP-dysferlin and EYFP-dysferlin in skeletal muscle fibers showed hyperbolic dependence on protein concentration, with a maximum efficiency of 35.3% and a very low KD. The observed FRET corresponds to a probe separation distance of 54.4 A, according to a computational model of dimeric FRET. Dysferlin dimerization was further confirmed by using fluorescence fluctuation spectroscopy with photon counting histogram analyses. Moreover, myoferlin, a mammalian orthologue of dysferlin, also forms dimer. The heterodimeric affinity between dysferlin and myoferlin is weaker than that of either homodimer. Finally, the transmembrane domain was identified to mediate the dimerization of dysferlin. Therefore, our data demonstrate that dysferlin forms a homodimer in cells, which may be a common feature for ferlin-1-like protein family.

Mitochondrial Fusion Dynamics in Adult Rat Skeletal Muscle

Skeletal muscle metabolism and physiology depends on mitochondria function. Impaired mitochondrial function is associated to myopathies and has been suspected to play a role in alcoholic myopathy. A recently recognized determinant of mitochondrial function is mitochondrial fusion-fission dynamics. Whether mitochondria undergo fusion events in adult skeletal muscle is unknown.We developed an assay to evaluate mitochondria fusion dynamics in adult rat FDB skeletal muscle fibers and satellite cells-derived myotubes expressing mitochondria targeted DsRed (mtDsRed) and photoactivable GFP (mtPAGFP). We used: (1) in vivo electroporated freshly isolated (2 to 24 h) fibers, (2) in vitro adenoviral infected (a) 4 days old fibers and (b) satellite cells-derived skeletal myotubes. Enzymatically isolated fibers co-expressing mtDsRed and mtPAGFP were imaged by confocal microscopy.Mito-DsRed fluorescence showed intermyofibillar mitochondria arranged in parallel pair rows following the transverse tubules direction. When we tagged the mitochondria in ∼5% of total cellular area with two photon photoactivated-GFP, rapid spreading of GFP fluorescence reveled subsets of interconnected mitochondria. Spreading and fusion events occurred mostly in longitudinal direction. Matrix fusion occurred with a frequency of 0.6±0.1 (n=14, mean ± SE) and 0.3±0.1 events/min/cell (n=12) in fresh and 4 days old fibers, respectively. Skeletal myotubes displayed 6.4±1.5 events/min/cell (n=4). We further evaluated the mitochondrial fusion dynamics in fresh fibers isolated from ethanol and pair-fed (6 month) rats. Fusion events number decreased 33% in fibers coming from ethanol-fed animals. In vitro incubation of fibers with ethanol (80mM, 48h) induced a 97% decrease in the fusion events number (n=8), regarding to control cells. Thus, adult skeletal muscle intermyofibrillar mitochondria undergo fusion that enables mixing of soluble matrix components. Skeletal muscle cells dynamics is dependent on the differentiation stage. Chronic ethanol exposure significantly suppresses fusion dynamics that might contribute to muscle mitochondria and contractile dysfunction.

Culture Methods and Initial Characterization of Calcium Homeostasis in 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 which allow monitoring of functional processes in living cultured fibers under controlled conditions. To date, such isolated fiber cultures have not been established for respiratory muscles, despite the critical function of these muscles and their role in mortality of many muscular diseases. We have developed an adherent culture system for single adult intercostal muscle fibers from the adult mouse. This system allows for monitoring functional properties of these living muscle fibers in culture exposed to various conditions (pharmacological agents, electrical field stimulation to drive muscle fiber contraction, etc.). We also provide initial characterization and demonstrate several common electrophysiological, Ca2+ imaging techniques and over-expression of foreign fluorescent fusion proteins in this new model system with comparisons to the established Flexor Digitorum Brevis muscle primary culture model. Supported by NIH-NIAMS Grants R01-AR055099, R01-AR056477 and T32-AR007592.

Caveolin-3 is a direct molecular partner of the Cav1.1 subunit of the skeletal muscle L-type calcium channel

Caveolin-3 is the striated muscle specific isoform of the scaffolding protein family of caveolins and has been shown to interact with a variety of proteins, including ion channels. Mutations in the human CAV3 gene have been associated with several muscle disorders called caveolinopathies and among these, the P104L mutation (Cav-3P104L) leads to limb girdle muscular dystrophy of type 1C characterized by the loss of sarcolemmal caveolin. There is still no clear-cut explanation as to specifically how caveolin-3 mutations lead to skeletal muscle wasting. Previous results argued in favor of a role for caveolin-3 in dihydropyridine receptor (DHPR) functional regulation and/or T-tubular membrane localization. It appeared worth closely examining such a functional link and investigating if it could result from the direct physical interaction of the two proteins. Transient expression of Cav-3P104L or caveolin-3 specific siRNAs in C2C12 myotubes both led to a significant decrease of the L-type Ca2+ channel maximal conductance. Immunolabeling analysis of adult skeletal muscle fibers revealed the colocalization of a pool of caveolin-3 with the DHPR within the T-tubular membrane. Caveolin-3 was also shown to be present in DHPR-containing triadic membrane preparations from which both proteins co-immunoprecipitated. Using GST-fusion proteins, the I–II loop of Cav1.1 was identified as the domain interacting with caveolin-3, with an apparent affinity of 60nM. The present study thus revealed a direct molecular interaction between caveolin-3 and the DHPR which is likely to underlie their functional link and whose loss might therefore be involved in pathophysiological mechanisms associated to muscle caveolinopathies.

Adherent Primary Cultures of Mouse Intercostal Muscle Fibers for Isolated Fiber Studies

Primary culture models of single adult skeletal muscle fibers dissociated from locomotor muscles adhered to glass coverslips are routine and allow monitoring of functional processes in living cultured fibers. To date, such isolated fiber cultures have not been established for respiratory muscles, despite the fact that dysfunction of core respiratory muscles leading to respiratory arrest is the most common cause of death in many muscular diseases. Here we present the first description of an adherent culture system for single adult intercostal muscle fibers from the adult mouse. This system allows for monitoring functional properties of these living muscle fibers in culture with or without electrical field stimulation to drive muscle fiber contraction at physiological or pathological respiratory firing patterns. We also provide initial characterization of these fibers, demonstrating several common techniques in this new model system in the context of the established Flexor Digitorum Brevis muscle primary culture model.

Chloride currents from the transverse tubular system in adult mammalian skeletal muscle fibers

Chloride fluxes are the main contributors to the resting conductance of mammalian skeletal muscle fibers. ClC-1, the most abundant chloride channel isoform in this preparation, is believed to be responsible for this conductance. However, the actual distribution of ClC-1 channels between the surface and transverse tubular system (TTS) membranes has not been assessed in intact muscle fibers. To investigate this issue, we voltageclamped enzymatically dissociated short fibers using a two-microelectrode configuration and simultaneously recorded chloride currents (ICl) and di-8-ANEPPS fluorescence signals to assess membrane potential changes in the TTS. Experiments were conducted in conditions that blocked all but the chloride conductance. Fibers were equilibrated with 40 or 70 mM intracellular chloride to enhance the magnitude of inward ICl, and the specific ClC-1 blocker 9-ACA was used to eliminate these currents whenever necessary. Voltage-dependent di-8-ANEPPS signals and ICl acquired before (control) and after the addition of 9-ACA were comparatively assessed. Early after the onset of stimulus pulses, di-8-ANEPPS signals under control conditions were smaller than those recorded in the presence of 9-ACA. We defined as attenuation the normalized time-dependent difference between these signals. Attenuation was discovered to be ICl dependent since its magnitude varied in close correlation with the amplitude and time course of ICl. While the properties of ICl, and those of the attenuation seen in optical records, could be simultaneously predicted by model simulations when the chloride permeability (PCl) at the surface and TTS membranes were approximately equal, the model failed to explain the optical data if PCl was precluded from the TTS membranes. Since the ratio between the areas of TTS membranes and the sarcolemma is large in mammalian muscle fibers, our results demonstrate that a significant fraction of the experimentally recorded ICl arises from TTS contributions.

FoxO1 regulates muscle fiber-type specification and inhibits calcineurin signaling during C2C12 myoblast differentiation

Adult skeletal muscle fibers can be categorized into slow-oxidative and fast-glycolytic subtypes based on specialized metabolic and contractile properties. The Forkhead box O1 (FoxO1) transcription factor governs muscle growth, metabolism, and cell differentiation, and has been shown to be involved in regulating muscle fiber type specification. However, to date, the mechanism behind FoxO1-mediated fiber type diversity is still unclear. In this article, FoxO1 being expressed preferentially in fast twitch fiber enriched muscles is reported. Moreover, the autors also detected that FoxO1 expression decreased in both fast and slow muscles from mice undergoing endurance exercise which induced a fast-to-slow fiber type transition. Using C2C12 myoblast, constitutively active FoxO1 mutant altered the proportion of muscle fiber type composition toward a fast-glycolytic phenotype and attenuated calcineurin phosphatase activity. In addition, a transcriptionally inactive FoxO1 by resveratrol triggered the expression of genes related to slow-oxidative muscle but not sufficient to induce a complete slow fiber transformation. Taken together, these results suggest that FoxO1 up-regulates fast fiber-type formation and down-regulates muscle oxidative capacity at least in part through inhibition of the calcineurin pathway.

DNA binding sites target nuclear NFATc1 to heterochromatin regions in adult skeletal muscle fibers

We have previously demonstrated that Ca²+/calcineurin-dependent dephosphorylation of the transcription factor nuclear factor of activated T cells subtype 1 (NFATc1) during repetitive skeletal muscle activity causes NFAT nuclear translocation and concentration in subnuclear NFAT foci. We now show that NFAT nuclear foci colocalize with heterochromatin regions of intense staining by DAPI or TO-PRO-3 that are present in the nucleus prior to NFATc1 nuclear entry. Nuclear NFATc1 also colocalizes with the heterochromatin markers trimethyl-histone H3 (Lys9) and heterochromatin protein 1α. Mutation of the NFATc1 DNA binding sites prevents entry and localization of NFATc1 in heterochromatin regions. However, fluorescence in situ hybridization shows that the NFAT-regulated genes for slow and fast myosin heavy chains are not localized within the heterochromatin regions. Fluorescence recovery after photobleaching shows that within a given nucleus, NFATc1 redistributes relatively rapidly (t(¹/₂) < 1 min) between NFAT foci. Nuclear export of an NFATc1 mutant not concentrated in NFAT foci is accelerated following nuclear entry during fiber activity, indicating buffering of free nuclear NFATc1 by NFATc1 within the NFAT foci. Taken together, our results suggest that NFAT foci serve as nuclear storage sites for NFATc1, allowing it to rapidly mobilize to other nuclear regions as required.

IP3-dependent, post-tetanic calcium transients induced by electrostimulation of adult skeletal muscle fibers

Tetanic electrical stimulation induces two separate calcium signals in rat skeletal myotubes, a fast one, dependent on Cav 1.1 or dihydropyridine receptors (DHPRs) and ryanodine receptors and related to contraction, and a slow signal, dependent on DHPR and inositol trisphosphate receptors (IP3Rs) and related to transcriptional events. We searched for slow calcium signals in adult muscle fibers using isolated adult flexor digitorum brevis fibers from 5–7-wk-old mice, loaded with fluo-3. When stimulated with trains of 0.3-ms pulses at various frequencies, cells responded with a fast calcium signal associated with muscle contraction, followed by a slower signal similar to one previously described in cultured myotubes. Nifedipine inhibited the slow signal more effectively than the fast one, suggesting a role for DHPR in its onset. The IP3R inhibitors Xestospongin B or C (5 µM) also inhibited it. The amplitude of post-tetanic calcium transients depends on both tetanus frequency and duration, having a maximum at 10–20 Hz. At this stimulation frequency, an increase of the slow isoform of troponin I mRNA was detected, while the fast isoform of this gene was inhibited. All three IP3R isoforms were present in adult muscle. IP3R-1 was differentially expressed in different types of muscle fibers, being higher in a subset of fast-type fibers. Interestingly, isolated fibers from the slow soleus muscle did not reveal the slow calcium signal induced by electrical stimulus. These results support the idea that IP3R-dependent slow calcium signals may be characteristic of distinct types of muscle fibers and may participate in the activation of specific transcriptional programs of slow and fast phenotype.

In vivo expression of G‐protein β1γ2 dimer in adult mouse skeletal muscle alters L‐type calcium current and excitation–contraction coupling

A number of G-protein-coupled receptors are expressed in skeletal muscle but their roles in muscle physiology and downstream effector systems remain poorly investigated. Here we explored the functional importance of the G-protein betagamma (Gbetagamma) signalling pathway on voltage-controlled Ca(2+) homeostasis in single isolated adult skeletal muscle fibres. A GFP-tagged Gbeta(1)gamma(2) dimer was expressed in vivo in mice muscle fibres. The GFP fluorescence pattern was consistent with a Gbeta(1)gamma(2) dimer localization in the transverse-tubule membrane. Membrane current and indo-1 fluorescence measurements performed under voltage-clamp conditions reveal a drastic reduction of both L-type Ca(2+) current density and of peak amplitude of the voltage-activated Ca(2+) transient in Gbeta(1)gamma(2)-expressing fibres. These effects were not observed upon expression of Gbeta(2)gamma(2), Gbeta(3)gamma(2) or Gbeta(4)gamma(2). Our data suggest that the G-protein beta(1)gamma(2) dimer may play an important regulatory role in skeletal muscle excitation-contraction coupling.

Skeletal Muscle Activity and the Fate of Myonuclei

Adult skeletal muscle fiber is a symplast multinuclear structure developed in ontogenesis by the fusion of the myoblasts (muscle progenitor cells). The nuclei of a muscle fiber (myonuclei) are those located at the periphery of fiber in the space between myofibrils and sarcolemma. In theory, a mass change in skeletal muscle during exercise or unloading may be associated with the altered myonuclear number, ratio of the transcription, and translation and proteolysis rates. Here we review the literature data related to the phenomenology and hypothetical mechanisms of the myonuclear number alterations during enhanced or reduced muscle contractile activity. In many cases (during severe muscle and systemic diseases and gravitational unloading), muscle atrophy is accompanied by a reduction in the amount of myonuclei. Such reduction is usually explained by the development of myonuclear apoptosis. A myonuclear number increase may be provided only by the satellite cell nuclei incorporation via cell fusion with the adjacent myofiber. It is believed that it is these cells which supply fiber with additional nuclei, providing postnatal growth, work hypertrophy, and repair processes. Here we discuss the possible mechanisms controlling satellite cell proliferation during exercise, functional unloading, and passive stretch.

Sodium Channel NaV1.5 Expression is Enhanced in Cultured Adult Rat Skeletal Muscle Fibers

This study analyzes changes in the distribution, electrophysiological properties, and proteic composition of voltage-gated sodium channels (Na(V)) in cultured adult rat skeletal muscle fibers. Patch clamp and molecular biology techniques were carried out in flexor digitorum brevis (FDB) adult rat skeletal muscle fibers maintained in vitro after cell dissociation with collagenase. After 4 days of culture, an increase of the Na(V)1.5 channel type was observed. This was confirmed by an increase in TTX-resistant channels and by Western blot test. These channels exhibited increased activation time constant (tau(m)) and reduced conductance, similar to what has been observed in denervated muscles in vivo, where the density of Na(V)1.5 was increasing progressively after denervation. By real-time polymerase chain reaction, we found that the expression of beta subunits was also modified, but only after 7 days of culture: increase in beta(1) without beta(4) modifications. beta(1) subunit is known to induce a negative shift of the inactivation curve, thus reducing current amplitude and duration. At day 7, tau(h) was back to normal and tau(m) still increased, in agreement with a decrease in sodium current and conductance at day 4 and normalization at day 7. Our model is a useful tool to study the effects of denervation in adult muscle fibers in vitro and the expression of sodium channels. Our data evidenced an increase in Na(V)1.5 channels and the involvement of beta subunits in the regulation of sodium current and fiber excitability.

Dihydrotestosterone activates the MAPK pathway and modulates maximum isometric force through the EGF receptor in isolated intact mouse skeletal muscle fibres

It is generally believed that steroid hormones have both genomic and non-genomic (rapid) actions. Although the latter form an important component of the physiological response of these hormones, little is known about the cellular signalling pathway(s) mediating these effects and their physiological functions in adult mammalian skeletal muscle fibres. Therefore, the primary aim of this study was to investigate the non-genomic actions of dihydrotestosterone (DHT) and their physiological role in isolated intact mammalian skeletal muscle fibre bundles. Our results show that treating the fibre bundles with physiological concentrations of DHT increases both twitch and tetanic contractions in fast twitch fibres. However, it decreases them in slow twitch fibres. These changes in force are accompanied by an increase in the phosphorylation of MAPK/ERK1/2 in both fibre types and that of regulatory myosin light chains in fast twitch fibres. Both effects were insensitive to inhibitors of Src kinase, androgen receptor, insulin-like growth factor 1 receptor and platelet-derived growth factor receptor. However, they were abolished by the MAPK/ERK1/2 kinase inhibitor PD98059 and the epidermal growth factor (EGF) receptor inhibitor tyrphostin AG 1478. In contrast, testosterone had no effect on force and increased the phosphorylation of ERK1/2 in slow twitch fibres only. From these results we conclude that sex steroids have non-genomic actions in isolated intact mammalian skeletal muscle fibres. These are mediated through the EGF receptor and one of their main physiological functions is the enhancement of force production in fast twitch skeletal muscle fibres.

Sarcoplasmic reticulum Ca2+ depletion in adult skeletal muscle fibres measured with the biosensor D1ER.

The endoplasmic/sarcoplasmic reticulum (ER/SR) plays a crucial role in cytoplasmic signalling in a variety of cells. It is particularly relevant to skeletal muscle fibres, where this organelle constitutes the main Ca2+ store for essential functions, such as contraction. In this work, we expressed the cameleon biosensor D1ER by in vivo electroporation in the mouse flexor digitorum brevis (FDB) muscle to directly assess SR Ca2+ depletion in response to electrical and pharmacological stimulation. The main conclusions are: (1) D1ER is expressed in the SR of FDB fibres according to both di-8-(amino naphthyl ethenyl pyridinium) staining experiments and reductions in the Förster resonance energy transfer signal consequent to SR Ca2+ release; (2) the amplitude of D1ER citrine/cyan fluorescent protein (CFP) ratio evoked by either 4-chloro-m-cresol (4-CmC) or electrical stimulation is directly proportional to the basal citrine/CFP ratio, which indicates that SR Ca2+ modulates ryanodine-receptor-isoform-1-mediated SR Ca2+ release in the intact muscle fibre; (3) SR Ca2+ release, measured as D1ER citrine/CFP signal, is voltage-dependent and follows a Boltzmann function; and (4) average SR Ca2+ depletion is 20% in response to 4-CmC and 6.4% in response to prolonged sarcolemmal depolarization. These results indicate that significantly depleting SR Ca2+ content under physiological conditions is difficult.

Expression of Functional Transgenic Alpha1s-DHPR Channels in Adult Mammalian Skeletal Muscle Fibers

Plasmids encoding for two variants of the α1sDHPR, tagged at the N-terminal with EGFP, were transfected into adult FDB muscles by in vivo electroporation. The wildtype variant of the channel (EGFP-α1sDHPR-wt) was rendered insensitive to dihydropyridines by site-directed mutagenesis (EGFP-α1sDHPR-T935Y). Standard and TPLSM fluorescence microscopy demonstrated that both variants were similarly expressed with high efficiency and targeted to the surface and TTS membranes of the muscle fibers. Functional evaluation of the efficiency of transgenic expression was carried out by characterizing Ca2+ currents and SR Ca2+ release in single fibers enzymatically isolated from transfected muscles. The fibers were voltage-clamped using a 2-microelectrode configuration and dialyzed internally with solutions containing 30 to 70 mM Cs-EGTA and 20 to 70 mM Cs-MOPS; they were externally bathed with isotonic TEA-Cl containing 2-12 mM Ca2+. Na and Cl currents were blocked with TTX and 9-ACA, respectively. The maximal Ca2+ conductance (gCamax), measured in 12 mM Ca2+, was 0.40 ± 0.04 (mean ± SD, n=6) in control fibers isolated from non-transfected animals. This parameter was not significantly different in fibers expressing EGFP-α1sDHPR-wt channels (0.42 ± 0.07, n=6). In contrast, fibers expressing EGFP-α1sDHPR-T935Y reported a significantly smaller gCamax of 0.27 ± 0.02 (n=5). Interestingly, after treatment with 1 μM of the specific DHPR blocker NP-200, the residual conductance was <5% in control and EGFP-α1sDHPR-wt transfected fibers, but 30-70% in fibers transfected with EGFP-α1sDHPR-T935Y. Our results suggest that, in adult skeletal muscle fibers, the functional expression of transgenic DHPR channels is done mostly at the expense of the expression of their endogenous counterparts. Supported by NIH grants Supported by NIH grants AR07664, and AR054816.

Exposure to High Glucose Alters Action Potential-Induced Ca2+ Transients and Enhances NFAT5 Expression and Nuclear Translocation in Cultured Adult Mouse Skeletal Muscle Fibers

Hyperosmolar non-ketotic syndrome (HNKS) is a life-threatening acute metabolic complication of uncontrolled type-2 diabetes characterized by severe hyperglycemia (600-900 mg/dL; 35-50 mM), dehydration, and muscle weakness; usually lasting for several days with a prognosis of 10-20% mortality. In the renal medulla, the transcription factor NFAT5 (or TonEBP) is a key regulator in protection from hypertonic stress. Here we evaluate the effects of sustained elevated extracellular glucose (50 mM of D or L-glucose for 48 hr) on Ca2+ signaling and endogenous NFAT5 activity in cultured adult murine FDB skeletal muscle fibers. Indo1-based ratiometric measurements showed no changes in resting Ca2+ in fibers challenged with high glucose. However, high glucose-exposed fibers displayed abnormal action potential-induced Ca2+ transients, manifest as a double Ca2+ spike upon a single 0.5 ms external field stimulus. Neither gadolinium (10 uM; a blocker of mechano-sensitive channels) nor apamin (1 uM; a Ca-dependent SK K+ channel blocker) reversed the effect of high glucose on AP-induced Ca2+ transients. Di-8-anepps staining revealed that the normally highly regular transverse-tubule morphology was noticeably disrupted in fibers exposed to high glucose. Thus, elevated glucose did not modify resting Ca2+ homeostasis, but affected AP-induced Ca2+ transients, possibly via a modification of T-tubule cable properties. Western blot and immunostaining assays indicated that endogenous NFAT5 was present in control FDB fibers. High glucose increased NFAT5 protein expression and augmented its nuclear translocation. NFAT-driven luciferase assays demonstrated an increase in NFAT-mediated transcriptional activity in fibers keep in high glucose conditions. In conclusion, increased expression and augmented transcriptional activity and abnormal Ca2+ signaling in skeletal muscle may play a role in the pathophysiology of the HNKS. Supported by NIH-NIAMS Grants R01-AR055099, R01-AR056477 and T32-AR007592.

Mitochondrial Superoxide Flashes in Skeletal Muscle are Linked to Metabolism

Reactive Oxygen Species (ROS) constitute important intracellular signalling molecules. Mitochondria are a well known source of ROS production, especially through the electron transport chain. In the present work, the Ca2+ biosensor mt-pericam, kindly provided by Pr Miyawaki (RIKEN, Saitama, Japan) was used as a mitochondria-targeted specific superoxide biosensor, by appropriate choice of the excitation wavelength. Mt-pericam was transfected in vivo into mouse flexor digitorum brevis adult muscles. Fibers were isolated and studied by confocal microscopy. Targeting of the biosensor was specific to the mitochondrial network with little, if any, cytosolic contamination. Fluorescence flashes were detected using excitation at 477 nm which was isosbestic for calcium. Flashes corresponded to superoxide production as shown from simultaneous records of either Mitosox (a mitochondrial superoxide probe) or Rhod-2 fluorescence signals. In addition, the superoxide scavenger Tiron decreased the flash frequency by 85%. On average, flashes have a 20 sec duration, and a F/F0 amplitude of 2. Flashes were recorded in subsarcolemmal, and intermyofibrillar mitochondria. Intermyofibrillar flashes presented three different spatial patterns: longitudinal, transversal or patch-shaped. Flash frequency was increased by application of glucose/pyruvate and decreased by inhibition of the electron transport chain with antimycin A. This is strong evidence for quantal superoxide production being intimately linked to mitochondrial metabolism. Superoxide flashes were also found to cause a decrease of the mitochondrial -potential and -free calcium, as shown by simultaneous measurements of TMRM and Rhod-2/X-Rhod-1, respectively. Together, my results show that superoxide flashes are a physiological phenomenon linked to mitochondrial metabolism that occurs in all subcellular populations of mitochondria in adult skeletal muscle fibers.

Allele Specific Gene Silencing in Autosomal-Dominant Skeletal Myopathies

Central Core Disease (CCD) and Malignant Hyperthermia (MH) are linked to single amino acid substitutions in the skeletal muscle Ca2+ release channel, the type 1 ryanodine receptor (RyR1). We focus on two autosomal dominant (AD) RyR1 mutations, Y522S (YS) and I4898T (IT), which cause MH and CCD, respectively. The AD mode of inheritance and data indicating knock-out of one RyR1 allele is well-tolerated in mice led us to hypothesize that allele-specific gene silencing (ASGS) of the mutant allele would rescue RyR1 functional defects in skeletal muscle cells from YS and IT knock-in mice.We evaluated the functional consequences of allele-specific silencing in YS and IT muscle cells using short interfering RNAs (siRNAs). To screen potential siRNAs for relative knockdown efficacy and allele specificity, we generated cDNAs encoding fusion proteins derived from wild type (WT) (Venus-Exons-3XFLAG) and either YS or IT mutation-containing (Cherry-Exons-3XHA) exons. Simultaneous transfection of these cDNAs and siRNAs into HEK293 cells and subsequent evaluation of mRNA (semi-quantitative RT-PCR) and protein levels (fluorescence microscopy and western blotting) was used to determine knockdown efficacy and allele-specificity prior to functional rescue experiments. Myotubes derived from heterozygous YS mice (YS/+) exhibit ∼4-fold increase in caffeine sensitivity (EC50 values were 0.5mM and 2.3mM for YS/+ and WT, respectively). Treatment with a YS-selective siRNA, normalized caffeine sensitivity (EC50 = 2.5mM) without decreasing peak caffeine-induced release. Similarly, YS-selective siRNA treatment rescued the increased voltage sensitivity of Ca2+ release in YS/+ myotubes determined in perforated-patch clamp experiments (VF1/2: WT = −18mV, YS/+ scrambled = −35mV, YS/+ YS-selective = −18mV). These results indicate that ASGS represents a promising approach for normalization of RyR1 function in MH and CCD. Similar functional rescue experiments in adult skeletal muscle fibers are currently underway.