Adult Mammalian Skeletal Muscle Research Articles

Mitochondrial glycerol 3-phosphate dehydrogenase promotes skeletal muscle regeneration.

While adult mammalian skeletal muscle is stable due to its post‐mitotic nature, muscle regeneration is still essential throughout life for maintaining functional fitness. During certain diseases, such as the modern pandemics of obesity and diabetes, the regeneration process becomes impaired, which leads to the loss of muscle function and contributes to the global burden of these diseases. However, the underlying mechanisms of the impairment are not well defined. Here, we identify mGPDH as a critical regulator of skeletal muscle regeneration. Specifically, it regulates myogenic markers and myoblast differentiation by controlling mitochondrial biogenesis via CaMKKβ/AMPK. mGPDH−/− attenuated skeletal muscle regeneration in vitro and in vivo, while mGPDH overexpression ameliorated dystrophic pathology in mdx mice. Moreover, in patients and animal models of obesity and diabetes, mGPDH expression in skeletal muscle was reduced, further suggesting a direct correlation between its abundance and muscular regeneration capability. Rescuing mGPDH expression in obese and diabetic mice led to a significant improvement in their muscle regeneration. Our study provides a potential therapeutic target for skeletal muscle regeneration impairment during obesity and diabetes.

Mitochondrial Glycerol 3-Phosphate Dehydrogenase Improves Skeletal Muscle Regeneration in Obesity and Diabetes

While adult mammalian skeletal muscle is stable due to its post-mitotic nature, muscle regeneration is still essential throughout life for maintaining functional fitness. During certain diseases, such as the modern pandemics of obesity and diabetes, the regeneration process becomes impaired, leading to the loss of muscle function, and contributing to the global burden of these diseases. However, the underlying mechanisms behind the impairment are not well defined. Here we identify mitochondrial glycerol 3-phosphate dehydrogenase (mGPDH) as a critical regulator of skeletal muscle regeneration. Specifically, it regulates myogenic markers and myoblast differentiation by controlling mitochondrial biogenesis via CaMKKβ/AMPK. mGPDH deletion (mGPDH-/-) attenuated skeletal muscle regeneration in vitro and in vivo while mGPDH overexpression improved the insufficient regeneration in mdx mice. Moreover, in patients and animal models of obesity and diabetes, mGPDH expression in skeletal muscle was reduced, further suggesting a direct correlation between its abundance and muscular regeneration capability. Rescuing mGPDH expression in obese (HFD, ob/ob) and diabetic (STZ) mice by AAV led to a significant improvement in their muscle regeneration (Figure 1). Our study provides a new mechanism and potential therapeutic target for skeletal muscle regeneration impairment. Disclosure H. Qu: None. Y. Zheng: None.

Functional Neuronal Differentiation of Injury-Induced Muscle-Derived Stem Cell-Like Cells with Therapeutic Implications

Mammalian skeletal muscles contain a number of heterogeneous cell populations. Our previous study characterized a unique population of myogenic lineage stem cells that can be isolated from adult mammalian skeletal muscles upon injury. These injury-induced muscle-derived stem cell-like cells (iMuSCs) displayed a multipotent state with sensitiveness and strong migration abilities. Here, we report that these iMuSCs have the capability to form neurospheres that represent multiple neural phenotypes. The induced neuronal cells expressed various neuron-specific proteins, their mRNA expression during neuronal differentiation recapitulated embryonic neurogenesis, they generated action potentials, and they formed functional synapses in vitro. Furthermore, the transplantation of iMuSCs or their cell extracts into the muscles of mdx mice (i.e., a mouse model of Duchenne Muscular Dystrophy [DMD]) could restore the morphology of their previously damaged neuromuscular junctions (NMJs), suggesting that the beneficial effects of iMuSCs may not be restricted to cell restoration alone, but also due to their transient paracrine actions. The current study reveals the essential role of iMuSCs in the restoration of NMJs related to injuries and diseases.

Ephrin-A3 promotes and maintains slow muscle fiber identity during postnatal development and reinnervation.

Each adult mammalian skeletal muscle has a unique complement of fast and slow myofibers, reflecting patterns established during development and reinforced via their innervation by fast and slow motor neurons. Existing data support a model of postnatal "matching" whereby predetermined myofiber type identity promotes pruning of inappropriate motor axons, but no molecular mechanism has yet been identified. We present evidence that fiber type-specific repulsive interactions inhibit innervation of slow myofibers by fast motor axons during both postnatal maturation of the neuromuscular junction and myofiber reinnervation after injury. The repulsive guidance ligand ephrin-A3 is expressed only on slow myofibers, whereas its candidate receptor, EphA8, localizes exclusively to fast motor endplates. Adult mice lacking ephrin-A3 have dramatically fewer slow myofibers in fast and mixed muscles, and misexpression of ephrin-A3 on fast myofibers followed by denervation/reinnervation promotes their respecification to a slow phenotype. We therefore conclude that Eph/ephrin interactions guide the fiber type specificity of neuromuscular interactions during development and adult life.

In vivo assessment of contractile strength distinguishes differential gene function in skeletal muscle of zebrafish larvae.

The accessible genetics and extensive skeletal musculature of the zebrafish make it a versatile and increasingly used model for studying muscle contraction. We here describe the development of an in vivo assay for measuring the contractile force of intact zebrafish at the larval stage. In addition, as proof of applicability, we have used this assay to quantify contractile strength of zebrafish larvae in a morphant model of deranged rbfox function. Average maximum tetanic (180 Hz) whole body forces produced by wild-type larvae at 2, 3, 4, and 5 days postfertilization amounted to 3.0, 7.2, 9.1, and 10.8 mN, respectively. To compare at potentially different stages of muscle development, we developed an immunohistological assay for empirically determining the cross-sectional area of larval trunk skeletal muscle to quantify muscle-specific force per cross-sectional area. At 4-5 days postfertilization, specific force amounts to ∼ 300 mN/mm(2), which is similar to fully developed adult mammalian skeletal muscle. We used these assays to measure contractile strength in zebrafish singly or doubly deficient for two rbfox paralogs, rbfox1l and rbfox2, which encode RNA-binding factors shown previously to modulate muscle function and muscle-specific splicing. We found rbfox2 morphants produce maximal tetanic forces similar to wild-type larvae, whereas rbfox1l morphants demonstrate significantly impaired function. rbfox1l/rbfox2 morphants are paralyzed, and their lack of contractile force production in our assay suggests that paralysis is a muscle-autonomous defect. These quantitative functional results allow measurement of muscle-specific phenotypes independent of neural input.

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.

Mammalian Non-CpG Methylation: Stem Cells and Beyond.

Although CpG dinucleotides remain the primary site for DNA methylation in mammals, there is emerging evidence that DNA methylation at non-CpG sites (CpA, CpT and CpC) is not only present in mammalian cells, but may play a unique role in the regulation of gene expression. For some time it has been known that non-CpG methylation is abundant in plants and present in mammalian embryonic stem cells, but non-CpG methylation was thought to be lost upon cell differentiation. However, recent publications have described a role for non-CpG methylation in adult mammalian somatic cells including the adult mammalian brain, skeletal muscle, and hematopoietic cells and new interest in this field has been stimulated by the availability of high throughput sequencing techniques that can accurately measure this epigenetic modification. Genome wide assays indicate that non-CpG methylation is negligible in human fetal brain, but abundant in human adult brain tissue. Genome wide measurement of non-CpG methylation coupled with RNA-Sequencing indicates that in the human adult brain non-CpG methylation levels are inversely proportional to the abundance of mRNA transcript at the associated gene. Additionally specific examples where alterations in non-CpG methylation lead to changes in gene expression have been described; in PGC1α in human skeletal muscle, IFN-γ in human T-cells and SYT11 in human brain, all of which contribute to the development of human disease.

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.

Inositol 1,4,5 Trisphosphate Receptor Type 1 (IP3R1) Activate Ryanodine Receptor (RyR1) to Mediate Ca2+ Spark Signaling in Adult Mammalian Skeletal Muscle

The functional coupling between IP3R and RyR1 in skeletal muscle have not been extensively studied. Ca2+ sparks, a RyR1-dependent Ca2+ release events are rarely observed during resting condition in adult mammalian skeletal muscle due to the inhibitory function of the EC-coupling machinery. Previously we showed that membrane deformation elicited by osmotic stress generated robust Ca2+ sparks that are restricted to the periphery of sarcolemma in skeletal muscle. Here we tested the hypothesis that lipid signaling mediated by membrane deformation activates Ca2+ release from IP3R that subsequently leads to activation of neighboring RyR, producing robust and localized Ca2+ spark events. Immunohistochemical staining showed that IP3R1 and IP3R2 were localized along the sub-sarcolemma region, coinciding with the distribution of Ca2+ sparks in skeletal muscle. It is known that transient exposure of cells to osmotic stress could elevate PI(4,5)P2 and IP3 levels inside the cell. Pharmacological interventions blocking the activation of IP3R suppress the osmotic-stress induced Ca2+ spark events in skeletal muscle. Using the IP3R2-/- mice, we showed that stress-induced Ca2+ sparks was not affected, suggesting that the type 2 IP3R is not involved in the onset of Ca2+ sparks in skeletal muscle. Specific silencing of the type 1 IP3R from the wild type or the IP3R2-/- mice led to complete ablation of the stress-induced Ca2+ sparks in skeletal muscle. Overall, our findings reveal a functional coupling between IP3R1and RyR1 in regulating intracellular Ca2+ signaling in skeletal muscle. The stress induced Ca2+ sparks likely involve a cross-talk between IP3R1 and RyR1, which must overcome the inhibitory action of the EC coupling machinery at resting condition that is essential for the tight control of muscle contraction.

Bmi1 Is Expressed in Postnatal Myogenic Satellite Cells, Controls Their Maintenance and Plays an Essential Role in Repeated Muscle Regeneration

Satellite cells are the resident stem cell population of the adult mammalian skeletal muscle and they play a crucial role in its homeostasis and in its regenerative capacity after injury. We show here that the Polycomb group (PcG) gene Bmi1 is expressed in both the Pax7 positive (+)/Myf5 negative (−) stem cell population as well as the Pax7+/Myf5+ committed myogenic progenitor population. Depletion of Pax7+/Myf5− satellite cells with reciprocal increase in Pax7+/Myf5+ as well as MyoD positive (+) cells is seen in Bmi1−/− mice leading to reduced postnatal muscle fiber size and impaired regeneration upon injury. Bmi1−/− satellite cells have a reduced proliferative capacity and fail to re-enter the cell cycle when stimulated by high serum conditions in vitro, in keeping with a cell intrinsic defect. Thus, both the in vivo and in vitro results suggest that Bmi1 plays a crucial role in the maintenance of the stem cell pool in postnatal skeletal muscle and is essential for efficient muscle regeneration after injury especially after repeated muscle injury.

Dihydrotestosterone stimulates amino acid uptake and the expression of LAT2 in mouse skeletal muscle fibres through an ERK1/2‐dependent mechanism

Dihydrotestosterone (DHT) has acute/non-genomic actions in adult mammalian skeletal muscles whose physiological functions are still poorly understood. Therefore, the primary aim of this study was to investigate the acute/non-genomic effects of DHT on amino acid uptake as well as the cellular signal transduction events underlying these actions in mouse fast- and slow-twitch skeletal muscle fibre bundles. 14C-Labelled amino acids were used to investigate the effects of DHT and testosterone (T) on amino acid uptake and pharmacological interventions were used to determine the cellular signal transduction events mediating these actions. While T had no effect on the uptake of isoleucine (Ile) and α-methylaminoisobutyric acid (MeAIB) in both fibre types, DHT increased their uptake in the fast-twitch fibre bundles. This effect was reversed by inhibitors of protein translation, the epidermal growth factor receptor (EGFR), system A, system L, mTOR and MEK. However, it was relatively insensitive to inhibitors of transcription, androgen receptors and PI3K/Akt. Additionally, DHT treatment increased the expression of LAT2 and the phosphorylation of the EGFR in the fast-twitch fibre bundles and that of ERK1/2, RSK1/2 and ATF2 in both fibre types. Also, it decreased the phosphorylation of eEF2 and increased the incorporation of Ile into proteins in both fibre types. Most of these effects were reversed by EGFR and MEK inhibitors. From these findings we suggest that another physiological function of the acute/non-genomic actions of DHT in isolated mammalian skeletal muscle fibres is to stimulate amino acid uptake. This effect is mediated through the EGFR and involves the activation of the MAPK pathway and an increase in LAT2 expression.

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

A Novel Assay of Muscle Energy Dynamics in Mechanically Loaded Enzymatically Isolated Adult Mammalian Skeletal Myocytes

Current methods and tools available to study bioenergetics in adult mammalian skeletal muscle are mostly limited to whole organ or fiber bundle preparations. However, single cell preparations remain attractive for studying mechanics and bioenergetics, because they are free from connective tissue and endothelium, allow for consistent and uniform oxygen and substrate availability and can be investigated using real-time imaging methods at high temporal and spatial resolution. The majority of research on single muscle cells is conducted in mechanically unloaded conditions in which the rate of ATP consumption is low. ATP production is generally matched to consumption and due to both cytosolic glycolysis and mitochondrial oxidative phosphorylation. Investigations of mechanically loaded muscle cells that may consume ATP at “normal” rates provide us with a preparation that enables us to explore issues related to mitochondrial function, ROS production as will as functional Ca2+ signaling and contractile performance. However, doing experiments with mechanically loaded single cells requires technically challenging manually dissection methods. We have recently developed an improvement that enables us to use enzymatically dissociated skeletal myocytes. Using MyoTak, a tenacious biological adhesive, we have been able to attach dissociated skeletal myocytes to both force transducer and length controller units. The myocytes thus prepared can be readily imaged using an inverted microscope fluorescence imaging system in confocal, widefield and multiphoton systems. We will show results that use this system to examine the force produced in enzymatically isolated mammalian skeletal myocytes with isometric contractions elicited by field stimulation, NADPH auto-fluorescence, ROS (reactive oxygen species) production during contraction.

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.

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.

Molecular Mechanism of Store Operated Ca2+ Entry in Adult Mammalian Skeletal Muscle

In skeletal muscle, store-operated Ca2+ entry (SOCE) is a trans-sarcolemmal Ca2+ influx pathway activated when sarcoplasmic reticulum (SR) Ca2+ stores are depleted. However, the mechanism of activation and physiological role of SOCE in adult skeletal muscle remains largely unknown. We recently reported that both STIM1 and Orai1 proteins are required for SOCE in skeletal myotubes. Here we investigated the mechanism of SOCE in adult skeletal muscles using mouse FDB fibers following electroporation of either cherry-tagged dnOrai1 cDNA or STIM1 siRNAs. Using a Mn2+ quench assay, we found that thapsigargin-induced SR Ca2+ store depletion activates a Ca2+ influx pathway in adult FDB fibers that is inhibited by: 1. multiple SOCE channel blockers (La3+, BTP-2 or SK&F96365) (3321±598 counts/sec, n=16 and 477±274 counts/sec, n=4 in the absence and presence of La3+, respectively), 2. expression of cherry-tagged dnOrai1 (571±130 counts/sec, n=12), or 3. STIM1 knockdown (7565±1590 counts/sec, n=3 and 217±247 counts/sec, n=5 in control and after STIM1 knockdown, respectively). To further assess the role of SOCE in muscle, we generated skeletal muscle-specific HA-tagged dominant negative Orai1 transgenic mice (HSAdnOrai) using a transgene driven by the human skeletal muscle actin (HSA) promoter (provided by Dr. J. Molkentin). HSAdnOrai mice survive beyond weaning and develop/breed normally. Western blot analysis using an HA antibody confirmed dnOrai1 transgene expression in skeletal muscle, but not in heart, lung, brain, spleen kidney, or liver. Primary myotubes derived from HSAdnOrai mice show significantly decreased SOCE following store depletion as assessed in Mn2+ quench (>92%) and Ca2+ influx (>95%) assays These results demonstrate that STIM1-Orai1 coupling mediates SOCE in adult skeletal muscle and that HSAdnOrai transgenic mice are a valuable tool for future studies of the physiological role of SOCE in skeletal muscle.

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.

Adaptive change in electrically stimulated muscle: A framework for the design of clinical protocols

Adult mammalian skeletal muscles have a remarkable capacity for adapting to increased use. Although this behavior is familiar from the changes brought about by endurance exercise, it is seen to a much greater extent in the response to long-term neuromuscular stimulation. The associated phenomena include a markedly increased resistance to fatigue, and this is the key to several clinical applications. However, a more rational basis is needed for designing regimes of stimulation that are conducive to an optimal outcome. In this review I examine relevant factors, such as the amount, frequency, and duty cycle of stimulation, the influence of force generation, and the animal model. From these considerations a framework emerges for the design of protocols that yield an overall functional profile appropriate to the application. Three contrasting examples illustrate the issues that need to be addressed clinically.

Calmodulin in adult mammalian skeletal muscle: localization and effect on sarcoplasmic reticulum Ca2+ release

Calmodulin is a ubiquitous Ca2+ binding protein that binds to ryanodine rectors (RyR) and is thought to modulate its activity. Here we evaluated the effects of recombinant calmodulin on the rate of occurrence and spatial properties of Ca2+ sparks as an assay of activation in saponin-permeabilized mouse myofibers. Control myofibers exhibited a time-dependent increase and subsequent decrease in spark frequency. Recombinant wild-type calmodulin prevented the time-dependent appearance of Ca2+ sparks and decreased the derived Ca2+ flux from the sarcoplasmic reticulum during a spark by approximately 37%. A recombinant Ca2+-insensitive form of calmodulin resulted in an instantaneous increase in spark frequency as well as an increase in the derived Ca2+ flux by approximately 24%. Endogenous calmodulin was found to primarily localize to the Z-line. Surprisingly, removal of endogenous calmodulin did not alter the time dependence of Ca2+ spark appearance. These results indicate that calmodulin may not be essential for RyR1-dependent Ca2+ release in adult mammalian skeletal muscle.