Enhanced capacity for CaMKII signaling mitigates calcium release related contractile fatigue with high intensity exercise.

BACKGROUND: We tested whether enhancing the capacity for calcium/calmodulin-dependent protein kinase type II (CaMKII) signaling would delay fatigue of excitation-induced calcium release and improve contractile characteristics of skeletal muscle during fatiguing exercise. METHODS: Fast and slow type muscle, gastrocnemius medialis (GM) and soleus (SOL), of rats and mouse interosseus (IO) muscle fibers, were transfected with pcDNA3-based plasmids for rat α and ß CaMKII or empty controls. Levels of CaMKII, its T287-phosphorylation (pT287-CaMKII), and phosphorylation of components of calcium release and re-uptake, ryanodine receptor 1 (pS2843-RyR1) and phospholamban (pT17-PLN), were quantified biochemically. Sarcoplasmic calcium in transfected muscle fibers was monitored microscopically during trains of electrical excitation based on Fluo-4 FF fluorescence (n = 5-7). Effects of low- (n = 6) and high- (n = 8) intensity exercise on pT287-CaMKII and contractile characteristics were studied in situ. RESULTS: Co-transfection with αCaMKII-pcDNA3/ßCaMKII-pcDNA3 increased α and ßCaMKII levels in SOL (+45.8 %, +250.5 %) and GM (+40.4 %, +89.9 %) muscle fibers compared to control transfection. High-intensity exercise increased pT287-ßCaMKII and pS2843-RyR1 levels in SOL (+269 %, +151 %) and GM (+354 %, +119 %), but decreased pT287-αCaMKII and p17-PLN levels in GM compared to SOL (-76 % vs. +166 %; 0 % vs. +128 %). α/ß CaMKII overexpression attenuated the decline of calcium release in muscle fibers with repeated excitation, and mitigated exercise-induced deterioration of rates in force production, and passive force, in a muscle-dependent manner, in correlation with pS2843-RyR1 and pT17-PLN levels (|r| > 0.7). CONCLUSION: Enhanced capacity for α/ß CaMKII signaling improves fatigue-resistance of active and passive contractile muscle properties in association with RyR1- and PLN-related improvements in sarcoplasmic calcium release.

A mechano- and heat-gated two-pore domain K+ channel controls excitability in adult zebrafish skeletal muscle.

TRAAK channels are mechano-gated two-pore-domain K+ channels. Up to now, activity of these channels has been reported in neurons but not in skeletal muscle, yet an archetype of tissue challenged by mechanical stress. Using patch clamp methods on isolated skeletal muscle fibers from adult zebrafish, we show here that single channels sharing properties of TRAAK channels, i.e., selective to K+ ions, of 56 pS unitary conductance in the presence of 5 mM external K+, activated by membrane stretch, heat, arachidonic acid, and internal alkaline pH, are present in enzymatically isolated fast skeletal muscle fibers from adult zebrafish. The kcnk4b transcript encoding for TRAAK channels was cloned and found, concomitantly with activity of mechano-gated K+ channels, to be absent in zebrafish fast skeletal muscles at the larval stage but arising around 1 mo of age. The transfer of the kcnk4b gene in HEK cells and in the adult mouse muscle, that do not express functional TRAAK channels, led to expression and activity of mechano-gated K+ channels displaying properties comparable to native zebrafish TRAAK channels. In whole-cell voltage-clamp and current-clamp conditions, membrane stretch and heat led to activation of macroscopic K+ currents and to acceleration of the repolarization phase of action potentials respectively, suggesting that heat production and membrane deformation associated with skeletal muscle activity can control muscle excitability through TRAAK channel activation. TRAAK channels may represent a teleost-specific evolutionary product contributing to improve swimming performance for escaping predators and capturing prey at a critical stage of development.

PCYT2-regulated lipid biosynthesis is critical to muscle health and ageing.

Muscle degeneration is the most prevalent cause for frailty and dependency in inherited diseases and ageing. Elucidation of pathophysiological mechanisms, as well as effective treatments for muscle diseases, represents an important goal in improving human health. Here, we show that the lipid synthesis enzyme phosphatidylethanolamine cytidyltransferase (PCYT2/ECT) is critical to muscle health. Human deficiency in PCYT2 causes a severe disease with failure to thrive and progressive weakness. pcyt2-mutant zebrafish and muscle-specific Pcyt2-knockout mice recapitulate the participant phenotypes, with failure to thrive, progressive muscle weakness and accelerated ageing. Mechanistically, muscle Pcyt2 deficiency affects cellular bioenergetics and membrane lipid bilayer structure and stability. PCYT2 activity declines in ageing muscles of mice and humans, and adeno-associated virus-based delivery of PCYT2 ameliorates muscle weakness in Pcyt2-knockout and old mice, offering a therapy for individuals with a rare disease and muscle ageing. Thus, PCYT2 plays a fundamental and conserved role in vertebrate muscle health, linking PCYT2 and PCYT2-synthesized lipids to severe muscle dystrophy and ageing.

Probenecid affects muscle Ca2+ homeostasis and contraction independently from pannexin channel block.

Tight control of skeletal muscle contractile activation is secured by the excitation-contraction (EC) coupling protein complex, a molecular machinery allowing the plasma membrane voltage to control the activity of the ryanodine receptor Ca2+ release channel in the sarcoplasmic reticulum (SR) membrane. This machinery has been shown to be intimately linked to the plasma membrane protein pannexin-1 (Panx1). We investigated whether the prescription drug probenecid, a widely used Panx1 blocker, affects Ca2+ signaling, EC coupling, and muscle force. The effect of probenecid was tested on membrane current, resting Ca2+, and SR Ca2+ release in isolated mouse muscle fibers, using a combination of whole-cell voltage-clamp and Ca2+ imaging, and on electrically triggered contraction of isolated muscles. Probenecid (1 mM) induces SR Ca2+ leak at rest and reduces peak voltage-activated SR Ca2+ release and contractile force by 40%. Carbenoxolone, another Panx1 blocker, also reduces Ca2+ release, but neither a Panx1 channel inhibitory peptide nor a purinergic antagonist affected Ca2+ release, suggesting that probenecid and carbenoxolone do not act through inhibition of Panx1-mediated ATP release and consequently altered purinergic signaling. Probenecid may act by altering Panx1 interaction with the EC coupling machinery, yet the implication of another molecular target cannot be excluded. Since probenecid has been used both in the clinic and as a masking agent for doping in sports, these results should encourage evaluation of possible effects on muscle function in treated individuals. In addition, they also raise the question of whether probenecid-induced altered Ca2+ homeostasis may be shared by other tissues.

Immune-Mediated Rippling Muscle Disease Associated With Thymoma and Anti-MURC/Cavin-4 Autoantibodies.

OBJECTIVES: Rippling muscle disease (RMD) is characterized by muscle stiffness, muscle hypertrophy, and rippling muscle induced by stretching or percussion. Hereditary RMD is due to sequence variants in the CAV3 and PTRF/CAVIN1 genes encoding Caveolin-3 or Cavin-1, respectively; a few series of patients with acquired autoimmune forms of RMD (iRMD) associated with AChR antibody-positive myasthenia gravis and/or thymoma have also been described. Recently, MURC/caveolae-associated protein 4 (Cavin-4) autoantibody was identified in 8 of 10 patients without thymoma, highlighting its potential both as a biomarker and as a triggering agent of this pathology. Here, we report the case of a patient with iRMD-AchR antibody negative associated with thymoma. METHODS: We suspected a paraneoplastic origin and investigated the presence of specific autoantibodies targeting muscle antigens through a combination of Western blotting and affinity purification coupled with mass spectrometry-based proteomic approaches. RESULTS: We identified circulating MURC/Cavin-4 autoantibodies and found strong similarities between histologic features of the patient's muscle and those commonly reported in caveolinopathies. Strikingly, MURC/Cavin-4 autoantibody titer strongly decreased after tumor resection and immunotherapy correlating with complete disappearance of the rippling phenotype and full patient remission. DISCUSSION: MURC/Cavin-4 autoantibodies may play a pathogenic role in paraneoplastic iRMD associated with thymoma.

Disrupted T-tubular network accounts for asynchronous calcium release in MTM1-deficient skeletal muscle.

In mammalian skeletal muscle, the propagation of surface membrane depolarization into the interior of the muscle fibre along the transverse (T) tubular network is essential for the synchronized release of calcium from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyRs) in response to the conformational change in the voltage-sensor dihydropyridine receptors. Deficiency in 3-phosphoinositide phosphatase myotubularin (MTM1) has been reported to disrupt T-tubules, resulting in impaired SR calcium release. Here confocal calcium transients recorded in muscle fibres of MTM1-deficient mice were compared with the results from a model where propagation of the depolarization along the T-tubules was modelled mathematically with disruptions in the network assumed to modify the access and transmembrane resistance as well as the capacitance. If, in simulations, T-tubules were assumed to be partially or completely inaccessible to the depolarization and RyRs at these points to be prime for calcium-induced calcium release, all the features of measured SR calcium release could be reproduced. We conclude that the inappropriate propagation of the depolarization into the fibre interior is the initial critical cause of severely impaired SR calcium release in MTM1 deficiency, while the Ca2+ -triggered opening of RyRs provides an alleviating support to the diseased process. KEY POINTS: Myotubular myopathy is a fatal disease due to genetic deficiency in the phosphoinositide phosphatase MTM1. Although the causes are known and corresponding gene therapy strategies are being developed, there is no mechanistic understanding of the disease-associated muscle function failure. Resolving this issue is of primary interest not only for a fundamental understanding of how MTM1 is critical for healthy muscle function, but also for establishing the related cellular mechanisms most primarily or stringently affected by the disease, which are thus of potential interest as therapy targets. The mathematical modelling approach used in the present work proves that the disease-associated alteration of the plasma membrane invagination network is sufficient to explain the dysfunctions of excitation-contraction coupling, providing the first integrated quantitative framework that explains the associated contraction failure.

Superfast excitation-contraction coupling in adult zebrafish skeletal muscle fibers.

The zebrafish has emerged as a very relevant animal model for probing the pathophysiology of human skeletal muscle disorders. This vertebrate animal model displays a startle response characterized by high-frequency swimming activity powered by contraction of fast skeletal muscle fibers excited at extremely high frequencies, critical for escaping predators and capturing prey. Such intense muscle performance requires extremely fast properties of the contractile machinery but also of excitation-contraction coupling, the process by which an action potential spreading along the sarcolemma induces a change in configuration of the dihydropyridine receptors, resulting in intramembrane charge movements, which in turn triggers the release of Ca2+ from the sarcoplasmic reticulum. However, thus far, the fastest Ca2+ transients evoked by vertebrate muscle fibers has been described in muscles used to produce sounds, such as those in the toadfish swim bladder, but not in muscles used for locomotion. By performing intracellular Ca2+ measurements under voltage control in isolated fast skeletal muscle fibers from adult zebrafish and mouse, we demonstrate that fish fast muscle fibers display superfast kinetics of action potentials, intramembrane charge movements, and action potential-evoked Ca2+ transient, allowing fusion and fused sustained Ca2+ transients at frequencies of excitation much higher than in mouse fast skeletal muscle fibers and comparable to those recorded in muscles producing sounds. The present study is the first demonstration of superfast kinetics of excitation-contraction coupling in skeletal muscle allowing superfast locomotor behaviors in a vertebrate.

Mice with muscle-specific deletion of Bin1 recapitulate centronuclear myopathy and acute downregulation of dynamin 2 improves their phenotypes.

Mutations in the BIN1 (Bridging Interactor 1) gene, encoding the membrane remodeling protein amphiphysin 2, cause centronuclear myopathy (CNM) associated with severe muscle weakness and myofiber disorganization and hypotrophy. There is no available therapy, and the validation of therapeutic proof of concept is impaired by the lack of a faithful and easy-to-handle mammalian model. Here, we generated and characterized the Bin1mck-/- mouse through Bin1 knockout in skeletal muscle. Bin1mck-/- mice were viable, unlike the constitutive Bin1 knockout, and displayed decreased muscle force and most histological hallmarks of CNM, including myofiber hypotrophy and intracellular disorganization. Notably, Bin1mck-/- myofibers presented strong defects in mitochondria and T-tubule networks associated with deficient calcium homeostasis and excitation-contraction coupling at the triads, potentially representing the main pathomechanisms. Systemic injection of antisense oligonucleotides (ASOs) targeting Dnm2 (Dynamin 2), which codes for dynamin 2, a BIN1 binding partner regulating membrane fission and mutated in other forms of CNM, improved muscle force and normalized the histological Bin1mck-/- phenotypes within 5 weeks. Overall, we generated a faithful mammalian model for CNM linked to BIN1 defects and validated Dnm2 ASOs as a first translatable approach to efficiently treat BIN1-CNM.

Pannexin-1 and CaV1.1 show reciprocal interaction during excitation-contraction and excitation-transcription coupling in skeletal muscle.

One of the most important functions of skeletal muscle is to respond to nerve stimuli by contracting. This function ensures body movement but also participates in other important physiological roles, like regulation of glucose homeostasis. Muscle activity is closely regulated to adapt to different demands and shows a plasticity that relies on both transcriptional activity and nerve stimuli. These two processes, both dependent on depolarization of the plasma membrane, have so far been regarded as separated and independent processes due to a lack of evidence of common protein partners or molecular mechanisms. In this study, we reveal intimate functional interactions between the process of excitation-induced contraction and the process of excitation-induced transcriptional activity in skeletal muscle. We show that the plasma membrane voltage-sensing protein CaV1.1 and the ATP-releasing channel Pannexin-1 (Panx1) regulate each other in a reciprocal manner, playing roles in both processes. Specifically, knockdown of CaV1.1 produces chronically elevated extracellular ATP concentrations at rest, consistent with disruption of the normal control of Panx1 activity. Conversely, knockdown of Panx1 affects not only activation of transcription but also CaV1.1 function on the control of muscle fiber contraction. Altogether, our results establish the presence of bidirectional functional regulations between the molecular machineries involved in the control of contraction and transcription induced by membrane depolarization of adult muscle fibers. Our results are important for an integrative understanding of skeletal muscle function and may impact our understanding of several neuromuscular diseases.

MACF1 controls skeletal muscle function through the microtubule-dependent localization of extra-synaptic myonuclei and mitochondria biogenesis.

Skeletal muscles are composed of hundreds of multinucleated muscle fibers (myofibers) whose myonuclei are regularly positioned all along the myofiber's periphery except the few ones clustered underneath the neuromuscular junction (NMJ) at the synaptic zone. This precise myonuclei organization is altered in different types of muscle disease, including centronuclear myopathies (CNMs). However, the molecular machinery regulating myonuclei position and organization in mature myofibers remains largely unknown. Conversely, it is also unclear how peripheral myonuclei positioning is lost in the related muscle diseases. Here, we describe the microtubule-associated protein, MACF1, as an essential and evolutionary conserved regulator of myonuclei positioning and maintenance, in cultured mammalian myotubes, in Drosophila muscle, and in adult mammalian muscle using a conditional muscle-specific knockout mouse model. In vitro, we show that MACF1 controls microtubules dynamics and contributes to microtubule stabilization during myofiber's maturation. In addition, we demonstrate that MACF1 regulates the microtubules density specifically around myonuclei, and, as a consequence, governs myonuclei motion. Our in vivo studies show that MACF1 deficiency is associated with alteration of extra-synaptic myonuclei positioning and microtubules network organization, both preceding NMJ fragmentation. Accordingly, MACF1 deficiency results in reduced muscle excitability and disorganized triads, leaving voltage-activated sarcoplasmic reticulum Ca2+ release and maximal muscle force unchanged. Finally, adult MACF1-KO mice present an improved resistance to fatigue correlated with a strong increase in mitochondria biogenesis.

Detection of Ca2+ transients near ryanodine receptors by targeting fluorescent Ca2+ sensors to the triad.

In intact muscle fibers, functional properties of ryanodine receptor (RYR)-mediated sarcoplasmic reticulum (SR) Ca2+ release triggered by activation of the voltage sensor CaV1.1 have so far essentially been addressed with diffusible Ca2+-sensitive dyes. Here, we used a domain (T306) of the protein triadin to target the Ca2+-sensitive probe GCaMP6f to the junctional SR membrane, in the immediate vicinity of RYR channels, within the triad region. Fluorescence of untargeted GCaMP6f was distributed throughout the muscle fibers and experienced large Ca2+-dependent changes, with obvious kinetic delays, upon application of voltage-clamp depolarizing pulses. Conversely, T306-GCaMP6f localized to the triad and generated Ca2+-dependent fluorescence transients of lower amplitude and faster kinetics for low and intermediate levels of Ca2+ release than those of untargeted GCaMP6f. By contrast, model simulation of the spatial gradients of Ca2+ following Ca2+ release predicted limited kinetic differences under the assumptions that the two probes were present at the same concentration and suffered from identical kinetic limitations. At the spatial level, T306-GCaMP6f transients within distinct regions of a same fiber yielded a uniform time course, even at low levels of Ca2+ release activation. Similar observations were made using GCaMP6f fused to the γ1 auxiliary subunit of CaV1.1. Despite the probe's limitations, our results point out the remarkable synchronicity of voltage-dependent Ca2+ release activation and termination among individual triads and highlight the potential of the approach to visualize activation or closure of single groups of RYR channels. We anticipate targeting of improved Ca2+ sensors to the triad will provide illuminating insights into physiological normal RYR function and its dysfunction under stress or pathological conditions.

In vivo RyR1 reduction in muscle triggers a core-like myopathy.

Mutations in the RYR1 gene, encoding the skeletal muscle calcium channel RyR1, lead to congenital myopathies, through expression of a channel with abnormal permeability and/or in reduced amount, but the direct functional whole organism consequences of exclusive reduction in RyR1 amount have never been studied. We have developed and characterized a mouse model with inducible muscle specific RYR1 deletion. Tamoxifen-induced recombination in the RYR1 gene at adult age resulted in a progressive reduction in the protein amount reaching a stable level of 50% of the initial amount, and was associated with a progressive muscle weakness and atrophy. Measurement of calcium fluxes in isolated muscle fibers demonstrated a reduction in the amplitude of RyR1-related calcium release mirroring the reduction in the protein amount. Alterations in the muscle structure were observed, with fibers atrophy, abnormal mitochondria distribution and membrane remodeling. An increase in the expression level of many proteins was observed, as well as an inhibition of the autophagy process. This model demonstrates that RyR1 reduction is sufficient to recapitulate most features of Central Core Disease, and accordingly similar alterations were observed in muscle biopsies from Dusty Core Disease patients (a subtype of Central Core Disease), pointing to common pathophysiological mechanisms related to RyR1 reduction.

Divalent cations permeation in a Ca2+ non-conducting skeletal muscle dihydropyridine receptor mouse model.

In response to excitation of skeletal muscle fibers, trains of action potentials induce changes in the configuration of the dihydropyridine receptor (DHPR) anchored in the tubular membrane which opens the Ca2+ release channel in the sarcoplasmic reticulum membrane. The DHPR also functions as a voltage-gated Ca2+ channel that conducts L-type Ca2+ currents routinely recorded in mammalian muscle fibers, which role was debated for more than four decades. Recently, to allow a closer look into the role of DHPR Ca2+ influx in mammalian muscle, a knock-in (ki) mouse model (ncDHPR) carrying mutation N617D (adjacent to domain II selectivity filter E) in the DHPRα1S subunit abolishing Ca2+ permeation through the channel was generated [Dayal et al., 2017]. In the present study, the Mn2+ quenching technique was initially intended to be used on voltage-clamped muscle fibers from this mouse to determine whether Ca2+ influx through a pathway distinct from DHPR may occur to compensate for the absence of DHPR Ca2+ influx. Surprisingly, while N617D DHPR muscle fibers of the ki mouse do not conduct Ca2+, Mn2+ entry and subsequent quenching did occur because Mn2+ was able to permeate and produce L-type currents through N617D DHPR. N617D DHPR was also found to conduct Ba2+ and Ba2+ currents were strongly blocked by external Ca2+. Ba2+ permeation was smaller, current kinetics slower and Ca2+ block more potent than in wild-type DHPR. These results indicate that residue N617 when replaced by the negatively charged residue D is suitably located at entrance of the pore to trap external Ca2+ impeding in this way permeation. Because Ba2+ binds with lower affinity to D, Ba2+ currents occur, but with reduced amplitudes as compared to Ba2+ currents through wild-type channels. We conclude that mutations located outside the selectivity filter influence channel permeation and possibly channel gating in a fully differentiated skeletal muscle environment.

Differential physiological roles for BIN1 isoforms in skeletal muscle development, function and regeneration.

Skeletal muscle development and regeneration are tightly regulated processes. How the intracellular organization of muscle fibers is achieved during these steps is unclear. Here, we focus on the cellular and physiological roles of amphiphysin 2 (BIN1), a membrane remodeling protein mutated in both congenital and adult centronuclear myopathies (CNM), that is ubiquitously expressed and has skeletal muscle-specific isoforms. We created and characterized constitutive muscle-specific and inducible Bin1 homozygous and heterozygous knockout mice targeting either ubiquitous or muscle-specific isoforms. Constitutive Bin1-deficient mice died at birth from lack of feeding due to a skeletal muscle defect. T-tubules and other organelles were misplaced and altered, supporting a general early role for BIN1 in intracellular organization, in addition to membrane remodeling. Although restricted deletion of Bin1 in unchallenged adult muscles had no impact, the forced switch from the muscle-specific isoforms to the ubiquitous isoforms through deletion of the in-frame muscle-specific exon delayed muscle regeneration. Thus, ubiquitous BIN1 function is necessary for muscle development and function, whereas its muscle-specific isoforms fine tune muscle regeneration in adulthood, supporting that BIN1 CNM with congenital onset are due to developmental defects, whereas later onset may be due to regeneration defects.

Preserved Ca2+ handling and excitation-contraction coupling in muscle fibres from diet-induced obese mice.

AIMS/HYPOTHESIS: Disrupted intracellular Ca2+ handling is known to play a role in diabetic cardiomyopathy but it has also been postulated to contribute to obesity- and type 2 diabetes-associated skeletal muscle dysfunction. Still, there is so far very limited functional insight into whether, and if so to what extent, muscular Ca2+ homeostasis is affected in this situation, so as to potentially determine or contribute to muscle weakness. In differentiated muscle, force production is under the control of the excitation-contraction coupling process: upon plasma membrane electrical activity, the CaV1.1 voltage sensor/Ca2+ channel in the plasma membrane triggers opening of the ryanodine receptor Ca2+ release channel in the sarcoplasmic reticulum (SR) membrane. Opening of the ryanodine receptor triggers the rise in cytosolic Ca2+, which activates contraction while Ca2+ uptake by the SR ATPase Ca2+-pump promotes relaxation. These are the core mechanisms underlying the tight control of muscle force by neuronal electrical activity. This study aimed at characterising their inherent physiological function in a diet-induced mouse model of obesity and type 2 diabetes. METHODS: Intact muscle fibres were isolated from mice fed either with a standard chow diet or with a high-fat, high-sucrose diet generating obesity, insulin resistance and glucose intolerance. Properties of muscle fibres were investigated with a combination of whole-cell voltage-clamp electrophysiology and confocal fluorescence imaging. The integrity and density of the plasma membrane network (transverse tubules) that carries the membrane excitation throughout the muscle fibres was assessed with the dye Di-8-ANEPPS. CaV1.1 Ca2+ channel activity was studied by measuring the changes in current across the plasma membrane elicited by voltage-clamp depolarising pulses of increasing amplitude. SR Ca2+ release through ryanodine receptors was simultaneously detected with the Ca2+-sensitive dye Rhod-2 in the cytosol. CaV1.1 voltage-sensing activity was separately characterised from the properties of intra-plasma-membrane charge movement produced by short voltage-clamp depolarising pulses. Spontaneous Ca2+ release at rest was assessed with the Ca2+-sensitive dye Fluo-4. The rate of SR Ca2+ uptake was assessed from the time course of cytosolic Ca2+ recovery after the end of voltage excitation using the Ca2+-sensitive dye Fluo-4FF. The response to a fatigue-stimulation protocol was determined from the time course of decline of the peak Fluo-4FF Ca2+ transients elicited by 30 trains of 5-ms-long depolarising pulses delivered at 100 Hz. RESULTS: The transverse tubule network architecture and density were well preserved in the fibres from the obese mice. The CaV1.1 Ca2+ current and voltage-sensing properties were also largely unaffected with mean values for maximum conductance and maximum amount of charge of 234 ± 12 S/F and 30.7 ± 1.6 nC/µF compared with 196 ± 13 S/F and 32.9 ± 2.0 nC/µF in fibres from mice fed with the standard diet, respectively. Voltage-activated SR Ca2+ release through ryanodine receptors also exhibited very similar properties in the two groups with mean values for maximum rate of Ca2+ release of 76.0 ± 6.5 and 78.1 ± 4.4 µmol l-1 ms-1, in fibres from control and obese mice, respectively. The response to a fatigue protocol was also largely unaffected in fibres from the obese mice, and so were the rate of cytosolic Ca2+ removal and the spontaneous Ca2+ release activity at rest. CONCLUSIONS/INTERPRETATION: The functional properties of the main mechanisms involved in the control of muscle Ca2+ homeostasis are well preserved in muscle fibres from obese mice, at the level of both the plasma membrane and of the SR. We conclude that intracellular Ca2+ handling and excitation-contraction coupling in skeletal muscle fibres are not primary targets of obesity and type 2 diabetes. Graphical abstract.

[Unraveling the pathophysiology of Bethlem Myopathy using a unique zebrafish model for the disease]. / Étude physiopathologique de la myopathie de Bethlem à l'aide d'un modèle de poisson zèbre - 16es JSFM : Prix Master 2018.

Bethlem myopathy (BM) is a neuromuscular disease characterized by joint contractures and muscle weakness. BM is caused by mutations in one of the genes encoding one of the three α-chains of collagen VI (COLVI), a component of the skeletal muscle extracellular matrix. Nowadays, an unresolved question is to understand how alteration of COLVI located outside the muscle cells leads to functional modifications in muscle fibers. The zebrafish model col6a1Δex14 is currently the unique animal model of the disease since it is the only model to reproduce a mutation that is the most frequently found in BM patients. In patient and col6a1Δex14 zebrafish muscles, the structure of the sarcoplasmic reticulum has been found to be altered, thus suggesting dysfunction in intracellular Ca2+ handling and/or in ion channels that are known to control Ca2+ homeostasis and to play pivotal roles in muscle function and pathogenesis. Therefore, our project aims at exploring the properties of ion channels and intracellular Ca2+ regulation using electrophysiological approaches and intracellular Ca2+ measurement at rest and during activity in isolated muscle fibers from col6a1Δex14 zebrafish. On one hand, this project should contribute to decipher how alteration in an extracellular matrix component transduces pathogenic signals within muscle fiber and should possibly lead to identify therapeutic targets for this currently incurable disease. On the other hand, because functional studies on zebrafish muscle cells are scarce, this project will provide a sound database on the electrophysiological properties of this cell model.

TITLE: Étude physiopathologique de la myopathie de Bethlem à l'aide d'un modèle de poisson zèbre - 16es JSFM : Prix Master 2018. ABSTRACT: La myopathie de Bethlem (BM) est une maladie caractérisée par des rétractions et une faiblesse musculaires. Cette pathologie résulte de mutations dans un des gènes codant l'une des trois chaînes α du collagène VI (COLVI), un composant de la matrice extracellulaire musculaire squelettique. Aujourd'hui, une question non résolue est de comprendre comment l'altération de COLVI présent à l'extérieur des cellules musculaires conduit à des modifications fonctionnelles dans les fibres musculaires. Le modèle poisson zèbre col6a1Δex14 est actuellement un modèle animal unique de la BM puisqu'il est le seul à reproduire spécifiquement l'une des mutations la plus fréquemment retrouvée chez les patients. Chez les patients et le poisson col6a1Δex14, la structure du réticulum sarcoplasmique est altérée, suggérant une perturbation de l'homéostasie calcique musculaire et/ou des canaux ioniques qui, en contrôlant cette homéostasie, jouent un rôle crucial dans la fonction et la pathogenèse musculaire. Notre projet vise ainsi à étudier à l'aide de techniques électrophysiologiques et de mesure de Ca2+ les propriétés des canaux ioniques et la régulation du Ca2+ intracellulaire au repos et en activité dans la fibre musculaire du poisson col6a1Δex14. Nos recherches devraient contribuer à mieux comprendre comment la perturbation de la matrice influe sur la fonction musculaire et conduire à terme à identifier des cibles thérapeutiques pour traiter cette maladie actuellement incurable. Enfin, du fait de la rareté des études fonctionnelles sur la cellule musculaire de poisson zèbre, ce projet permettra de constituer une base de données de référence sur les propriétés électrophysiologiques de ce modèle.

Molecular determinants of homo- and heteromeric interactions of Junctophilin-1 at triads in adult skeletal muscle fibers.

In adult skeletal muscles, 2 junctophilin isoforms (JPH1 and JPH2) tether the sarcoplasmic reticulum (SR) to transverse tubule (T-tubule) membranes, generating stable membrane contact sites known as triads. JPHs are anchored to the membrane of the SR by a C-terminal transmembrane domain (TMD) and bind the T-tubule membrane through their cytosolic N-terminal region, which contains 8 lipid-binding (MORN) motifs. By combining expression of GFP-JPH1 deletion mutants in skeletal muscle fibers with in vitro biochemical experiments, we investigated the molecular determinants of JPH1 recruitment at triads in adult skeletal muscle fibers. We found that MORN motifs bind PI(4,5)P2 in the sarcolemma, but do not mediate the selective localization of JPH1 at the T-tubule compartment of triads. On the contrary, fusion proteins containing only the TMD of JPH1 were able to localize at the junctional SR compartment of the triad. Bimolecular fluorescence complementation experiments indicated that the TMD of JPH1 can form dimers, suggesting that the observed localization at triads may result from dimerization with the TMDs of resident JPH1. A second domain, capable of mediating homo- and heterodimeric interactions between JPH1 and JPH2 was identified in the cytosolic region. FRAP experiments revealed that removal of either one of these 2 domains in JPH1 decreases the association of the resulting mutant proteins with triads. Altogether, these results suggest that the ability to establish homo- and heterodimeric interactions with resident JPHs may support the recruitment and stability of newly synthesized JPHs at triads in adult skeletal muscle fibers.

Ca2+-induced sarcoplasmic reticulum Ca2+ release in myotubularin-deficient muscle fibers.

Skeletal muscle deficiency in the 3-phosphoinositide (PtdInsP) phosphatase myotubularin (MTM1) causes myotubular myopathy which is associated with severe depression of voltage-activated sarcoplasmic reticulum Ca2+ release through ryanodine receptors. In the present study we aimed at further understanding how Ca2+ release is altered in MTM1-deficient muscle fibers, at rest and during activation. While in wild-type muscle fibers, SR Ca2+ release exhibits fast stereotyped kinetics of activation and decay throughout the voltage range of activation, Ca2+ release in MTM1-deficient muscle fibers exhibits slow and unconventional kinetics at intermediate voltages, suggestive of partial loss of the normal control of ryanodine receptor Ca2+ channel activity. In addition, the diseased muscle fibers at rest exhibit spontaneous elementary Ca2+ release events at a frequency 30 times greater than that of control fibers. Eighty percent of the events have spatiotemporal properties of archetypal Ca2+ sparks while the rest take either the form of lower amplitude, longer duration Ca2+ release events or of a combination thereof. The events occur at preferred locations in the fibers, indicating spatially uneven distribution of the parameters determining spontaneous ryanodine receptor 1 opening. Spatially large Ca2+ release sources were obviously involved in some of these events, suggesting that opening of ryanodine receptors in one cluster can activate opening of ryanodine receptors in a neighboring one. Overall results demonstrate that opening of Ca2+-activated ryanodine receptors is promoted both at rest and during excitation-contraction coupling in MTM1-deficient muscle fibers. Because access to this activation mode is denied to ryanodine receptors in healthy skeletal muscle, this may play an important role in the associated disease situation.

Mammalian skeletal muscle does not express functional voltage-gated H+ channels.

High metabolic activity and existence of a large transmembrane inward electrochemical gradient for H+ at rest promote intracellular acidification of skeletal muscle. Exchangers and cotransports efficiently contend against accumulation of intracellular H+ and associated deleterious effects on muscle functions. Voltage-gated H+ channels have also been found to represent another H+ extrusion pathway in cultured muscle cells. Up to now, the skeletal muscle cell was therefore the unique vertebrate excitable cell in which voltage-gated H+ currents have been described. In this study, we show that, unlike cultured cells, single mouse muscle fibers do not generate H+ currents in response to depolarization. In contrast, expression of human voltage-gated H+ channels in mouse muscle gives rise to robust outward voltage-gated H+ currents. This result excludes that inappropriate experimental conditions may have failed to reveal voltage-gated H+ currents in control muscle. This work therefore demonstrates that fully differentiated mammalian muscle fibers do not express functional voltage-gated H+ channels and consequently can no longer be considered as the only vertebrate excitable cells exhibiting voltage-gated H+ currents.