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.

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.

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.

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.

[Pilot study about the effectivity of an intervention based on games in nutritional status and muscle strength on children]. / Estudio piloto de la efectividad de una intervención basada en juegos sobre el estado nutricional y la fuerza muscular en niños.

INTRODUCTION: The overnutrition is a constant on developing countries; Chile is not an exception because it has a marked tendency to overweight and obesity in schoolchildren. The muscular strength has been associated with cardiovascular and metabolic health status in scholars. Effective interventions using games are needed to improve the nutritional status and physical fitness in school children. OBJECTIVE: To assess the intervention effectiveness based on games played at school time to improve the nutritional status and physical fitness in schoolchildren. METHOD: 156 students aged between 7 to 15 years, attending to two public schools with full school day, to which a pilot program was applied. This pilot program was based on dynamic recreational games during 45 minutes from monday to friday for 3 months in the largest playtime of the school day. RESULTS: At the end of the intervention, we observed a significant modification on children nutritional status, which highlights an increase in the number of children that reached the normal nutritional status (p < 0.001). We also observed a significant number of obese children who reached overweight nutritional status (p < 0.001). We also observed a decrease of leg muscular strength at the end of the study. CONCLUSIONS: We found a positive effect of a program based on dynamic recreational games in the largest school playtime, improving nutritional status. However, we didn't observed modifications in the muscular strength.

INTRODUCCIÓN: La malnutrición por exceso es una constante en países en vías de desarrollo, Chile no es una excepción ya que existe una marcada tendencia hacia el sobrepeso y obesidad en la población escolar. La fuerza muscular ha sido asociada con un adecuado estado de salud cardiovascular y metabólica en la población escolar. Se necesitan intervenciones efectivas, que utilicen herramientas lúdicas y que permitan mejorar el estado nutricional y la capacidad física de los escolares. OBJETIVO: Valorar la efectividad de una intervención basada en juegos realizados dentro de la jornada escolar de los niños para mejorar el estado nutricional y la fuerza muscular. MÉTODO: 156 escolares de 7 a 15 años, pertenecientes a dos colegios municipalizados con jornada escolar completa, participaron de un programa piloto basado en juegos recreativos dinámico durante 45 minutos de lunes a viernes durante 3 meses, en el recreo más extenso de la jornada escolar. RESULTADOS: Al finalizar la intervención se observó una modificación estadísticamente significativa en el estado nutricional, donde destaca un aumento de niños que alcanza el estado nutricional normal. En aquellos que fueron clasificados previamente como obesos se logra modificar su estado nutricional hacia sobrepeso (p < 0,001). Se observó una disminución de la fuerza muscular del tren inferior al término del estudio (p < 0,001). CONCLUSIONES: Se demostró el impacto positivo de una intervención basada en juegos recreativos dinámicos durante los recreos escolares, con modificación positiva en el estado nutricional, pero sin mejoría de la fuerza muscular de los escolares.

Estudio piloto de la efectividad de una intervención basada en juegos sobre el estado nutricional y la fuerza muscular en niños / Pilot study about the effectivity of an intervention based on games in nutritional status and muscle strength on children

Introducción: La malnutrición por exceso es una constante en países en vías de desarrollo, Chile no es una excepción ya que existe una marcada tendencia hacia el sobrepeso y obesidad en la población escolar. La fuerza muscular ha sido asociada con un adecuado estado de salud cardiovascular y metabólica en la población escolar. Se necesitan intervenciones efectivas, que utilicen herramientas lúdicas y que permitan mejorar el estado nutricional y la capacidad física de los escolares. Objetivo: Valorar la efectividad de una intervención basada en juegos realizados dentro de la jornada escolar de los niños para mejorar el estado nutricional y la fuerza muscular. Método: 156 escolares de 7 a 15 años, pertenecientes a dos colegios municipalizados con jornada escolar completa, participaron de un programa piloto basado en juegos recreativos dinámico durante 45 minutos de lunes a viernes durante 3 meses, en el recreo más extenso de la jornada escolar. Resultados: Al finalizar la intervención se observó una modificación estadísticamente significativa en el estado nutricional, donde destaca un aumento de niños que alcanza el estado nutricional normal. En aquellos que fueron clasificados previamente como obesos se logra modificar su estado nutricional hacia sobrepeso (p < 0,001). Se observó una disminución de la fuerza muscular del tren inferior al término del estudio (p < 0,001). Conclusiones: Se demostró el impacto positivo de una intervención basada en juegos recreativos dinámicos durante los recreos escolares, con modificación positiva en el estado nutricional, pero sin mejoría de la fuerza muscular de los escolares (AU)

Introduction: The overnutrition is a constant on developing countries; Chile is not an exception because it has a marked tendency to overweight and obesity in schoolchildren. The muscular strength has been associated with cardiovascular and metabolic health status in scholars. Effective interventions using games are needed to improve the nutritional status and physical fitness in school children. Objective: To assess the intervention effectiveness based on games played at school time to improve the nutritional status and physical fitness in schoolchildren. Method: 156 students aged between 7 to 15 years, attending to two public schools with full school day, to which a pilot program was applied. This pilot program was based on dynamic recreational games during 45 minutes from monday to friday for 3 months in the largest playtime of the school day. Results: At the end of the intervention, we observed a significant modification on children nutritional status, which highlights an increase in the number of children that reached the normal nutritional status (p < 0.001). We also observed a significant number of obese children who reached overweight nutritional status (p < 0.001). We also observed a decrease of leg muscular strength at the end of the study. Conclusions: We found a positive effect of a program based on dynamic recreational games in the largest school playtime, improving nutritional status. However, we didn’t observed modifications in the muscular strength (AU)