Inter-individual variability in muscle responses to mechanical stress during exercise is poorly understood. Therefore, new cell culture scaffolds are needed to gain deeper insights into the cellular mechanisms underlying the influence of mechanical stress on human myogenic progenitor cells behavior. To this end, we propose the first in vitro model involving uniaxial mechanical stress applied to aligned human primary muscle-derived cells, employing a biocompatible organic-inorganic photostructurable hybrid material (OIPHM) covalently attached to a stretchable PDMS support. Using a laser printing technique with an additive photolithographic process, we optimally micropatterned the PDMS support to create longitudinal microgrooves, achieving well-aligned muscle fibers without significantly affecting their diameter. This support was biofunctionalized with peptide sequences from the ECM, which interact with cellular adhesion receptors and prevent myotube detachment induced by stretching. X-ray photoelectron spectroscopy (XPS) of biofunctionalized PDMS with RGD-derived peptide deposition revealed a significant increase in nitrogen compared to silicon, associated with the presence of a 380 nm thick layer measured by atomic force microscopy (AFM). Upon cell culture, we observed that functionalization with an RGD peptide had a beneficial impact on cell fusion rate and myotube area compared to bare PDMS. At the initiation of the stretching protocol, we observed a three-fold rapid and transient increase in RNA expression for the mechanosensitive ion channel protein piezo and a decrease in the ratio of nuclei expressing myogenin relative to the total nuclei count (43 ± 16% vs. 6 ± 6%, p < 0.01). Compared to day 0 of differentiation, stretching the myotubes induced MHC and Titin colocalization (0.66 ± 0.13 vs. 0.93 ± 0.05, p < 0.01), favoring sarcomere organization and maturation. In this study, we propose and validate an optimized protocol for culturing human primary muscle-derived cells, allowing standardized uniaxial mechanical stress with a biocompatible OIPHM covalently linked to PDMS biofunctionalized with an ECM-derived peptide, to better characterize the behavior of myogenic progenitor cells under mechanical stress in future studies.

IntroductionMuscle modeling is getting more and more attention for a better understanding of numerous pathologies. In vivo models are the most accurate but due to financial and ethical considerations, ex vivo and in vitro models tend to draw more attention. In vitro models usually focus on specific caracteristics like reproducing the Extra Cellular Matrix (ECM), inducing the alignement of the myotubes and co-culturing with other cell types. Our aim was to propose an in vitro model of differentiated and aligned human skeletal muscle fibers.MethodsTo this end, we describe the micropatterning on a PDMS [poly(dimethylsiloxane)] substrate of EETMOS [2-(3.4 epoxycyclohexylethyltrimethoxysilane)] an organic-inorganic hybrid polymer, to produce a network of parallel lines that can be stretchable to optimise différenciation. EETMOS-based resin was synthesized by sol-gel process and polymerized using UV-photolithography. Human primary myoblasts were seeded onto the microstructurated substrate to be, after proliferation, stretched to differentiate into aligned myotubes. The effect of the spacing between the parallel lines was assessed by immunofluorescence.ResultsAfter an optimization of the model, we ended up with 30μm large myotubes reached with a 75μm spacing. Then we functionalized the silicone with the use of silylated peptide ligands derived from extracellular matrix adhesion proteins to avoid detachment of the fibers from their support, during the stretching protocol (10% from L0). An improvement in the expression of sarcomere proteins was observed with stretching in relation to better differentiation of the myogenic progeniteur.ConclusionWe successfully produced aligned myotubes which afterwards, reacted to mechanical stretching. The in vitro model that we propose would be a very useful tool to evaluate in a patient, from a microbiopsy, his muscular responses to mechanical stress.

How cells respond to mechanical forces by converting them into biological signals underlie crucial cellular processes. Our understanding of mechanotransduction has been hindered by technical barriers, including limitations in our ability to effectively apply low range piconewton forces to specific mechanoreceptors on cell membranes without laborious and repetitive trials. To overcome these challenges we introduce the Nano-winch, a robust, easily assembled, programmable DNA origami-based molecular actuator. The Nano-winch is designed to manipulate multiple mechanoreceptors in parallel by exerting fine-tuned, low- piconewton forces in autonomous and remotely activated modes via adjustable single- and double-stranded DNA linkages, respectively. Nano-winches in autonomous mode can land and operate on the cell surface. Targeting the device to integrin stimulated detectable downstream phosphorylation of focal adhesion kinase, an indication that Nano-winches can be applied to study cellular mechanical processes. Remote activation mode allowed finer extension control and greater force exertion. We united remotely activated Nano-winches with single-channel bilayer experiments to directly observe the opening of a channel by mechanical force in the force responsive gated channel protein, BtuB. This customizable origami provides an instrument-free approach that can be applied to control and explore a diversity of mechanotransduction circuits on living cells.

A good quality of life requires maintaining adequate skeletal muscle mass and strength, but therapeutic agents are lacking for this. We developed a bioassay-guided fractionation approach to identify molecules with hypertrophy-promoting effect in human skeletal muscle cells. We found that extracts from rosemary leaves induce muscle cell hypertrophy. By bioassay-guided purification we identified the phenolic diterpene carnosol as the compound responsible for the hypertrophy-promoting activity of rosemary leaf extracts. We then evaluated the impact of carnosol on the different signaling pathways involved in the control of muscle cell size. We found that activation of the NRF2 signaling pathway by carnosol is not sufficient to mediate its hypertrophy-promoting effect. Moreover, carnosol inhibits the expression of the ubiquitin ligase E3 Muscle RING Finger protein-1 that plays an important role in muscle remodeling, but has no effect on the protein synthesis pathway controlled by the protein kinase B/mechanistic target of rapamycin pathway. By measuring the chymotrypsin-like activity of the proteasome, we found that proteasome activity was significantly decreased by carnosol and Muscle RING Finger 1 inactivation. These results strongly suggest that carnosol can induce skeletal muscle hypertrophy by repressing the ubiquitin-proteasome system-dependent protein degradation pathway through inhibition of the E3 ubiquitin ligase Muscle RING Finger protein-1.

Many pathologies affecting muscles (muscular dystrophies, sarcopenia, cachexia, renal insufficiency, obesity, diabetes type 2, etc.) are now clearly linked to mechanisms involving oxidative stress. In this context, there is a growing interest in exploring plants to find new natural antioxidants to prevent the appearance and the development of these muscle disorders. In this study, we investigated the antioxidant properties of Arctium lappa leaves in a model of primary human muscle cells exposed to H2O2 oxidative stress. We identified using bioassay-guided purification, onopordopicrin, a sesquiterpene lactone as the main molecule responsible for the antioxidant activity of A. lappa leaf extract. According to our findings, onopordopicrin inhibited the H2O2-mediated loss of muscle cell viability, by limiting the production of free radicals and abolishing DNA cellular damages. Moreover, we showed that onopordopicrin promoted the expression of the nuclear factor-erythroid-2-related factor 2 (Nrf2) downstream target protein heme oxygenase-1 (HO-1) in muscle cells. By using siRNA, we demonstrated that the inhibition of the expression of Nrf2 reduced the protective effect of onopordopicrin, indicating that the activation of the Nrf2/HO-1 signaling pathway mediates the antioxidant effect of onopordopicrin in primary human muscle cells. Therefore, our results suggest that onopordopicrin may be a potential therapeutic molecule to fight against oxidative stress in pathological specific muscle disorders.

Lentinus edodes (= Lentinula edodes) is an edible mushroom grown and marketed for centuries due to its nutritional and medicinal properties. L. edodes has multiple pharmacological activities as an antioxidant and anti-inflammatory. Few studies were performed taking into account the influence of culture conditions to optimize the biological properties of L. edodes on human health. Our work focused on the comparison of antioxidant capacity and anti-inflammatory activity of L. edodes fruit bodies cultivated by three mushroom producers in the French Occitanie region using the same strain in various growing conditions (organic and nonorganic). Sequential extraction was performed on freeze-dried fungal materials. All extracts have a quantifiable but moderate antioxidant activity measured via DPPH and ORAC tests. The anti-inflammatory activity of the ethanol and aqueous extracts was evaluated on a model of inflammatory macrophages. The ethanol extracts inhibit NO production in a dose-dependent manner when the cells are pretreated for 4 h with a 24 h stimulation time.

Pain, increase of body temperature, skin redness and swelling are the main symptoms of the inflammatory process. Inflammation is a well-known contributing factor to many agerelated chronic diseases. Mycotherapy is one strategy to prevent or suppress inflammation.Lentinula edodes (Shiitake) is an edible mushroom grown and marketed for centuries mainly in Asian countries due to its nutritional and medicinal properties [1-2]. Shiitake has multiple pharmacological activities as antitumor, antioxidant and anti-inflammatory effects [3-4].

Human ether-a-gogo related gene (hERG) product is the membrane potassium channel Kv11.1, which is involved in the electrical activity of the heart. As such, it is a key player in the toxicity of many drug candidates. Therefore, having this protein at hand during earlier stages of drug discovery is important for preventing later toxicity. Furthermore, having a fair quantity of functional channels may help in the development of the necessary techniques for gaining insight in this channel structure. Thus, we performed a comparative study of methods for over-expressing a mutated but functional, hERG in different orthologous hosts, such as yeast, bacteria, insect and human cell lines. We also engineered the protein to test various constructs of a functional channel. We obtained a significant amount of a functional mutant channel from HEK cells that we thoroughly characterized. The present work paves the way for the expression of large amounts of this protein, with which protein crystallization or cryo-electronic microscopy will be attempted. This will be a way to gain information on the structure of the hERG active site and its modelization to obtain data on the pauses of various reference compounds from the pharmacopeia, as well as to gain information about the thermodynamics of the hERG/ligand relationship.

Research on rosemary antioxidant activity and its potential use in human health and food applications is focused on rosemary leaves and two main bioactive compounds carnosic acid and carnosol. However, many other, not-yet identified molecules could be present, especially in rosemary by-products. In this study, we first showed that rosemary stem extract was the most efficient in protecting human skeletal muscle cells against oxidation. Then, using bioassay-guided fractionation, we identified taxodione, an abietane diterpene, as the main bioactive molecule in the rosemary stem extract. We demonstrated that taxodione protects skeletal muscle cells from hydrogen peroxide-induced cytotoxic damage (by monitoring ROS production, H2AX phosphorylation and CHOP gene expression). Moreover, we showed that taxodione reduces lipid and protein oxidation in post-mortem mice and beef muscles during refrigerated storage. In conclusion, our results indicate that taxodione extracted from rosemary stems, a cheap and unused resource of natural antioxidants, limits oxidation in muscle tissue.

The mechanical and cellular relationships between systole and diastole during left ventricular (LV) dysfunction remain to be established. LV contraction-relaxation coupling was examined during LV hypertrophy induced by chronic hypertension. Chronically instrumented pigs received angiotensin II infusion for4weeks to induce chronic hypertension (133 ± 7 mmHg vs 98 ± 5 mmHg for mean arterial pressure at Day 28 vs 0, respectively) and LV hypertrophy. LV function was investigated with the instrumentation and echocardiography for LV twist-untwist assessment before and after dobutamine infusion. The cellular mechanisms were investigated by exploring the intracellular Ca2+ handling. At Day 28, pigs exhibited LV hypertrophy with LV diastolic dysfunction (impaired LV isovolumic relaxation, increased LV end-diastolic pressure, decreased and delayed LV untwisting rate) and LV systolic dysfunction (impaired LV isovolumic contraction and twist) although LV ejection fraction was preserved. Isolated cardiomyocytes exhibited altered shortening and lengthening. Interestingly, contraction-relaxation coupling remained preserved both in vivo and in vitro during LV hypertrophy. LV systolic and diastolic dysfunctions were associated to post-translational remodeling and dysfunction of the type 2 cardiac ryanodine receptor/Ca2+ release channel (RyR2), i.e., PKA hyperphosphorylation of RyR2, depletion of calstabin 2 (FKBP12.6), RyR2 leak and hypersensitivity of RyR2 to cytosolic Ca2+ during both contraction and relaxation phases. In conclusion, LV contraction-relaxation coupling remained preserved during chronic hypertension despite LV systolic and diastolic dysfunctions. This implies that LV diastolic dysfunction is accompanied by LV systolic dysfunction. At the cellular level, this is linked to sarcoplasmic reticulum Ca2+ leak through PKA-mediated RyR2 hyperphosphorylation and depletion of its stabilizing partner.

Respiratory muscle contractile inactivity during mechanical ventilation (MV) induces diaphragm muscle weakness, a condition referred to as ventilator-induced diaphragmatic dysfunction (VIDD). Although VIDD pathophysiological mechanisms are still not fully understood, it has been recently suggested that remodeling of the sarcoplasmic reticulum (SR) calcium release channel/ryanodine receptors (RyR1) in the diaphragm is a proximal mechanism of VIDD. Here, we used piglets, a large animal model of VIDD that is more relevant to human pathophysiology, to determine whether RyR1 alterations are observed in the presence of diaphragm weakness. In piglets, diaphragm weakness induced by 72 h of respiratory muscle unloading was associated with SR RyR1 remodeling and abnormal resting SR Ca2+ leak in the diaphragm. Specifically, following controlled mechanical ventilation, diaphragm contractile function was reduced. Moreover, RyR1 macromolecular complexes were more oxidized, S-nitrosylated and phosphorylated at Ser-2844 and depleted of the stabilizing subunit calstabin1 compared with controls on adaptive support ventilation that maintains diaphragmatic contractile activity. Our study strongly supports the hypothesis that RyR1 is a potential therapeutic target in VIDD and the interest of using small molecule drugs to prevent RyR1-mediated SR Ca2+ leak induced by respiratory muscle unloading in patients who require controlled mechanical ventilation.

The mechanisms underlying ryanodine receptor (RyR) dysfunction associated with Alzheimer disease (AD) are still not well understood. Here, we show that neuronal RyR2 channels undergo post-translational remodeling (PKA phosphorylation, oxidation, and nitrosylation) in brains of AD patients, and in two murine models of AD (3 × Tg-AD, APP +/− /PS1 +/−). RyR2 is depleted of calstabin2 (KFBP12.6) in the channel complex, resulting in endoplasmic reticular (ER) calcium (Ca2+) leak. RyR-mediated ER Ca2+ leak activates Ca2+-dependent signaling pathways, contributing to AD pathogenesis. Pharmacological (using a novel RyR stabilizing drug Rycal) or genetic rescue of the RyR2-mediated intracellular Ca2+ leak improved synaptic plasticity, normalized behavioral and cognitive functions and reduced Aβ load. Genetically altered mice with congenitally leaky RyR2 exhibited premature and severe defects in synaptic plasticity, behavior and cognitive function. These data provide a mechanism underlying leaky RyR2 channels, which could be considered as potential AD therapeutic targets.

Significance Ventilator-induced diaphragmatic dysfunction (VIDD) refers to the diaphragm muscle weakness that follows prolonged controlled mechanical ventilation, impeding recovery from respiratory failure. The mechanisms underlying VIDD are still not fully understood. Using human samples and murine models of VIDD, we identify here a pathophysiological pathway involving structural and functional impairment of the ryanodine receptor (RyR1), the main sarcoplasmic reticulum (SR) Ca 2+ release channel. We demonstrate that RyR1 defects, which contribute to diaphragm muscle weakness, induced by controlled mechanical ventilation are the result of oxidative stress associated to sympathetic nervous system activation. Thus, preventing RyR1-mediated SR Ca 2+ leak may provide a novel therapeutic approach in controlled mechanical ventilation.

Long chain fatty acids bind to carnitine and form long chain acyl carnitine (LCAC), to enter into the mitochondria. They are oxidized in the mitochondrial matrix. LCAC accumulates rapidly under metabolic disorders, such as acute cardiac ischemia, chronic heart failure or diabetic cardiomyopathy. LCAC accumulation is associated with severe cardiac arrhythmia including ventricular tachycardia or fibrillation. We thus hypothesized that palmitoyl-carnitine (PC), alters mitochondrial function leading to Ca2 + dependent-arrhythmia. In isolated cardiac mitochondria from C57Bl/6 mice, application of 10 μM PC decreased adenine nucleotide translocase (ANT) activity without affecting mitochondrial permeability transition pore (mPTP) opening. Mitochondrial reactive oxygen species (ROS) production, measured with MitoSOX Red dye in isolated ventricular cardiomyocytes, increased significantly under PC application. Inhibition of ANT by bongkrekic acid (20 μM) prevented PC-induced mitochondrial ROS production. In addition, PC increased type 2 ryanodine receptor (RyR2) oxidation, S-nitrosylation and dissociation of FKBP12.6 from RyR2, and therefore increased sarcoplasmic reticulum (SR) Ca2 + leak. ANT inhibition or anti-oxidant strategy (N-acetylcysteine) prevented SR Ca2 + leak, FKBP12.6 depletion and RyR2 oxidation/S-nitrosylation induced by PC. Finally, both bongkrekic acid and NAC significantly reduced spontaneous Ca2 + wave occurrences under PC. Altogether, these results suggest that an elevation of PC disturbs ANT activity and alters Ca2 + handling in a ROS-dependent pathway, demonstrating a new pathway whereby altered FA metabolism may contribute to the development of ventricular arrhythmia in pathophysiological conditions.

Omp85 transporters mediate protein insertion into, or translocation across, membranes. They have a conserved architecture, with POTRA domains that interact with substrate proteins, a 16-stranded transmembrane β barrel, and an extracellular loop, L6, folded back in the barrel pore. Here using electrophysiology, in vivo biochemical approaches and electron paramagnetic resonance, we show that the L6 loop of the Omp85 transporter FhaC changes conformation and modulates channel opening. Those conformational changes involve breaking the conserved interaction between the tip of L6 and the inner β-barrel wall. The membrane-proximal POTRA domain also exchanges between several conformations, and the binding of FHA displaces this equilibrium. We further demonstrate a dynamic, physical communication between the POTRA domains and L6, which must take place via the β barrel. Our findings thus link all three essential components of Omp85 transporters and indicate that they operate in a concerted fashion in the transport cycle.

SummaryFhaC is an integral outer membrane protein of the whooping cough agent Bordetella pertussis that mediates the transport to the cell surface of a major virulence factor, the filamentous haemagglutinin adhesin FHA. The FHA/FhaC pair is a prototypic TpsA/TpsB system of the widespread ‘Two‐Partner Secretion’ pathway, dedicated to the transport of long extracellular proteins in various pathogenic and environmental Gram‐negative bacteria. FhaC belongs to the ubiquitous Omp85 superfamily of protein transporters. The X‐ray structure of FhaC shows that the transmembrane β‐barrel channel hypothesized to serve as the FHA‐conducting pore is obstructed by two structural elements conserved among TpsB transporters, an N‐terminal α helix and an extracellular loop. Here, we provide evidence for conformational dynamics of FhaC related to the secretion mechanism. Using paramagnetic electron resonance, electrophysiology and in vivo approaches, we showed that FhaC exchanges between open and closed conformations. The interaction with its secretory partner FHA alters this distribution of conformations. The open conformation of FhaC implies a large displacement from the channel of the N‐terminal ‘plug’ helix, which remains in the periplasm during FHA secretion. The membrane environment favours the dynamics of the TpsB transporter.

The cardiac ryanodine receptor (RyR2) is the main channel for the release of intracellular calcium during excitation-contraction coupling in cardiac muscle. Calstabin2 (i.e. FKBP12.6), a member of the FK506-binding protein family, has high affinity to RyR2, stabilizing RyR2 in the closed state and reducing its activity. The goal of the present work was first to examine the ion channel properties of RyR2 from calstabin2-/- mice reconstituted into lipid bilayers in resting condition. The second aim was to evaluate the effect of adding recombinant calstabin2 on RyR2 ion channel properties. RyR2 channels from 9 months old calstabin2 deficient mice exhibited an increase of opening probability compared to RyR2 from wild-type mice in conditions of low free cytosolic Ca2+ of 150 nM to approximate the conditions in the heart during diastole. The activity of RyR2 calstabin2-/- channels was also higher with 350 nmol/L and 700 nmol/L cytosolic Ca2+. When channels were activated by higher cytosolic Ca2+ concentration (> 700 nmol/L free Ca2+), no differences in open probability were found between RyR2 calstabin2-/- and WT channels. Addition of 1 mmol/L Na2-ATP increased the number of long openings to the maximum conductance in both native and calstabin2-/- channels with a prevalent sub-conductance activity for the depleted calstabin2 RyR2 channels. Addition of calstabin2 did neither increase nor decrease RyR2 calstabin2-/- open probability and had no effect in preventing the sub-conductance activity. This suggests that in vitro addition of calstabin2 is not sufficient to restore the RyR2/calstabin2 complex. This conclusion should further supported by the biochemical analyzes of RyR2 macromolecular complex remodeling, performed along with age-dependency.