Piezo1 mechanosensitive ion channels convert mechanical stimuli into biochemical signals across diverse tissues, yet their role in the contractile function of adult skeletal muscle remains unclear. Here, we demonstrate that Piezo1 regulates skeletal muscle mechanics through a channel-independent mechanism that tunes the length-tension relationship. We examined the effects of pharmacological modulation using the Piezo1 agonist Yoda1 and antagonist Dooku1 in individual muscle fibers from wild-type mice and from muscles with reduced Piezo1 expression (anti-Piezo1 shRNA) using calcium influx and electrophysiological assays. Ex vivo force measurements were performed on these muscles and compared with the dystrophic mdx model. Piezo1 activation had no effect on force at resting length, whereas its inhibition significantly reduced contractile force at stretched lengths, indicating a selective role in length-dependent force regulation. This effect was independent of extracellular calcium and diminished by Piezo1 knockdown. This reduction was absent in mdx muscle, demonstrating dependence on an intact dystrophin-associated cytoskeleton. These findings identify Piezo1 as a previously unrecognized regulator of muscle mechanical performance that operates independently of ion conduction. Our results uncover a mechanobiological interface between Piezo1 and cytoskeletal integrity, expanding current concepts of muscle mechanoregulation and highlighting Piezo1 as a potential therapeutic target for improving muscle function.

Cell fusion requires the activity of several phagocytic receptors and the temporary exposure of phosphatidylserine (PS) on the surface of viable myoblasts. Recently, we reported that these receptors turn myoblasts into potent phagocytic cells. Since cell fusion and phagocytosis share many molecules and mechanisms in myoblasts, we aimed to investigate how myoblasts choose between the two pathways during fusion. To prevent accidental uptake, viable cells express "don't eat-me" signals. By analyzing RNA sequencing data, we found that differentiation affected the expression of multiple "don't eat-me" genes in the C2C12 mouse myoblast cells, including upregulation of Sirpα, a receptor for CD47. The same was observed in differentiating myoblasts in vivo following cardiotoxin-induced injury in mouse skeletal muscle. Treatment of differentiating C2C12 cells with anti-CD47 antibody significantly reduced cell fusion but did not affect cell survival or differentiation. Both CD47 and SIRPα appeared at contact points of fusing myoblasts. Blocking CD47 signaling increased the uptake of viable red blood cells but only slightly increased the uptake of viable myoblasts. Blocking thrombospondin-1, another CD47 ligand, also inhibited fusion. Inhibiting CD47 signaling did not impact the engulfment of apoptotic cells. However, long-term exposure to continuously PS-expressing apoptotic cells disrupted myotube formation by inhibiting PIEZO1 activation, leading to syncytia formation. Overall, our data show that differentiating myoblasts upregulate CD47 to avoid accidental phagocytosis of live cells but mainly to promote myoblast fusion. Therefore, the activity of this signaling pathway contributes to the decision-making between the two processes that would compete with each other during myoblast differentiation.

Skeletal muscle dysfunction during spaceflight arises from combined mechanical and non-gravitational stressors. Here, we modeled solar particle event-like proton irradiation in C2C12 myogenic cells to examine radiation-induced membrane and mechanosensitive channel alterations. Proton exposure increased membrane rigidity in a dose-dependent manner and significantly reduced the pharmacological activatability of Piezo1 channels, identifying membrane-mediated mechanotransduction as a potential contributor to radiation-induced muscle dysfunction.

Context Increased orbital tissue volume due to matrix expansion, orbital fibroblast (OF) proliferation, and adipocyte differentiation are the hallmarks of thyroid eye disease (TED). Their combination with the presence of hyaluronan-bound excess water in the constrains of the bony orbit results in increased intraorbital pressure. High intraorbital pressure, along with changes in the mechanical properties of orbital tissues, may lead to the activation of mechanosensitive receptors. The expression and role of the Piezo1 mechanoreceptor has not been investigated in TED.Objective We aimed to verify the expression of Piezo1 in OFs, and to study the effect of in vitro Piezo1 activation by its synthetic agonist Yoda1 on adipocyte differentiation.Methods OF cultures established using orbital connective tissues from patients with TED and controls were studied in the presence or absence of adipogenic stimuli. Piezo1 expression was confirmed by Western Blot and immunofluorescent imaging, and its function was verified by intracellular Ca2+ measurement. Adipogenic differentiation was characterized using Oil Red O staining for lipid accumulation, real-time polymerase chain reaction for gene and Western blot for protein expressions indicative in adipogenesis.Results OFs express functional Piezo1 channels. Differentiation into adipocytes is inherent to TED OFs. Piezo1 activation by Yoda1 inhibits the expressions of early (CEBP beta, CEBP delta) and main (PPAR gamma, CEBP alpha) transcription factors, and the terminal marker FABP4 during adipogenesis, resulting in markedly lower intracytoplasmic lipid accumulation.Conclusion Piezo1 channels are expressed and functional in OFs. Modeling orbital pressure by in vitro Piezo1 activation reduces de novo adipogenesis of OFs derived from TED orbits.

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

BACKGROUND:Malignant hyperthermia susceptibility (MHS) and acute pancreatitis (AP) share a common cellular pathomechanism that is Ca2+-overload of the muscle fiber and the pancreatic acinar cell (PAC). In the muscle, gain-of-function mutations of the ryanodine receptor (RyR1) make the Ca2+-release mechanism hypersensitive to certain ligands, including Ca2+, volatile anaesthetics and succinylcholine, creating a medical emergency when the patient is exposed to these drugs. As RyR1 was shown to contribute to Ca2+-overload in PAC, we presumed that pancreata of MHS individuals are more prone to AP. Accordingly, a recent case study reported coincidence of MHS with recurrent AP, indicating a pathological link between the two diseases. METHODS:We tested if MHS poses a risk for AP in mice carrying the Y522S MHS mutation. Fluorescent Ca2+ imaging was performed in PACs. Conventional histopathological analysis and plazma amylase measurement was performed using a cerulein-induced pancreatitis mouse model. RESULTS:The intracellular Ca2+-signals of PACs from MHS mice were slightly bigger then in wild type when stimulated with 0.2 and 2 μM carbachol (cch) or with 1 and 5 mM bile acid (taurocholic acid). Store-operated-Ca2+-entry was also higher in PACs from MHS mice. Nevertheless, histopathological analysis and plasma amylase levels did not indicate more severe AP in MHS. CONCLUSIONS:These results suggest that the Y522S RyR1 mutation alter the Ca2+-homeostasis in PACs, but not as much as to cause or aggravate AP.

Here, we investigated the mechanisms by which aging-related reductions of the levels of Numb in skeletal muscle fibers contribute to loss of muscle strength and power, two critical features of sarcopenia. Numb is an adaptor protein best known for its critical roles in development, including asymmetric cell division, cell-type specification, and termination of intracellular signaling. Numb expression is reduced in old humans and mice. We previously showed that, in mouse skeletal muscle fibers, Numb is localized to sarcomeres where it is concentrated near triads; conditional inactivation of Numb and a closely related protein Numb -like ( Numbl ) in mouse myofibers caused weakness, disorganization of sarcomeres, and smaller mitochondria with impaired function. Here, we found that a single knockout of Numb in myofibers causes reduction in tetanic force comparable to a double Numb , Numbl knockout. We found by proteomics analysis of protein complexes isolated from C2C12 myotubes by immunoprecipitation using antibodies against Numb that Septin 7 is a potential Numb-binding partner. Septin 7 is a member of the family of GTP-binding proteins that organize into filaments, sheets, and rings, and is considered part of the cytoskeleton. Immunofluorescence evaluation revealed a partial overlap of staining for Numb and Septin 7 in myofibers. Conditional, inducible knockouts of Numb led to disorganization of Septin 7 staining in myofibers. These findings indicate that Septin 7 is a Numb-binding partner and suggest that interactions between Numb and Septin 7 are critical for structural organization of the sarcomere and muscle contractile function.

Piezo1 mechanosensitive cation channels detect mechanical stimuli and convert it into physiological signals. The role of mechanosensitive channels may be especially important in skeletal muscle compared to non-contractile cell types. There is some proof on its role in muscle regeneration and in muscle satellite cell function, but there is still very little known on the effect of the channel on muscle performance and adult muscle calcium homeostasis. Here we analyzed the role of Piezo1 in skeletal muscle by studying the effect of pharmacological activator and inhibitor in mice.

Basic leucine zipper (bZIP) transcription factors are crucial components of differentiation, cellular homeostasis and the environmental stress defense of eukaryotes. In this work, we further studied the consequence of gene deletion and overexpression of two bZIP transcription factors, NapA and RsmA, on superoxide production, mitochondrial morphology and hyphal diameter of Aspergillus nidulans . We have found that reactive oxygen species production was influenced by both gene deletion and overexpression of napA under tert -butylhydroperoxide ( t BOOH) elicited oxidative stress. Furthermore, gene expression of napA negatively correlated with mitochondrial volumetric ratio as well as sterigmatocystin production of A. nidulans . High rsmA expression was accompanied with elevated relative superoxide ratio in the second hyphal compartment. A negative correlation between the expression of rsmA and catalase enzyme activity or mitochondrial volumetric ratio was also confirmed by statistical analysis. Hyphal diameter was independent on either rsmA and napA expression as well as 0.2 mM t BOOH treatment.

Skeletal muscle has the role to maintain body posture against a constant gravitational load. During aging, in low physical activity or even in spaceflight, the muscle mass is decreasing, resulting in impaired myogenesis and regeneration. The cytoskeleton, mechanosensitive ion channels, and calcium homeostasis play crucial role in maintaining normal myogenic processes. Simulated microgravity (SM) with 3D clinorotation is one of the accepted techniques to model gravitational unloading in vitro. C2C12 mouse myoblast cell line is a verified in vitro model system of myogenesis. Using RPM 2.0, a Random Positioning Machine as a partial g simulator, by randomly rotating the accommodated experiment package around the Earth's gravity vector, we are able to investigate alteration of physiological processes caused by SM. In our experiments, territorial gravity is compared to 0 g, respectively. However, fusion of myoblasts was preserved with SM, the formation of myotubes was different as compared to the control environment. Expression of Myosin Heavy Chain 2 (MYH2) protein, which is an important structural protein and a marker molecule of myotube differentiation program, was significantly reduced in samples kept in RPM. Immunocytochemistry of myogenic cultures revealed decreased size and average nuclei content in differentiated myotubes. The expression level of Piezo1 channels, which are mechanically activated cation channels are also decreased under SM. Altered calcium transients evoked by KCl have been observed in myotubes formed under simulated microgravity. Our results are in coherence with the recently published data. Our future goal is to analyze the architecture of cytoskeletal Septin7 protein and investigate how intracellular calcium concentrations and mechanotransduction is regulated by Piezo1 channels under microgravity condition.

Septin7 as a unique member of the GTP binding protein family, is widely expressed in the eukaryotic cells and considered to be essential in the formation of hetero-oligomeric septin complexes. As a cytoskeletal component, Septin7 is involved in many important cellular processes. However, its contribution in striated muscle physiology is poorly described. In skeletal muscle, a highly orchestrated process of migration is crucial in the development of functional fibers and in regeneration. Here, we describe the pronounced appearance of Septin7 filaments and a continuous change of Septin7 protein architecture during the migration of myogenic cells. In Septin7 knockdown C2C12 cultures, the basic parameters of migration are significantly different, and the intracellular calcium concentration change in migrating cells are lower compared to that of scrambled cultures. Using a plant cytokinin, forchlorfenuron, to dampen septin dynamics, the altered behavior of the migrating cells is described, where Septin7-depleted cells are more resistant to the treatment. These results indicate the functional relevance of Septin7 in the migration of myoblasts, implying its contribution to muscle myogenesis and regeneration.

Septins are considered the fourth component of the cytoskeleton with the septin7 isoform playing a critical role in the formation of diffusion barriers in phospholipid bilayers and intra- and extracellular scaffolds. While its importance has already been confirmed in different intracellular processes, very little is known about its role in skeletal muscle. Muscle regeneration was studied in a Sept7 conditional knock-down mouse model to prove the possible role of septin7 in this process. Sterile inflammation in skeletal muscle was induced which was followed by regeneration resulting in the upregulation of septin7 expression. Partial knock-down of Sept7 resulted in an increased number of inflammatory cells and myofibers containing central nuclei. Taken together, our data suggest that partial knock-down of Sept7 hinders the kinetics of muscle regeneration, indicating its crucial role in skeletal muscle functions.

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.

Since the recent discovery of the mechanosensitive Piezo1 channels, many studies have addressed the role of the channel in various physiological or even pathological processes of different organs. Although the number of studies on their effects on the musculoskeletal system is constantly increasing, we are still far from a precise understanding. In this review, the knowledge available so far regarding the musculoskeletal system is summarized, reviewing the results achieved in the field of skeletal muscles, bones, joints and cartilage, tendons and ligaments, as well as intervertebral discs.

Vascular calcification (VC) is associated with a number of cardiovascular diseases, as well as chronic kidney disease. The role of smooth muscle cells (SMC) has already been widely explored in VC, as has the role of intracellular Ca2+ in regulating SMC function. Increased intracellular calcium concentration ([Ca2+]i) in vascular SMC has been proposed to stimulate VC. However, the contribution of the non-selective Piezo1 mechanosensitive cation channels to the elevation of [Ca2+]i, and consequently to the process of VC has never been examined. In this work the essential contribution of Piezo1 channels to arterial medial calcification is demonstrated. The presence of Piezo1 was proved on human aortic smooth muscle samples using immunohistochemistry. Quantitative PCR and Western blot analysis confirmed the expression of the channel on the human aortic smooth muscle cell line (HAoSMC). Functional measurements were done on HAoSMC under control and calcifying condition. Calcification was induced by supplementing the growth medium with inorganic phosphate (1.5 mmol/L, pH 7.4) and calcium (CaCl2, 0.6 mmol/L) for 7 days. Measurement of [Ca2+]i using fluorescent Fura-2 dye upon stimulation of Piezo1 channels (either by hypoosmolarity, or Yoda1) demonstrated significantly higher calcium transients in calcified as compared to control HAoSMCs. The expression of mechanosensitive Piezo1 channel is augmented in calcified arterial SMCs leading to a higher calcium influx upon stimulation. Activation of the channel by Yoda1 (10 μmol/L) enhanced calcification of HAoSMCs, while Dooku1, which antagonizes the effect of Yoda1, reduced this amplification. Application of Dooku1 alone inhibited the calcification. Knockdown of Piezo1 by siRNA suppressed the calcification evoked by Yoda1 under calcifying conditions. Our results demonstrate the pivotal role of Piezo1 channels in arterial medial calcification.

Obscurin is a giant sarcomeric protein expressed in striated muscles known to establish several interactions with other proteins of the sarcomere, but also with proteins of the sarcoplasmic reticulum and costameres. Here, we report experiments aiming to better understand the contribution of obscurin to skeletal muscle fibers, starting with a detailed characterization of the diaphragm muscle function, which we previously reported to be the most affected muscle in obscurin (Obscn) KO mice. Twitch and tetanus tension were not significantly different in the diaphragm of WT and Obscn KO mice, while the time to peak (TTP) and half relaxation time (HRT) were prolonged. Differences in force-frequency and force-velocity relationships and an enhanced fatigability are observed in an Obscn KO diaphragm with respect to WT controls. Voltage clamp experiments show that a sarcoplasmic reticulum's Ca2+ release and SERCA reuptake rates were decreased in muscle fibers from Obscn KO mice, suggesting that an impairment in intracellular Ca2+ dynamics could explain the observed differences in the TTP and HRT in the diaphragm. In partial contrast with previous observations, Obscn KO mice show a normal exercise tolerance, but fiber damage, the altered sarcomere ultrastructure and M-band disarray are still observed after intense exercise.

The endocannabinoid system (ECS) refers to a widespread signaling system and its alteration is implicated in a growing number of human diseases. Cannabinoid receptors (CBRs) are highly expressed in the central nervous system and many peripheral tissues. Evidence suggests that CB1Rs are expressed in human and murine skeletal muscle mainly in the cell membrane, but a subpopulation is present also in the mitochondria. However, very little is known about the latter population. To date, the connection between the function of CB1Rs and the regulation of intracellular Ca2+ signaling has not been investigated yet. Tamoxifen-inducible skeletal muscle-specific conditional CB1 knock-down (skmCB1-KD, hereafter referred to as Cre+/−) mice were used in this study for functional and morphological analysis. After confirming CB1R down-regulation on the mRNA and protein level, we performed in vitro muscle force measurements and found that peak twitch, tetanus, and fatigue were decreased significantly in Cre+/− mice. Resting intracellular calcium concentration, voltage dependence of the calcium transients as well as the activity dependent mitochondrial calcium uptake were essentially unaltered by Cnr1 gene manipulation. Nevertheless, we found striking differences in the ultrastructural architecture of the mitochondrial network of muscle tissue from the Cre+/− mice. Our results suggest a role of CB1Rs in maintaining physiological muscle function and morphology. Targeting ECS could be a potential tool in certain diseases, including muscular dystrophies where increased endocannabinoid levels have already been described.

Septins are considered as the fourth component of the cytoskeleton, with septin-7 isoform playing a critical role in myogenic cell division and fusion. Skeletal muscle regeneration is a highly orchestrated process that requires many steps, including proper cell division to achieve functional recovery. Here, the role of septin-7 was investigated in this complex process. To this end, muscle injury was induced in wild type BL6/C57 and septin-7-conditional (mer-Cre-mer) knock-down mice by in vivo BaCl2 injection to the left m. tibialis anterior muscle (TA) of the mice (the right m. tibialis anterior muscle was nontreated control). Mice were sacrificed 4 and 14 d later to reflect the early (monitored by PAX7 level) and late (monitored by myogenin level) phases of muscle regeneration. Western blotting was used to follow the changes of septin-7, PAX7, and myogenin expression at the protein level, while changes of mRNA were detected by qPCR. Morphological differences were visualized by HE staining. Levels of septin-7 protein increased 4 and 14 d after injury in BL6/C57 mice and mRNA expression of SEPT7 showed significant elevation both 4 and 14 d after injection in Cre+ mice only, considered to be a compensatory increase of mRNA expression of SEPT7 in order to ensure the appropriate regeneration process. Furthermore, up-regulation of septin-7 protein was more pronounced on day 14 in both Cre- and Cre+ mice, which may indicate its importance in the later phase of regeneration. Level of PAX7 and myogenin were also increased 4 and 14 d after injury in BL6/C57, Cre-, and Cre+ mice, respectively. Taken together, our data suggest the importance of septin-7 in skeletal muscle regeneration.

Today septins are considered as the fourth component of the cytoskeleton, with the Septin7 isoform playing a critical role in the formation of higher-order structures. While its importance has already been confirmed in several intracellular processes of different organs, very little is known about its role in skeletal muscle. Here, using Septin7 conditional knockdown (KD) mouse model, the C2C12 cell line, and enzymatically isolated adult muscle fibers, the organization and localization of septin filaments are revealed, and an ontogenesis-dependent expression of Septin7 is demonstrated. KD mice displayed a characteristic hunchback phenotype with skeletal deformities, reduction in in vivo and in vitro force generation, and disorganized mitochondrial networks. Furthermore, knockout of Septin7 in C2C12 cells resulted in complete loss of cell division while KD cells provided evidence that Septin7 is essential for proper myotube differentiation. These and the transient increase in Septin7 expression following muscle injury suggest that it may be involved in muscle regeneration and development.