Periodic Stretching of Cultured Myotubes Enhances Myofibril Assembly.

The effects of mechanical stress on cultured muscle cells were examined with particular interest in myofibril assembly by using a cell-stretching system. We observed that formation and maintenance of cross-striated myofibrils in chick muscle cell cultures was suppressed in the media containing higher concentration of KCl, tetrodotoxin, or ML-9 (an inhibitor of myosin light chain kinase), but periodic stretching of myotubes for several days enabled formation of striated myofibrils just as in standard muscle cultures. However, ryanodine (a blocker of the Ca2 + channel in sarcoplasmic reticulum) and BDM (an inhibitor of myosin-actin interaction) suppressed the stretch-induced myofibrillogenesis. We further found that stretching of myotubes causes quick and transient elevation of the intracellular Ca2 + concentration and this elevation is disturbed by inhibition of Ca2 + channels of sarcoplasmic reticulum and suppression of Ca2 + influx from culture medium. These observations indicate that periodic stretching induces elevation of intracellular Ca2 + concentration and that this elevation may be due to release of Ca2 + from sarcoplasmic reticulum and Ca2 + influx from outside of the cells. The increased Ca2 + may activate actin-myosin interaction by interacting with troponin that is located along actin filaments and/or inducing phosphorylation of myosin light chains and thereby promote myofibril assembly.

Cessation of electrically-induced muscle contraction activates autophagy in cultured myotubes.

Exercise is known to improve skeletal muscle function. The mechanism involves muscle contraction-induced activation of the mTOR pathway, which plays a central role in protein synthesis. However, mTOR activation blocks autophagy, a recycling mechanism with a critical role in cellular maintenance/homeostasis. These two responses to muscle contraction look contradictory to the functional improvement of exercise. Herein, we investigate these paradoxical muscle responses in a series of active-inactive phases in a cultured myotube model receiving electrical stimulation to induce intermittent muscle contraction. Our model shows that (1) contractile activity induces mTOR activation and muscle hypertrophy but blocks autophagy, resulting in the accumulation of damaged proteins, while (2) cessation of muscle contraction rapidly activates autophagy, removing damaged protein, yet a prolonged inactive state results in muscle atrophy. Our findings provide new insights into muscle biology and suggest that not only muscle contraction, but also the subsequent cessation of contraction plays a substantial role for the improvement of skeletal muscle function.

Planar compression of extracellular substrates induces S phase arrest via ATM-independent CHK2 activation.

Cell proliferation is regulated not only by soluble chemical factors but also by mechanical cues surrounding cells. Mechanical stretch of extracellular substrates is known to promote cell proliferation by driving exit from the G0 phase and entry into the S phase. Here, we report that planer compression of extracellular substrates induces cell cycle arrest in the S phase. The compression-induced S phase arrest is mediated by the checkpoint kinase 2 (CHK2)-p53 pathway. In contrast to the canonical S phase checkpoint pathway activated by DNA damage, CHK2 activation by the substrate compression is independent of ataxia telangiectasia mutated (ATM). We further find that disassembly of the actin cytoskeleton is required for the compression-induced S phase arrest. Notably, cancer cells do not exhibit S phase arrest upon the substrate compression. Our results suggest a novel mechanism for homeostatic control of cell growth under mechanical perturbations.

Actin-interacting Protein 1 Promotes Disassembly of Actin-depolymerizing Factor/Cofilin-bound Actin Filaments in a pH-dependent Manner.

Actin-interacting protein 1 (AIP1) is a conserved WD repeat protein that promotes disassembly of actin filaments when actin-depolymerizing factor (ADF)/cofilin is present. Although AIP1 is known to be essential for a number of cellular events involving dynamic rearrangement of the actin cytoskeleton, the regulatory mechanism of the function of AIP1 is unknown. In this study, we report that two AIP1 isoforms from the nematode Caenorhabditis elegans, known as UNC-78 and AIPL-1, are pH-sensitive in enhancement of actin filament disassembly. Both AIP1 isoforms only weakly enhance disassembly of ADF/cofilin-bound actin filaments at an acidic pH but show stronger disassembly activity at neutral and basic pH values. However, a severing-defective mutant of UNC-78 shows pH-insensitive binding to ADF/cofilin-decorated actin filaments, suggesting that the process of filament severing or disassembly, but not filament binding, is pH-dependent. His-60 of AIP1 is located near the predicted binding surface for the ADF/cofilin-actin complex, and an H60K mutation of AIP1 partially impairs its pH sensitivity, suggesting that His-60 is involved in the pH sensor for AIP1. These biochemical results suggest that pH-dependent changes in AIP1 activity might be a novel regulatory mechanism of actin filament dynamics.

Training at non-damaging intensities facilitates recovery from muscle atrophy.

INTRODUCTION: Resistance training promotes recovery from muscle atrophy, but optimum training programs have not been established. We aimed to determine the optimum training intensity for muscle atrophy. METHODS: Mice recovering from atrophied muscles after 2 weeks of tail suspension underwent repeated isometric training with varying joint torques 50 times per day. RESULTS: Muscle recovery assessed by maximal isometric contraction and myofiber cross-sectional areas (CSAs) were facilitated at 40% and 60% maximum contraction strength (MC), but at not at 10% and 90% MC. At 60% and 90% MC, damaged and contained smaller diameter fibers were observed. Activation of myogenic satellite cells and a marked increase in myonuclei were observed at 40%, 60%, and 90% MC. CONCLUSIONS: The increases in myofiber CSAs were likely caused by increased myonuclei formed through fusion of resistance-induced myofibers with myogenic satellite cells. These data indicate that resistance training without muscle damage facilitates efficient recovery from atrophy. Muscle Nerve 55: 243-253, 2017.

Single-molecule imaging and kinetic analysis of cooperative cofilin-actin filament interactions.

The actin filament-severing protein actin depolymerizing factor (ADF)/cofilin is ubiquitously distributed among eukaryotes and modulates actin dynamics. The cooperative binding of cofilin to actin filaments is crucial for the concentration-dependent unconventional modulation of actin dynamics by cofilin. In this study, the kinetic parameters associated with the cooperative binding of cofilin to actin filaments were directly evaluated using a single-molecule imaging technique. The on-rate of cofilin binding to the actin filament was estimated to be 0.06 µM(-1)⋅s(-1) when the cofilin concentration was in the range of 30 nM to 1 µM. A dwell time histogram of cofilin bindings decays exponentially to give an off-rate of 0.6 s(-1). During long-term cofilin binding events (>0.4 s), additional cofilin bindings were observed in the vicinity of the initial binding site. The on-rate for these events was 2.3-fold higher than that for noncontiguous bindings. Super-high-resolution image analysis of the cofilin binding location showed that the on-rate enhancement occurred within 65 nm of the original binding event. By contrast, the cofilin off-rate was not affected by the presence of prebound cofilin. Neither decreasing the temperature nor increasing the viscosity of the test solution altered the on-rates, off-rates, or the cooperative parameter (ω) of the binding. These results indicate that cofilin binding enhances additional cofilin binding in the vicinity of the initial binding site (ca. 24 subunits), but it does not affect the off-rate, which could be the molecular mechanism of the cooperative binding of cofilin to actin filaments.

Non-channel mechanosensors working at focal adhesion-stress fiber complex.

Mechanosensitive ion channels (MSCs) have long been the only established molecular class of cell mechanosensors; however, in the last decade, a variety of non-channel type mechanosensor molecules have been identified. Many of them are focal adhesion-associated proteins that include integrin, talin, and actin. Mechanosensors must be non-soluble molecules firmly interacting with relatively rigid cellular structures such as membranes (in terms of lateral stiffness), cytoskeletons, and adhesion structures. The partner of MSCs is the membrane in which MSC proteins efficiently transduce changes in the membrane tension into conformational changes that lead to channel opening. By contrast, the integrin, talin, and actin filament form a linear complex of which both ends are typically anchored to the extracellular matrices via integrins. Upon cell deformation by forces, this structure turns out to be a portion that efficiently transduces the generated stress into conformational changes of composite molecules, leading to the activation of integrin (catch bond with extracellular matrices) and talin (unfolding to induce vinculin bindings). Importantly, this structure also serves as an "active" mechanosensor to detect substrate rigidity by pulling the substrate with contraction of actin stress fibers (SFs), which may induce talin unfolding and an activation of MSCs in the vicinity of integrins. A recent study demonstrates that the actin filament acts as a mechanosensor with unique characteristics; the filament behaves as a negative tension sensor in which increased torsional fluctuations by tension decrease accelerate ADF/cofilin binding, leading to filament disruption. Here, we review the latest progress in the study of those non-channel mechanosensors and discuss their activation mechanisms and physiological roles.

Force- and Ca²âº-dependent internalization of integrins in cultured endothelial cells.

The effects of mechanical force applied to the integrin clusters at focal contacts were examined in cultured human umbilical vein endothelial cells. When a fibronectin-coated glass bead was attached to the apical cell surface, focal contacts formed beneath the bead that became linked to focal contacts at the basal cell membrane by actin stress fibers in 5 minutes. Integrin dynamics at the basal focal contacts were monitored in live cells in response to a localized mechanical stimulus generated by displacing the glass bead. Traction force transmitted to the basal focal contacts through the stress fibers was monitored by measuring the deformation of the polyacrylamide gel substratum. The force declined in a few seconds, probably owing to decreases in the elastic modulus of the stress fibers. This transient mechanical stimulus caused the dephosphorylation of paxillin and disassembly of integrin clusters at the basal cell membrane in 20 minutes. The disassembly was mediated mainly by clathrin-dependent endocytosis of integrins. The integrin internalization was inhibited in Ca(2+)- and K(+)-free solution, and by phenylarsine oxide, a phosphatase inhibitor. These results suggest that a transient mechanical stimulus applied to focal contacts induces Ca(2+)-dependent dephosphorylation of some proteins, including paxillin, and facilitates clathrin-dependent endocytosis of integrins.

Involvement of PI3K/Akt/TOR pathway in stretch-induced hypertrophy of myotubes.

Skeletal muscle cells are hypertrophied by mechanical stresses, but the underlying molecular mechanisms are not fully understood. Two signaling pathways, phosphatidylinositol 3-kinase (PI3K)/Akt to target of rapamycin (TOR) and extracellular signal-regulated kinase kinase (MEK) to extracellular signal-regulated kinase (ERK), have been proposed to be involved in muscle hypertrophy. In this study we examined the involvement of these pathways in primary cultures of chick skeletal myotubes subjected to passive cyclic stretching for 72 hours, a time that was sufficient to induce significant hypertrophy in our preparations. Hypertrophy was largely suppressed by wortmannin or rapamycin, inhibitors of PI3K or mTOR, respectively. Furthermore, phosphorylation of Akt was enhanced by stretching and suppressed by wortmannin. The MEK inhibitor, U0126, exerted a minimal influence on stretch-induced hypertrophy. We found that cyclic stretching of myotubes activates the PI3K/Akt/TOR pathway, resulting in muscle hypertrophy. The MEK/ERK pathway may contribute negatively to spontaneous hypertrophy.

Real-Time Single-Molecule Kinetic Analyses of AIP1-Enhanced Actin Filament Severing in the Presence of Cofilin.

Rearrangement of actin filaments by polymerization, depolymerization, and severing is important for cell locomotion, membrane trafficking, and many other cellular functions. Cofilin and actin-interacting protein 1 (AIP1; also known as WDR1) are evolutionally conserved proteins that cooperatively sever actin filaments. However, little is known about the biophysical basis of the actin filament severing by these proteins. Here, we performed single-molecule kinetic analyses of fluorescently labeled AIP1 during the severing process of cofilin-decorated actin filaments. Results demonstrated that binding of a single AIP molecule was sufficient to enhance filament severing. After AIP1 binding to a filament, severing occurred with a delay of 0.7 s. Kinetics of binding and dissociation of a single AIP1 molecule to/from actin filaments followed a second-order and a first-order kinetics scheme, respectively. AIP1 binding and severing were detected preferentially at the boundary between the cofilin-decorated and bare regions on actin filaments. Based on the kinetic parameters explored in this study, we propose a possible mechanism behind the enhanced severing by AIP1.

Stand-up exercise training facilitates muscle recovery from disuse atrophy by stimulating myogenic satellite cell proliferation in mice.

Determining the cellular and molecular recovery processes in inactivity - or unloading -induced atrophied muscles should improve rehabilitation strategies. We assessed the effects of stand-up exercise (SE) training on the recovery of atrophied skeletal muscles in male mice. Mice were trained to stand up and press an elevated lever in response to a light-tone cue preceding an electric foot shock and then subjected to tail suspension (TS) for 2 weeks to induce disuse atrophy in hind limb muscles. After release from TS, mice were divided into SE-trained (SE cues: 25 times per set, two sets per day) and non-SE-trained groups. Seven days after the training, average myofiber cross-sectional area (CSA) of the soleus muscle was significantly greater in the SE-trained group than in the non-SE-trained group (1843 ± 194 µm(2) vs. 1315 ± 153 µm(2)). Mean soleus muscle CSA in the SE trained group was not different from that in the CON group subjected to neither TS nor SE training (2005 ± 196 µm(2)), indicating that SE training caused nearly complete recovery from muscle atrophy. The number of myonuclei per myofiber was increased by ~60% in the SE-trained group compared with the non-SE-trained and CON groups (0.92 ± 0.03 vs. 0.57 ± 0.03 and 0.56 ± 0.11, respectively). The number of proliferating myonuclei, identified by 5-ethynyl-2'-deoxyuridine staining, increased within the first few days of SE training. Thus, it is highly likely that myogenic satellite cells proliferated rapidly in atrophied muscles in response to SE training and fused with existing myofibers to reestablish muscle mass.

Mechano-sensing by actin filaments and focal adhesion proteins.

Mechanosensitive ion channels have long been the only established molecular class of cell mechanosensors with known molecular entities. However, recent advances in the state-of-the-art techniques, including single-molecule manipulation and imaging, have enabled an investigation of non-channel type cell mechanosensors and the underlying biophysical mechanisms of their activation. To date, two focal adhesion proteins, talin and p130Cas, have been postulated to act as putative mechanosensors, acting through mechano-induced unfolding of their particular soft domain(s) susceptible to phosphorylation. More recently, the actin filament has been demonstrated to act as a mechanosensor in the presence of the soluble actin-severing protein, cofilin. The cofilin severing activity negatively depends on the tension in the actin filament through tension-dependent binding/unbinding of cofilin to/from the actin filament. As a result, relaxed actin filaments are severed, while tensed ones are either not severed or severed after a long delay. Here we review the latest progress in the mechanosensing by non-channel type proteins and discuss the possible physiological roles of the mechanosensing performed by actin filaments in the course of cell migration.

Actin filaments function as a tension sensor by tension-dependent binding of cofilin to the filament.

Intracellular and extracellular mechanical forces affect the structure and dynamics of the actin cytoskeleton. However, the underlying molecular and biophysical mechanisms, including how mechanical forces are sensed, are largely unknown. Actin-depolymerizing factor/cofilin proteins are actin-modulating proteins that are ubiquitously distributed in eukaryotes, and they are the most likely candidate as proteins to drive stress fiber disassembly in response to changes in tension in the fiber. In this study, we propose a novel hypothesis that tension in an actin filament prevents the filament from being severed by cofilin. To test this, we placed single actin filaments under tension using optical tweezers. When a fiber was tensed, it was severed after the application of cofilin with a significantly larger delay in comparison with control filaments suspended in solution. The binding rate of cofilin to an actin bundle decreased when the bundle was tensed. These results suggest that tension in an actin filament reduces the cofilin binding, resulting in a decrease in its effective severing activity.

Repetitive stretch suppresses denervation-induced atrophy of soleus muscle in rats.

This study was conducted to examine whether stretch-related mechanical loading on skeletal muscle can suppress denervation-induced muscle atrophy, and if so, to depict the underlying molecular mechanism. Denervated rat soleus muscle was repetitively stretched (every 5 s for 15 min/day) for 2 weeks. Histochemical analysis showed that the cross-sectional area of denervated soleus muscle fibers with repetitive stretching was significantly larger than that of control denervated muscle (P<0.05). We then examined the involvement of the Akt/mammalian target of the rapamycin (mTOR) cascade in the suppressive effects of repetitive stretching on muscle atrophy. Repetitive stretching significantly increased the Akt, p70S6K, and 4E-BP1 phosphorylation in denervated soleus muscle compared to controls (P<0.05). Furthermore, repetitive stretching-induced suppression of muscle atrophy was fully inhibited by rapamycin, a potent inhibitor of mTOR. These results indicate that denervation-induced muscle atrophy is significantly suppressed by stretch-related mechanical loading of the muscle through upregulation of the Akt/mTOR signal pathway.

Actin stress fibers transmit and focus force to activate mechanosensitive channels.

Mechanosensitive (MS) channels are expressed in various cells in a wide range of phylogenetic lineages from bacteria to humans. Understanding the molecular and biophysical mechanisms of their activation is an important research pursuit. It is controversial whether eukaryotic MS channels need accessory proteins -- typically cytoskeletal structures -- for activation, because MS channel activities are modulated by pharmacological treatments that affect the cytoskeleton. Here we demonstrate that direct mechanical stimulation (stretching) of an actin stress fiber using optical tweezers can activate MS channels in cultured human umbilical vein endothelial cells (HUVECs). Furthermore, by using high-speed total internal reflection microscopy, we visualized spots of Ca(2+) influx across individual MS channels distributed near focal adhesions in the basal surface of HUVECs. This study provides the first direct evidence that the cytoskeleton works as a force-transmitting and force-focusing molecular device to activate MS channels in eukaryotic cells.

Large Na(+) influx and high Na(+), K (+)-ATPase activity in mitochondria-rich epithelial cells of the inner ear endolymphatic sac.

Fluid in the mammalian endolymphatic sac (ES) is connected to the endolymph in the cochlea and the vestibule. Since the dominant ion in the ES is Na(+), it has been postulated that Na(+) transport is essential for regulating the endolymph pressure. This study focused on the cellular mechanism of Na(+) transport in ES epithelial cells. To evaluate the Na(+) transport capability of the ES epithelial cells, changes in intracellular Na(+) concentration ([Na(+)](i)) of individual ES cells were measured with sodium-binding benzofurzan isophthalate in a freshly dissected ES sheet and in dissociated ES cells in response to either the K(+)-free or ouabain-containing solution. Analysis of the [Na(+)](i) changes by the Na(+) load and mitochondrial staining with rhodamine 123 showed that the ES cells were classified into two groups; one exhibited an intensive [Na(+)](i) increase, higher Na(+), K(+)-ATPase activity, and intensive mitochondrial staining (mitochondria-rich cells), and the other exhibited a moderate [Na(+)](i) increase, lower Na(+), K(+)-ATPase activity, and moderate mitochondrial staining (filament-rich cells). These results suggest that mitochondria-rich ES epithelial cells (ca. 30% of ES cells) endowed with high Na(+) permeability and Na(+), K(+)-ATPase activity potentially contribute to the transport of Na(+) outside of the endolymphatic sac.

Uniaxial cyclic stretch-stimulated glucose transport is mediated by a ca-dependent mechanism in cultured skeletal muscle cells.

OBJECTIVE: Mechanical stimuli such as stretch increase glucose transport and glycogen metabolism in skeletal muscle. However, the molecular mechanisms involved in the mechanotransduction events are poorly understood. The present study was conducted in order to determine whether the signaling mechanism leading to mechanical stretch-stimulated glucose transport is similar to, or distinct from, the signaling mechanisms leading to insulin- and contraction-stimulated glucose transport in cultured muscle cells. METHODS: Cultured C2C12 myotubes were stretched, after which the 2-deoxy-D-glucose (2-DG) uptake was measured. RESULTS: Following cyclic stretch, C2C12 myotubes showed a significant increase in 2-DG uptake, and this effect was not prevented by inhibiting phosphatidylinositol 3-kinase or 5'-AMP-activated protein kinase and by extracellular Ca(2+) chelation. Conversely, the stretch-stimulated 2-DG uptake was completely prevented by dantrolene (an inhibitor of Ca(2+) release from sarcoplasmic reticulum). Furthermore, the stretch-stimulated 2-DG uptake was prevented by the Ca(2+)/calmodulin-dependent kinase inhibitor KN93 which did not prevent the insulin-stimulated 2-DG uptake. CONCLUSIONS: These results suggest that the effects of stretch-stimulated glucose transport are independent of the insulin-signaling pathway. By contrast, following mechanical stretch in skeletal muscle, the signal transduction pathway leading to glucose transport may require the participation of cytosolic Ca(2+) and Ca(2+)/calmodulin kinase, but not 5'-AMP-activated protein kinase.