Mitochondrial fission links ECM mechanotransduction to metabolic redox homeostasis and metastatic chemotherapy resistance.

Metastatic breast cancer cells disseminate to organs with a soft microenvironment. Whether and how the mechanical properties of the local tissue influence their response to treatment remains unclear. Here we found that a soft extracellular matrix empowers redox homeostasis. Cells cultured on a soft extracellular matrix display increased peri-mitochondrial F-actin, promoted by Spire1C and Arp2/3 nucleation factors, and increased DRP1- and MIEF1/2-dependent mitochondrial fission. Changes in mitochondrial dynamics lead to increased production of mitochondrial reactive oxygen species and activate the NRF2 antioxidant transcriptional response, including increased cystine uptake and glutathione metabolism. This retrograde response endows cells with resistance to oxidative stress and reactive oxygen species-dependent chemotherapy drugs. This is relevant in a mouse model of metastatic breast cancer cells dormant in the lung soft tissue, where inhibition of DRP1 and NRF2 restored cisplatin sensitivity and prevented disseminated cancer-cell awakening. We propose that targeting this mitochondrial dynamics- and redox-based mechanotransduction pathway could open avenues to prevent metastatic relapse.

Mechanical disruption of E-cadherin complexes with epidermal growth factor receptor actuates growth factor-dependent signaling.

Increased intercellular tension is associated with enhanced cell proliferation and tissue growth. Here, we present evidence for a force-transduction mechanism that links mechanical perturbations of epithelial (E)-cadherin (CDH1) receptors to the force-dependent activation of epidermal growth factor receptor (EGFR, ERBB1)-a key regulator of cell proliferation. Here, coimmunoprecipitation studies first show that E-cadherin and EGFR form complexes at the plasma membrane that are disrupted by either epidermal growth factor (EGF) or increased tension on homophilic E-cadherin bonds. Although force on E-cadherin bonds disrupts the complex in the absence of EGF, soluble EGF is required to mechanically activate EGFR at cadherin adhesions. Fully quantified spectral imaging fluorescence resonance energy transfer further revealed that E-cadherin and EGFR directly associate to form a heterotrimeric complex of two cadherins and one EGFR protein. Together, these results support a model in which the tugging forces on homophilic E-cadherin bonds trigger force-activated signaling by releasing EGFR monomers to dimerize, bind EGF ligand, and signal. These findings reveal the initial steps in E-cadherin-mediated force transduction that directly link intercellular force fluctuations to the activation of growth regulatory signaling cascades.

Mouse lung organoid responses to reduced, increased, and cyclic stretch.

Most lung development occurs in the context of cyclic stretch. Alteration of the mechanical microenvironment is a common feature of many pulmonary diseases, with congenital diaphragmatic hernia (CDH) and fetal tracheal occlusion (FETO, a therapy for CDH) being extreme examples with changes in lung structure, cell differentiation, and function. To address limitations in cell culture and in vivo mechanotransductive models, we developed two mouse lung organoid (mLO) mechanotransductive models using postnatal day 5 (PND5) mouse lung CD326-positive cells and fibroblasts subjected to increased, decreased, and cyclic strain. In the first model, mLOs were exposed to forskolin (FSK) and/or disrupted (DIS) and evaluated at 20 h. mLO cross-sectional area changed by +59%, +24%, and -68% in FSK, control, and DIS mLOs, respectively. FSK-treated organoids had twice as many proliferating cells as other organoids. In the second model, 20 h of 10.25% biaxial cyclic strain increased the mRNAs of lung mesenchymal cell lineages compared with static stretch and no stretch. Cyclic stretch increased TGF-ß and integrin-mediated signaling, with upstream analysis indicating roles for histone deacetylases, microRNAs, and long noncoding RNAs. Cyclic stretch mLOs increased αSMA-positive and αSMA-PDGFRα-double-positive cells compared with no stretch and static stretch mLOs. In this PND5 mLO mechanotransductive model, cell proliferation is increased by static stretch, and cyclic stretch induces mesenchymal gene expression changes important in postnatal lung development.

Endothelial upregulation of mechanosensitive channel Piezo1 in pulmonary hypertension.

Piezo is a mechanosensitive cation channel responsible for stretch-mediated Ca2+ and Na+ influx in multiple types of cells. Little is known about the functional role of Piezo1 in the lung vasculature and its potential pathogenic role in pulmonary arterial hypertension (PAH). Pulmonary arterial endothelial cells (PAECs) are constantly under mechanic stretch and shear stress that are sufficient to activate Piezo channels. Here, we report that Piezo1 is significantly upregulated in PAECs from patients with idiopathic PAH and animals with experimental pulmonary hypertension (PH) compared with normal controls. Membrane stretch by decreasing extracellular osmotic pressure or by cyclic stretch (18% CS) increases Ca2+-dependent phosphorylation (p) of AKT and ERK, and subsequently upregulates expression of Notch ligands, Jagged1/2 (Jag-1 and Jag-2), and Delta like-4 (DLL4) in PAECs. siRNA-mediated downregulation of Piezo1 significantly inhibited the stretch-mediated pAKT increase and Jag-1 upregulation, whereas downregulation of AKT by siRNA markedly attenuated the stretch-mediated Jag-1 upregulation in human PAECs. Furthermore, the mRNA and protein expression level of Piezo1 in the isolated pulmonary artery, which mainly contains pulmonary arterial smooth muscle cells (PASMCs), from animals with severe PH was also significantly higher than that from control animals. Intraperitoneal injection of a Piezo1 channel blocker, GsMTx4, ameliorated experimental PH in mice. Taken together, our study suggests that membrane stretch-mediated Ca2+ influx through Piezo1 is an important trigger for pAKT-mediated upregulation of Jag-1 in PAECs. Upregulation of the mechanosensitive channel Piezo1 and the resultant increase in the Notch ligands (Jag-1/2 and DLL4) in PAECs may play a critical pathogenic role in the development of pulmonary vascular remodeling in PAH and PH.

Contribution of tetrodotoxin-sensitive, voltage-gated sodium channels (NaV1) to action potential discharge from mouse esophageal tension mechanoreceptors.

Action potentials depend on voltage-gated sodium channels (NaV1s), which have nine α subtypes. NaV1 inhibition is a target for pathologies involving excitable cells such as pain. However, because NaV1 subtypes are widely expressed, inhibitors may inhibit regulatory sensory systems. Here, we investigated specific NaV1s and their inhibition in mouse esophageal mechanoreceptors-non-nociceptive vagal sensory afferents that are stimulated by low threshold mechanical distension, which regulate esophageal motility. Using single fiber electrophysiology, we found mechanoreceptor responses to esophageal distension were abolished by tetrodotoxin. Single-cell RT-PCR revealed that esophageal-labeled TRPV1-negative vagal neurons expressed multiple tetrodotoxin-sensitive NaV1s: NaV1.7 (almost all neurons) and NaV1.1, NaV1.2, and NaV1.6 (in ∼50% of neurons). Inhibition of NaV1.7, using PF-05089771, had a small inhibitory effect on mechanoreceptor responses to distension. Inhibition of NaV1.1 and NaV1.6, using ICA-121341, had a similar small inhibitory effect. The combination of PF-05089771 and ICA-121341 inhibited but did not eliminate mechanoreceptor responses. Inhibition of NaV1.2, NaV1.6, and NaV1.7 using LSN-3049227 inhibited but did not eliminate mechanoreceptor responses. Thus, all four tetrodotoxin-sensitive NaV1s contribute to action potential initiation from esophageal mechanoreceptors terminals. This is different to those NaV1s necessary for vagal action potential conduction, as demonstrated using GCaMP6s imaging of esophageal vagal neurons during electrical stimulation. Tetrodotoxin-sensitive conduction was abolished in many esophageal neurons by PF-05089771 alone, indicating a critical role of NaV1.7. In summary, multiple NaV1 subtypes contribute to electrical signaling in esophageal mechanoreceptors. Thus, inhibition of individual NaV1s would likely have minimal effect on afferent regulation of esophageal motility.

Tethering Cells via Enzymatic Oxidative Crosslinking Enables Mechanotransduction in Non-Cell-Adhesive Materials.

Cell-matrix interactions govern cell behavior and tissue function by facilitating transduction of biomechanical cues. Engineered tissues often incorporate these interactions by employing cell-adhesive materials. However, using constitutively active cell-adhesive materials impedes control over cell fate and elicits inflammatory responses upon implantation. Here, an alternative cell-material interaction strategy that provides mechanotransducive properties via discrete inducible on-cell crosslinking (DOCKING) of materials, including those that are inherently non-cell-adhesive, is introduced. Specifically, tyramine-functionalized materials are tethered to tyrosines that are naturally present in extracellular protein domains via enzyme-mediated oxidative crosslinking. Temporal control over the stiffness of on-cell tethered 3D microniches reveals that DOCKING uniquely enables lineage programming of stem cells by targeting adhesome-related mechanotransduction pathways acting independently of cell volume changes and spreading. In short, DOCKING represents a bioinspired and cytocompatible cell-tethering strategy that offers new routes to study and engineer cell-material interactions, thereby advancing applications ranging from drug delivery, to cell-based therapy, and cultured meat.

Disrupting biological sensors of force promotes tissue regeneration in large organisms.

Tissue repair and healing remain among the most complicated processes that occur during postnatal life. Humans and other large organisms heal by forming fibrotic scar tissue with diminished function, while smaller organisms respond with scarless tissue regeneration and functional restoration. Well-established scaling principles reveal that organism size exponentially correlates with peak tissue forces during movement, and evolutionary responses have compensated by strengthening organ-level mechanical properties. How these adaptations may affect tissue injury has not been previously examined in large animals and humans. Here, we show that blocking mechanotransduction signaling through the focal adhesion kinase pathway in large animals significantly accelerates wound healing and enhances regeneration of skin with secondary structures such as hair follicles. In human cells, we demonstrate that mechanical forces shift fibroblasts toward pro-fibrotic phenotypes driven by ERK-YAP activation, leading to myofibroblast differentiation and excessive collagen production. Disruption of mechanical signaling specifically abrogates these responses and instead promotes regenerative fibroblast clusters characterized by AKT-EGR1.

Peripheral antinociceptive effects of a bifunctional µ and δ opioid receptor ligand in rat model of inflammatory bladder pain.

There is a need to develop a novel analgesic for pain associated with interstitial cystitis/painful bladder syndrome (IC/PBS). The use of the conventional µ-opioid receptor agonists to manage IC/PBS pain is controversial due to adverse CNS effects. These effects are attenuated in benzylideneoxymorphone (BOM), a low-efficacy µ-opioid receptor agonist/δ-opioid receptor antagonist that attenuates thermal pain and is devoid of reinforcing effects. We hypothesize that BOM will inhibit bladder pain by attenuating responses of urinary bladder distension (UBD)-sensitive afferent fibers. Therefore, the effect of BOM was tested on responses of UBD-sensitive afferent fibers in L6 dorsal root from inflamed and non-inflamed bladder of rats. Immunohistochemical (IHC) examination reveals that following the induction of inflammation there were significant high expressions of µ, δ, and µ-δ heteromer receptors in DRG. BOM dose-dependently (1-10 mg/kg, i.v) attenuated mechanotransduction properties of these afferent fibers from inflamed but not from non-inflamed rats. In behavioral model of bladder pain, BOM significantly attenuated visceromotor responses (VMRs) to UBD only in inflamed group of rats when injected either systemically (10 mg/kg, i.v.) or locally into the bladder (0.1 ml of 10 mg/ml). Furthermore, oxymorphone (OXM), a high-efficacy µ-opioid receptor agonist, attenuated responses of mechanosensitive bladder afferent fibers and VMRs to UBD. Naloxone (10 mg/kg, i.v.) significantly reversed the inhibitory effects of BOM and OXM on responses of bladder afferent fibers and VMRs suggesting µ-opioid receptor-related analgesic effects of these compounds. The results reveal that a low-efficacy, bifunctional opioid-based compound can produce analgesia by attenuating mechanotransduction functions of afferent fibers innervating the urinary bladder.

Starvation induces shrinkage of the bacterial cytoplasm.

Environmental fluctuations are a common challenge for single-celled organisms; enteric bacteria such as Escherichia coli experience dramatic changes in nutrient availability, pH, and temperature during their journey into and out of the host. While the effects of altered nutrient availability on gene expression and protein synthesis are well known, their impacts on cytoplasmic dynamics and cell morphology have been largely overlooked. Here, we discover that depletion of utilizable nutrients results in shrinkage of E. coli's inner membrane from the cell wall. Shrinkage was accompanied by an ∼17% reduction in cytoplasmic volume and a concurrent increase in periplasmic volume. Inner membrane retraction after sudden starvation occurred almost exclusively at the new cell pole. This phenomenon was distinct from turgor-mediated plasmolysis and independent of new transcription, translation, or canonical starvation-sensing pathways. Cytoplasmic dry-mass density increased during shrinkage, suggesting that it is driven primarily by loss of water. Shrinkage was reversible: upon a shift to nutrient-rich medium, expansion started almost immediately at a rate dependent on carbon source quality. A robust entry into and recovery from shrinkage required the Tol-Pal system, highlighting the importance of envelope coupling during shrinkage and recovery. Klebsiella pneumoniae also exhibited shrinkage when shifted to carbon-free conditions, suggesting a conserved phenomenon. These findings demonstrate that even when Gram-negative bacterial growth is arrested, cell morphology and physiology are still dynamic.

DNA-Based Microparticle Tension Sensors (µTS) for Measuring Cell Mechanics in Non-planar Geometries and for High-Throughput Quantification.

Mechanotransduction, the interplay between physical and chemical signaling, plays vital roles in many biological processes. The state-of-the-art techniques to quantify cell forces employ deformable polymer films or molecular probes tethered to glass substrates. However, the applications of these assays in fundamental and clinical research are restricted by the planar geometry and low throughput of microscopy readout. Herein, we develop a DNA-based microparticle tension sensor, which features a spherical surface and thus allows for investigation of mechanotransduction at curved interfaces. The micron-scale of µTS enables flow cytometry readout, which is rapid and high throughput. We applied the method to map and measure T-cell receptor forces and platelet integrin forces at 12 and 56 pN thresholds. Furthermore, we quantified the inhibition efficiency of two anti-platelet drugs providing a proof-of-concept demonstration of µTS to screen drugs that modulate cellular mechanics.

Mechanotransduction channel Piezo is widely expressed in the spider, Cupiennius salei, mechanosensory neurons and central nervous system.

Mechanosensory neurons use mechanotransduction (MET) ion channels to detect mechanical forces and displacements. Proteins that function as MET channels have appeared multiple times during evolution and occur in at least four different families: the DEG/ENaC and TRP channels, as well as the TMC and Piezo proteins. We found twelve putative members of MET channel families in two spider transcriptomes, but detected only one, the Piezo protein, by in situ hybridization in their mechanosensory neurons. In contrast, probes for orthologs of TRP, ENaC or TMC genes that code MET channels in other species did not produce any signals in these cells. An antibody against C. salei Piezo detected the protein in all parts of their mechanosensory cells and in many neurons of the CNS. Unspecific blockers of MET channels, Ruthenium Red and GsMTx4, had no effect on the mechanically activated currents of the mechanosensory VS-3 neurons, but the latter toxin reduced action potential firing when these cells were stimulated electrically. The Piezo protein is expressed throughout the spider nervous system including the mechanosensory neurons. It is possible that it contributes to mechanosensory transduction in spider mechanosensilla, but it must have other functions in peripheral and central neurons.

Identification of a series of hair-cell MET channel blockers that protect against aminoglycoside-induced ototoxicity.

To identify small molecules that shield mammalian sensory hair cells from the ototoxic side effects of aminoglycoside antibiotics, 10,240 compounds were initially screened in zebrafish larvae, selecting for those that protected lateral-line hair cells against neomycin and gentamicin. When the 64 hits from this screen were retested in mouse cochlear cultures, 8 protected outer hair cells (OHCs) from gentamicin in vitro without causing hair-bundle damage. These 8 hits shared structural features and blocked, to varying degrees, the OHC's mechano-electrical transducer (MET) channel, a route of aminoglycoside entry into hair cells. Further characterization of one of the strongest MET channel blockers, UoS-7692, revealed it additionally protected against kanamycin and tobramycin and did not abrogate the bactericidal activity of gentamicin. UoS-7692 behaved, like the aminoglycosides, as a permeant blocker of the MET channel; significantly reduced gentamicin-Texas red loading into OHCs; and preserved lateral-line function in neomycin-treated zebrafish. Transtympanic injection of UoS-7692 protected mouse OHCs from furosemide/kanamycin exposure in vivo and partially preserved hearing. The results confirmed the hair-cell MET channel as a viable target for the identification of compounds that protect the cochlea from aminoglycosides and provide a series of hit compounds that will inform the design of future otoprotectants.

CB2 cannabinoid receptor agonist selectively inhibits the mechanosensitivity of mucosal afferents in the guinea pig bladder.

Bladder afferents play a pivotal role in bladder function such as urine storage and micturition as well as conscious sensations such as urgency and pain. Endocannabinoids are ligands of cannabinoid 1 and 2 (CB1 and CB2) receptors but can influence the activity of a variety of G protein-coupled receptors as well as ligand-gated and voltage-gated channels. It is still not known which classes of bladder afferents are influenced by CB1 and CB2 receptor agonists. This study aimed to determine the role of CB2 receptors in two major classes of afferents in the guinea pig bladder: mucosal and muscular-mucosal. The mechanosensitivity of these two classes was determined by an ex vivo extracellular electrophysiological recording technique. A stable analog of endocannabinoid anandamide, methanandamide (mAEA), potentiated the mechanosensitivity of mucosal bladder afferents in response to stroking. In the presence of a transient receptor potential vanilloid 1 antagonist (capsazepine), the effect of mAEA switched from excitatory to inhibitory. A selective CB2 receptor agonist, 4-quinolone-3-carboxyamide (4Q3C), significantly inhibited the mechanosensitivity of mucosal bladder afferents to stroking. In the presence of a CB2 receptor antagonist, the inhibitory effect of 4Q3C was lost. mAEA and 4Q3C did not affect responses to stretch and/or mucosal stroking of muscular-mucosal afferents. Our findings revealed that agonists of CB2 receptors selectively inhibited the mechanosensitivity of capsaicin-sensitive mucosal bladder afferents but not muscular-mucosal afferents. This may have important implications for understanding of the role of endocannabinoids in modulating bladder function and sensation in health and diseases.NEW & NOTEWORTHY This article describes, for the first time, to our knowledge, the direct inhibitory effect of cannabinoid 2 receptor agonists on guinea pig mucosal bladder afferents. The cannabinoid 2 receptor is involved in pain and inflammation, suggesting that this may be a viable target for treatment of bladder disorders such as cystitis.

Angiotensin II type 1 receptor is involved in flow-induced vasomotor responses of isolated middle cerebral arteries: role of oxidative stress.

This study aimed to determine the mechanosensing role of angiotensin II type 1 receptor (AT1R) in flow-induced dilation (FID) and oxidative stress production in middle cerebral arteries (MCA) of Sprague-Dawley rats. Eleven-week old, healthy male Sprague-Dawley rats on a standard diet were given the AT1R blocker losartan (1 mg/mL) in drinking water (losartan group) or tap water (control group) ad libitum for 7 days. Blockade of AT1R attenuated FID and acetylcholine-induced dilation was compared with control group. Nitric oxide (NO) synthase inhibitor Nω-nitro-l-arginine methyl ester (l-NAME) and cyclooxygenase inhibitor indomethacin (Indo) significantly reduced FID in control group. The attenuated FID in losartan group was further reduced by Indo only at Δ100 mmHg, whereas l-NAME had no effect. In losartan group, Tempol (a superoxide scavenger) restored dilatation, whereas Tempol + l-NAME together significantly reduced FID compared with restored dilatation with Tempol alone. Direct fluorescence measurements of NO and reactive oxygen species (ROS) production in MCA, in no-flow conditions revealed significantly reduced vascular NO levels with AT1R blockade compared with control group, whereas in flow condition increased the NO and ROS production in losartan group and had no effect in the control group. In losartan group, Tempol decreased ROS production in both no-flow and flow conditions. AT1R blockade elicited increased serum concentrations of ANG II, 8-iso-PGF2α, and TBARS, and decreased antioxidant enzyme activity (SOD and CAT). These results suggest that in small isolated cerebral arteries: 1) AT1 receptor maintains dilations in physiological conditions; 2) AT1R blockade leads to increased vascular and systemic oxidative stress, which underlies impaired FID.NEW & NOTEWORTHY The AT1R blockade impaired the endothelium-dependent, both flow- and acetylcholine-induced dilations of MCA by decreasing vascular NO production and increasing the level of vascular and systemic oxidative stress, whereas it mildly influenced the vascular wall inflammatory phenotype, but had no effect on the systemic inflammatory response. Our data provide functional and molecular evidence for an important role of AT1 receptor activation in physiological conditions, suggesting that AT1 receptors have multiple biological functions.

Mechanical stiffness augments ligand-dependent progesterone receptor B activation via MEK 1/2 and Rho/ROCK-dependent signaling pathways in uterine fibroid cells.

OBJECTIVE: To test whether mechanical substrate stiffness would influence progesterone receptor B (PRB) signaling in fibroid cells. Uterine fibroids feature an excessive extracellular matrix, increased stiffness, and altered mechanical signaling. Fibroid growth is stimulated by progestins and opposed by anti-progestins, but a functional interaction between progesterone action and mechanical signaling has not been evaluated. DESIGN: Laboratory studies. SETTING: Translational science laboratory. PATIENT(S)/ANIMAL(S): Human fibroid cell lines and patient-matched fibroid and myometrial cell lines. INTERVENTION(S): Progesterone receptor B-dependent reporter assays and messenger RNA quantitation in cells cultured on stiff polystyrene plates (3GPa) or soft silicone plates (930KPa). Pharmacologic inhibitors of extracellular signal-related protein kinase (ERK) kinase 1/2 (MEK 1/2; PD98059), p38 mitogen-activated protein kinase (SB202190), receptor tyrosine kinases (RTKs; nintedanib), RhoA (A13), and Rho-associated coiled-coil kinase (ROCK; Y27632). MAIN OUTCOME MEASURE(S): Progesterone-responsive reporter activation. RESULT(S): Fibroid cells exhibited higher PRB-dependent reporter activity with progesterone (P4) in cells cultured on stiff vs. soft plates. Mechanically induced PRB activation with P4 was decreased 62% by PD98059, 78% by nintedanib, 38% by A13, and 50% by Y27632. Overexpression of the Rho-guanine nucleotide exchange factor (Rho-GEF), AKAP13, significantly increased PRB-dependent reporter activity. Collagen 1 messenger RNA levels were higher in fibroid cells grown on stiff vs. soft plates with P4. CONCLUSION(S): Cells cultured on mechanically stiff substrates had enhanced PRB activation via a mechanism that required MEK 1/2 and AKAP13/RhoA/ROCK signaling pathways. These studies provide a framework to explore the mechanisms by which mechanical stiffness affects progesterone receptor activation.

High dietary salt amplifies osmoresponsiveness in vasopressin-releasing neurons.

High dietary salt increases arterial pressure partly through activation of magnocellular neurosecretory cells (MNCVP) that secrete the antidiuretic and vasoconstrictor hormone vasopressin (VP) into the circulation. Here, we show that the intrinsic and synaptic excitation of MNCVP caused by hypertonicity are differentially potentiated in two models of salt-dependent hypertension in rats. One model combined salty chow with a chronic subpressor dose of angiotensin II (AngII-salt), the other involved replacing drinking water with 2% NaCl (salt loading, SL). In both models, we observed a significant increase in the quantal amplitude of EPSCs on MNCVP. However, model-specific changes were also observed. AngII-salt increased the probability of glutamate release by osmoreceptor afferents and increased overall excitatory network drive. In contrast, SL specifically increased membrane stiffness and the intrinsic osmosensitivity of MNCVP. These results reveal that dietary salt increases the excitability of MNCVP through effects on the cell-autonomous and synaptic osmoresponsiveness of MNCVP.

Theophylline alleviates gentamicin-induced cytotoxicity to sensory hair cells by maintaining HDAC2 expression.

Sensorineural hearing loss is a health problem with global prevalence. Aminoglycoside antibiotics, for instance gentamicin, may cause ototoxicity in mammals as a result of apoptosis and elevated oxidative stress in cochlear hair cells. Our study aimed to examine the potential effects of theophylline, an HDAC2 agonist, on gentamicin-induced cytotoxicity to sensory hair cells. Mouse cochlear explants and HEI-OC1 cells were in vitro cultured and challenged by gentamicin to induce ototoxicity, with or without theophylline. Cochlear hair cells were evaluated by fluorescent microscopy, and their mechanotransduction was assessed by electrophysiology. Expression levels of HDAC2 and apoptosis pathway factors were also evaluated following gentamicin and theophylline treatments. The functional role of HDAC2 in this setting was investigated by siRNA targeted silencing. Theophylline protected cochlear hair cells from ototoxicity induced by gentamicin, in terms of preserving cochlear structure and mechanotransduction ability, and preventing the activation of the intrinsic apoptosis pathway dose-dependently. HDAC2 expression was downregulated by gentamicin, which could be restored by theophylline. HDAC2 silencing in HEI-OC1 cells negated the beneficial effect of theophylline against gentamicin-induced growth defect and apoptosis activation. Theophylline protects sensory hair cells from gentamicin ototoxicity by maintaining HDAC2 expression. Our study thereby discovers a critical role of HDAC2 in gentamicin-induced ototoxicity, which could shine light on potential therapeutic options for treatment against sensorineural hearing loss.

Mechanical stretch promotes hypertrophic scar formation through mechanically activated cation channel Piezo1.

Hypertrophic scar (HS) formation is a skin fibroproliferative disease that occurs following a cutaneous injury, leading to functional and cosmetic impairment. To date, few therapeutic treatments exhibit satisfactory outcomes. The mechanical force has been shown to be a key regulator of HS formation, but the underlying mechanism is not completely understood. The Piezo1 channel has been identified as a novel mechanically activated cation channel (MAC) and is reportedly capable of regulating force-mediated cellular biological behaviors. However, the mechanotransduction role of Piezo1 in HS formation has not been investigated. In this work, we found that Piezo1 was overexpressed in myofibroblasts of human and rat HS tissues. In vitro, cyclic mechanical stretch (CMS) increased Piezo1 expression and Piezo1-mediated calcium influx in human dermal fibroblasts (HDFs). In addition, Piezo1 activity promoted HDFs proliferation, motility, and differentiation in response to CMS. More importantly, intradermal injection of GsMTx4, a Piezo1-blocking peptide, protected rats from stretch-induced HS formation. Together, Piezo1 was shown to participate in HS formation and could be a novel target for the development of promising therapies for HS formation.

Visualization of the mechanosensitive ion channel MscS under membrane tension.

Mechanosensitive channels sense mechanical forces in cell membranes and underlie many biological sensing processes1-3. However, how exactly they sense mechanical force remains under investigation4. The bacterial mechanosensitive channel of small conductance, MscS, is one of the most extensively studied mechanosensitive channels4-8, but how it is regulated by membrane tension remains unclear, even though the structures are known for its open and closed states9-11. Here we used cryo-electron microscopy to determine the structure of MscS in different membrane environments, including one that mimics a membrane under tension. We present the structures of MscS in the subconducting and desensitized states, and demonstrate that the conformation of MscS in a lipid bilayer in the open state is dynamic. Several associated lipids have distinct roles in MscS mechanosensation. Pore lipids are necessary to prevent ion conduction in the closed state. Gatekeeper lipids stabilize the closed conformation and dissociate with membrane tension, allowing the channel to open. Pocket lipids in a solvent-exposed pocket between subunits are pulled out under sustained tension, allowing the channel to transition to the subconducting state and then to the desensitized state. Our results provide a mechanistic underpinning and expand on the 'force-from-lipids' model for MscS mechanosensation4,11.

Mechanical Stress Induces Ca2+-Dependent Signal Transduction in Erythroblasts and Modulates Erythropoiesis.

Bioreactors are increasingly implemented for large scale cultures of various mammalian cells, which requires optimization of culture conditions. Such upscaling is also required to produce red blood cells (RBC) for transfusion and therapy purposes. However, the physiological suitability of RBC cultures to be transferred to stirred bioreactors is not well understood. PIEZO1 is the most abundantly expressed known mechanosensor on erythroid cells. It is a cation channel that translates mechanical forces directly into a physiological response. We investigated signaling cascades downstream of PIEZO1 activated upon transitioning stationary cultures to orbital shaking associated with mechanical stress, and compared the results to direct activation of PIEZO1 by the chemical agonist Yoda1. Erythroblasts subjected to orbital shaking displayed decreased proliferation, comparable to incubation in the presence of a low dose of Yoda1. Epo (Erythropoietin)-dependent STAT5 phosphorylation, and Calcineurin-dependent NFAT dephosphorylation was enhanced. Phosphorylation of ERK was also induced by both orbital shaking and Yoda1 treatment. Activation of these pathways was inhibited by intracellular Ca2+ chelation (BAPTA-AM) in the orbital shaker. Our results suggest that PIEZO1 is functional and could be activated by the mechanical forces in a bioreactor setup, and results in the induction of Ca2+-dependent signaling cascades regulating various aspects of erythropoiesis. With this study, we showed that Yoda1 treatment and mechanical stress induced via orbital shaking results in comparable activation of some Ca2+-dependent pathways, exhibiting that there are direct physiological outcomes of mechanical stress on erythroblasts.