Activation Of Mechanosensitive Channels Research Articles

Dissecting cell membrane tension dynamics and its effect on Piezo1-mediated cellular mechanosensitivity using force-controlled nanopipettes

The dynamics of cellular membrane tension and its role in mechanosensing, which is the ability of cells to respond to physical stimuli, remain incompletely understood, mainly due to the lack of appropriate tools. Here, we report a force-controlled nanopipette-based method that combines fluidic force microscopy with fluorescence imaging for precise manipulation of the cellular membrane tension while monitoring the impact on single-cell mechanosensitivity. The force-controlled nanopipette enables control of the indentation force imposed on the cell cortex as well as of the aspiration pressure applied to the plasma membrane. We show that this setup can be used to concurrently monitor the activation of Piezo1 mechanosensitive ion channels via calcium imaging. Moreover, the spatiotemporal behavior of the tension propagation is assessed with the fluorescent membrane tension probe Flipper-TR, and further dissected using molecular dynamics modeling. Finally, we demonstrate that aspiration and indentation act independently on the cellular mechanobiological machinery, that indentation induces a local pre-tension in the membrane, and that membrane tension stays confined by links to the cytoskeleton.

Piezo1 stretch-activated channel activity differs between murine bone marrow-derived and cardiac tissue-resident macrophages.

Macrophages (MΦ) play pivotal roles in tissue homeostasis and repair. Their mechanical environment has been identified as a key modulator of various cell functions, and MΦ mechanosensitivity is likely to be critical - in particular in a rhythmically contracting organ such as the heart. Cultured MΦ, differentiated in vitro from bone marrow (MΦBM), form a popular research model. This study explores the activity of mechanosensitive ion channels (MSC) in murine MΦBM and compares it to MSC activity in MΦ enzymatically isolated from cardiac tissue (tissue-resident MΦ; MΦTR). We show that MΦBM and MΦTR have stretch-induced currents, indicating the presence of functional MSC in their plasma membrane. The current profiles in MΦBM and in MΦTR show characteristics of cation non-selective MSC such as Piezo1 or transient receptor potential channels. While Piezo1 ion channel activity is detectable in the plasma membrane of MΦBM using the patch-clamp technique, or by measuring cytosolic calcium concentration upon perfusion with the Piezo1 channel agonist Yoda1, no Piezo1 channel activity was observed in MΦTR. The selective transient receptor potential vanilloid 4 (TRPV4) channel agonist GSK1016790A induces calcium entry in MΦTR and in MΦBM. In MΦ isolated from left-ventricular scar tissue 28days after cryoablation, stretch-induced current characteristics are not significantly different compared to non-injured control tissue, even though scarred ventricular tissue is expected to be mechanically remodelled and to contain an altered composition of pre-existing cardiac and circulation-recruited MΦ. Our data suggest that the in vitro differentiation protocols used to obtain MΦBM generate cells that differ from MΦ recruited from the circulation during tissue repair in vivo. Further investigations are needed to explore MSC identity in lineage-traced MΦ in scar tissue, and to compare mechanosensitivity of circulating monocytes with that of MΦBM. KEY POINTS: Bone marrow-derived (MΦBM) and tissue resident (MΦTR) macrophages have stretch-induced currents, indicating expression of functional mechanosensitive channels (MSC) in their plasma membrane. Stretch-activated current profiles show characteristics of cation non-selective MSC; and mRNA coding for MSC, including Piezo1 and TRPV4, is expressed in murine MΦBM and in MΦTR. Calcium entry upon pharmacological activation of TRPV4 confirms functionality of the channel in MΦTR and in MΦBM. Piezo1 ion channel activity is detected in the plasma membrane of MΦBM but not in MΦTR, suggesting that MΦBM may not be a good model to study the mechanotransduction of MΦTR. Stretch-induced currents, Piezo1mRNA expression and response to pharmacological activation are not significantly changed in cardiac MΦ 28days after cryoinjury compared to sham operated mice.

Calcium influx promotes PLEKHG4B localization to cell-cell junctions and regulates the integrity of junctional actin filaments.

PLEKHG4B is a Cdc42-targeting guanine-nucleotide exchange factor implicated in forming epithelial cell-cell junctions. Here we explored the mechanism regulating PLEKHG4B localization. PLEKHG4B localized to the basal membrane in normal Ca2+ medium but accumulated at cell-cell junctions upon ionomycin treatment. Ionomycin-induced junctional localization of PLEKHG4B was suppressed upon disrupting its annexin-A2 (ANXA2)-binding ability. Thus, Ca2+ influx and ANXA2 binding are crucial for PLEKHG4B localization to cell-cell junctions. Treatments with low Ca2+ or BAPTA-AM (an intracellular Ca2+ chelator) suppressed PLEKHG4B localization to the basal membrane. Mutations of the phosphoinositide-binding motif in the pleckstrin homology (PH) domain of PLEKHG4B or masking of membrane phosphatidylinositol-4,5-biphosphate [PI(4,5)P2] suppressed PLEKHG4B localization to the basal membrane, indicating that basal membrane localization of PLEKHG4B requires suitable intracellular Ca2+ levels and PI(4,5)P2 binding of the PH domain. Activation of mechanosensitive ion channels (MSCs) promoted PLEKHG4B localization to cell-cell junctions, and their inhibition suppressed it. Moreover, similar to the PLEKHG4B knockdown phenotypes, inhibition of MSCs or treatment with BAPTA-AM disturbed the integrity of actin filaments at cell-cell junctions. Taken together, our results suggest that Ca2+ influx plays crucial roles in PLEKHG4B localization to cell-cell junctions and the integrity of junctional actin organization, with MSCs contributing to this process.

Tunneling nanotube-transmitted mechanical signal and its cellular response

Tunneling nanotubes (TNTs) are elastic tubular structures that physically link cells, facilitating the intercellular transfer of organelles, chemical signals, and electrical signals. Despite TNTs serving as a multifunctional pathway for cell-cell communication, the transmission of mechanical signals through TNTs and the response of TNT-connected cells to these forces remain unexplored. In this study, external mechanical forces were applied to induce TNT bending between rat kidney (NRK) cells using micromanipulation. These forces, transmitted via TNTs, induced reduced curvature of the actin cortex and increased membrane tension at the TNT-connected sites. Additionally, TNT bending results in an elevation of intracellular calcium levels in TNT-connected cells, a response attenuated by gadolinium ions, a non-selective mechanosensitive calcium channel blocker. The degree of TNT deflection positively correlated with decreased actin cortex curvature and increased calcium levels. Furthermore, stretching TNT due to the separation of TNT-connected cells resulted in decreased actin cortex curvature and increased intracellular calcium in TNT-connected cells. The levels of these cellular responses depended on the length changes of TNTs. Moreover, TNT connections influence cell migration by regulating cell rotation, which involves the activation of mechanosensitive calcium channels. In conclusion, our study revealed the transmission of mechanical signals through TNTs and the subsequent responses of TNT-connected cells, highlighting a previously unrecognized communication function of TNTs. This research provides valuable insights into the role of TNTs in long-distance intercellular mechanical signaling.

Ca2+ -activated K+ channels regulate cell volume in human glioblastoma cells.

Glioblastoma (GBM), the most lethal form of brain tumors, bases its malignancy on the strong ability of its cells to migrate and invade the narrow spaces of healthy brain parenchyma. Cell migration and invasion are both critically dependent on changes in cell volume and shape driven by the transmembrane transport of osmotically important ions such as K+ and Cl- . However, while the Cl- channels participating in cell volume regulation have been clearly identified, the precise nature of the K+ channels involved is still uncertain. Using a combination of electrophysiological and imaging approaches in GBM U87-MG cells, we found that hypotonic-induced cell swelling triggered the opening of Ca2+ -activated K+ (KCa ) channels of large and intermediate conductance (BKCa and IKCa , respectively), both highly expressed in GBM cells. The influx of Ca2+ mediated by the hypotonic-induced activation of mechanosensitive channels was found to be a key step for opening both the BKCa and the IKCa channels. Finally, the activation of both KCa channels mediated by mechanosensitive channels was found to be essential for the development of the regulatory volume decrease following hypotonic shock. Taken together, these data indicate that KCa channels are the main K+ channels responsible for the volume regulation in U87-MG cells.

Epithelial cells sense local stiffness via Piezo1 mediated cytoskeletal reorganization.

Local substrate stiffness is one of the major mechanical inputs for tissue organization during its development and remodeling. It is widely recognized that adherent cells use transmembrane proteins (integrins) at focal adhesions to translate ECM mechanical cues into intracellular bioprocess. Here we show that epithelial cells respond to substrate stiffening primarily via actin cytoskeleton organization, that requires activation of mechanosensitive Piezo1 channels. Piezo1 Knockdown cells eliminated the actin stress fibers that formed on stiff substrates, while it had minimal effect on cell morphology and spreading area. Inhibition of Piezo1 channels with GsMTx4 also significantly reduced stiffness-induced F-actin reorganization, suggesting Piezo1 mediated cation current plays a role. Activation of Piezo1 channels with specific agonist (Yoda1) resulted in thickening of F-actin fibers and enlargement of FAs on stiffer substrates, whereas it did not affect the formation of nascent FAs that facilitate spreading on the soft substrates. These results demonstrate that Piezo1 functions as a force sensor that couples with actin cytoskeleton to distinguish the substrate stiffness and facilitate epithelial adaptive remodeling.

Fibrous Viscoelastic Extracellular Matrix Assists Precise Neuronal Connectivity

AbstractSynapse formation in complex neuronal network is a pivotal process for normal functioning of nervous system. Although intense research has been conducted, how neurons and axons are guided toward the target remains largely unclear. In traditional opinions, axons are directed through chemotaxis, while recently mechanotaxis has been brought up as a potential complementary mechanism, as it can provide delicately controlled signals in addition to the random diffusive chemical cues. To further explore the path‐finding mechanism, a quasi‐3D in vitro model for neuronal cells is constructed by integrating hydrogel collagen I as extracellular matrix (ECM), and primary mouse cortical neurons and PC12 cells are tested. It is strikingly found out that axons and neuronal cells can be precisely guided toward target neurites via ECM. By developing a label‐free traction force microscopy technique, the force networks among neurons are presented, validating that the fibrous matrix‐transmitted paratensile signals can assist the axon pathfinding. This precise axon guidance is related to the activation of mechanosensitive ion channels, calcium signaling, and probably the following F‐actin assembly. This mechanism can potentially assist developing clinical applications and designing biomaterials in near future.

Membrane stretch due to hypoosmolality leads to the development of various pulmonary and cardiovascular diseases

High blood pressures over a long period of time can lead to the development of diseases such as congestive heart failure(CHF). Congestive heart failure also commonly leads to the development of conditions such as hyponatremia, a common electrolyte disorder which increases the body’s water levels, creating hypoosmotic conditions for cells; these hypoosmotic conditions cause cell swelling, which subsequently causes mechanical stress on the cells. In these hypoosmotic environments, specific mechanosensitive ion channels which are activated by these mechanical stresses on cells may play a critical role in the progression of conditions resulting from these hypoosmotic environments, such as pulmonary arterial hypertension (PAH) and CHF. This study aims to look at the connection between the effects of hypoosmotic conditions in cells on the pathogenesis of the development of chronic diseases such as PAH and CHF. Mechanical actions on specific mechanosensitive ion channels such as Piezo1 have already been shown to play a critical role in regulating intracellular Ca2+homeostasis, which play an important part in the regulation of the vascular tone in places such as the pulmonary arteries; mechanical stimulation from hypotonic environments such as those seen in hyponatremia lead to Piezo1-mediated increases of intracellular Ca2+, which are subsequently linked to the increased contraction and proliferation of pulmonary arterial endothelial cells(PAECs). The proliferation of PAECs contribute to the pathogenesis of diseases, such as pulmonary arterial hypertension(PAH) or CHF, which there is currently no cure for. Patch-clamp experiments were conducted to examine the activity of mechanosensitive cation currents; graphed results showed that the activity of mechanosensitive cation currents were significantly increased (p<0.05) in PAECs treated with a hypoosmotic solution when compared with PAECs treated in an isosmotic control solution. Digital fluorescence microscopy using fura-2 AM, a membrane-permeable Ca2+ sensitive fluorescent indicator, and a Nikon digital imaging fluorescent microscopy system was also used to measure intracellular Ca2+ concentration in PAECs; graphed data indicated that the change in intracellular calcium concentration in PAECs was significantly increased(p<0.05) when placed in a hypoosmotic solution, than when placed in an isosmotic solution. The collective data indicates that hypoosmotic conditions increase the activity of mechanosensitive ion channels, as well as intracellular Ca2+ levels, which may prove the role of mechanosensitive ion channels like Piezo1 and TRPC6 in the development of diseases such as PAH and CHF. This work is supported in part by the National Heart, Lung, and Blood Institute of the National Institutes of Health Grants HL135807 and HL146764. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The Fifth Bioelectronic Medicine Summit Hosted by the Feinstein Institutes for Medical Research (Manhasset, NY) and Columbia Engineering (NY, NY) at The Garden City Hotel, Garden City, NY 11530 on October 11-12th, 2022- Bioelectronic Medicine: Today’s Tools, Tomorrow’s Therapies

The Fifth Bioelectronic Medicine Summit Hosted by the Feinstein Institutes for Medical Research (Manhasset, NY) and Columbia Engineering (NY, NY) at The Garden City Hotel, Garden City, NY 11530 on October 11-12th, 2022- Bioelectronic Medicine: Today’s Tools, Tomorrow’s Therapies

In silico analysis of the contribution of cardiomyocyte-fibroblast electromechanical interaction to the arrhythmia.

Although fibroblasts are about 5-10times smaller than cardiomyocytes, their number in the ventricle is about twice that of cardiomyocytes. The high density of fibroblasts in myocardial tissue leads to a noticeable effect of their electromechanical interaction with cardiomyocytes on the electrical and mechanical functions of the latter. Our work focuses on the analysis of the mechanisms of spontaneous electrical and mechanical activity of the fibroblast-coupled cardiomyocyte during its calcium overload, which occurs in a variety of pathologies, including acute ischemia. For this study, we developed a mathematical model of the electromechanical interaction between cardiomyocyte and fibroblasts and used it to simulate the impact of overloading cardiomyocytes. In contrast to modeling only the electrical interaction between cardiomyocyte and fibroblasts, the following new features emerge in simulations with the model that accounts for both electrical and mechanical coupling and mechano-electrical feedback loops in the interacting cells. First, the activity of mechanosensitive ion channels in the coupled fibroblasts depolarizes their resting potential. Second, this additional depolarization increases the resting potential of the coupled myocyte, thus augmenting its susceptibility to triggered activity. The triggered activity associated with the cardiomyocyte calcium overload manifests itself in the model either as early afterdepolarizations or as extrasystoles, i.e., extra action potentials and extra contractions. Analysis of the model simulations showed that mechanics contribute significantly to the proarrhythmic effects in the cardiomyocyte overloaded with calcium and coupled with fibroblasts, and that mechano-electrical feedback loops in both the cardiomyocyte and fibroblasts play a key role in this phenomenon.

Magnetic Ion Channel Activation (MICA)-Enabled Screening Assay: A Dynamic Platform for Remote Activation of Mechanosensitive Ion Channels.

This study reports results of a mechanical platform-based screening assay (MICA) to evaluate the remote activation of mechanosensitive ion channels. Here, we studied ERK pathway activation and the elevation in intracellular Ca2+ levels in response to the MICA application using the Luciferase assay and Fluo-8AM assay, respectively. Functionalised magnetic nanoparticles (MNPs) targeting membrane-bound integrins and mechanosensitive TREK1 ion channels were studied with HEK293 cell lines under MICA application. The study demonstrated that active targeting of mechanosensitive integrins via RGD (Arginylglycylaspartic acid) motifs or TREK1 (KCNK2, potassium channel subfamily K member 2) ion channels can stimulate the ERK pathway and intracellular calcium levels compared to non-MICA controls. This screening assay offers a powerful tool, which aligns with existing high-throughput drug screening platforms for use in the assessment of drugs that interact with ion channels and influence ion channel-modulated diseases.

Quantifying pressure and ultrasound activation of the mechanosensitive K+ channel TRAAK.

The study of mechanosensitive ion channels depends upon electrophysiological recordings coupled with mechanical stimulation by cell poking, pillar arrays, or pressure application via to excised membrane patches. In each of these environments, mechanical energy is deposited into the membrane and causes the membrane to stretch, inducing tension. Unlike other assays, pressure stimulation with simultaneous imaging of patched membranes permits tension, the relevant gating stimulus for most mechanosensitive channels, to be calculated. Here, we quantify the mechanical tuning of the mechanosensitive K+ channel TRAAK and compare it to the bacterial mechanosensitive channel MscS. We find TRAAK is activated at lower tension values and over a wider range of tension compared to MscS. We further show that ultrasound activates TRAAK and MscS in excised patches. Changes in membrane curvature induced by ultrasound or pressure stimulation resulted in the same degree of channel activation. This suggests ultrasound activation of mechanosensitive ion channels is due to increased membrane tension rather than alternative mechanisms posited for transfer of acoustic energy to channels.

“Force-From-Lipids” Dependence of the MscCG Mechanosensitive Channel Gating on Anionic Membranes

Mechanosensory transduction in Corynebacterium glutamicum plays a major role in glutamate efflux for industrial MSG, whose production depends on the activation of MscCG-type mechanosensitive channels. Dependence of the MscCG channel activation by membrane tension on the membrane lipid content has to date not been functionally characterized. Here, we report the MscCG channel patch clamp recording from liposomes fused with C. glutamicum membrane vesicles as well as from proteoliposomes containing the purified MscCG protein. Our recordings demonstrate that mechanosensitivity of MscCG channels depends significantly on the presence of negatively charged lipids in the proteoliposomes. MscCG channels in liposome preparations fused with native membrane vesicles exhibited the activation threshold similar to the channels recorded from C. glutamicum giant spheroplasts. In comparison, the activation threshold of the MscCG channels reconstituted into azolectin liposomes was higher than the activation threshold of E. coli MscL, which is gated by membrane tension close to the bilayer lytic tension. The spheroplast-like activation threshold was restored when the MscCG channels were reconstituted into liposomes made of E. coli polar lipid extract. In liposomes made of polar lipids mixed with synthetic phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin, the activation threshold of MscCG was significantly reduced compared to the activation threshold recorded in azolectin liposomes, which suggests the importance of anionic lipids for the channel mechanosensitivity. Moreover, the micropipette aspiration technique combined with patch fluorometry demonstrated that membranes containing anionic phosphatidylglycerol are softer than membranes containing only polar non-anionic phosphatidylcholine and phosphatidylethanolamine. The difference in mechanosensitivity between C. glutamicum MscCG and canonical MscS of E. coli observed in proteoliposomes explains the evolutionary tuning of the force from lipids sensing in various bacterial membrane environments.

Dynamically Reconstructed Collagen Fibers for Transmitting Mechanical Signals to Assist Macrophages Tracing Breast Cancer Cells

AbstractConstructing proper in vitro tumor immune microenvironment (TIME) is important for cancer immune‐therapy studies, while the selection of biomaterials is critical. As innate immune cells, macrophages can target and kill cancer cells in vivo at the early stage of tumor development. However, this targeting phenomenon has not been observed in vitro. Herein, a quasi‐3D in vitro cell culture model is constructed to mimic TIME by integrating hydrogel collagen as extracellular matrix for cells. In the collagen‐based quasi‐3D in vitro system, for the first time, it is found that macrophages can be attracted toward cancer cells along the dynamically reconstructed collagen fibers. By combining traction force microscopy and customized micro‐manipulator system, it is revealed that the collagen matrix‐transmitted tensile force signaling precisely guides the migration of macrophages toward cancer cells. The mechano‐responsiveness mechanism is related to the activation of mechanosensitive ion channels, and the induced local increase of calcium signal, which is proved to enhance the F‐actin assembly and to guide the cell migration. This novel mechanism advances the understanding of the role of collagen fibers in mechanotaxis of macrophages. Taken together, it has great potential for assisting biomaterial designs in developing new drug‐screening models and clinical strategies for cancer immune‐therapy.

Analysis of Possible Mechanisms of Endometrial Stem Cell Migration Suppression by Selective Chemical Activation of Piezo1 Mechanosensitive Channels

Analysis of Possible Mechanisms of Endometrial Stem Cell Migration Suppression by Selective Chemical Activation of Piezo1 Mechanosensitive Channels

Periodontal ligaments enhance neurite outgrowth in trigeminal ganglion neurons through Wnt5a production induced by mechanical stimulation.

The peripheral sensory nerve must be maintained to perceive environmental changes. Daily physiological mechanical stimulations, like gravity, floor reaction force, and occlusal force, influence the nerve homeostasis directly or indirectly. Although the direct axonal membrane stretch enhances axon outgrowth via mechanosensitive channel activation, the indirect mechanisms remain to be elucidated. In this study, we identified the indirect pathways where Wnt5a was a molecular cue released by mechanically stimulated rat periodontal ligament (rPDL) cells. qRT-PCR and ELISA showed that mechanically stimulated rPDL cells enhanced Wnt5a expression level and Wnt5a protein in a Ca2+-dependent manner. The inhibitors of PI3K (LY294002) and MEK1/2 (U0126) suppressed the Akt/PKB and ERK1/2 phosphorylation, respectively, in Western blotting analysis and consequently abolished the increase in Wnt5a expression. Similarly, PF573228, a focal adhesion kinase inhibitor, attenuated Akt- and ERK1/2-phosphorylation and Wnt5a expression. Importantly, the culture medium of stretched PDL cells enhanced neurite elongation, sprouting, and branching in trigeminal ganglion neurons that project to PDL. Moreover, treatment with an anti-Wnt5a antibody (to neutralize Wnt5a activity), AP7677a (anti-Ryk antibody, to block Ryk receptor activity), or strictinin (Ror1 inhibitor) suppressed the morphological changes. These findings reveal the indirect mechanisms that Wnt5a, released from the connective tissues in response to mechanical stimulation, enhances the outgrowth of the peripheral nerves. Our study suggests that the peripheral connective tissues regulate peripheral nerve homeostasis and that Wnt5a signaling could be targeted for the treatment of peripheral nerve disorders.

Activation of Mechanosensitive Ion Channels by Ultrasound.

Mechanosensitive channels (MSCs) play an important role in how cells transduce mechanical stimuli into electrical or chemical signals, which provides an interventional possibility through the manipulation of ion channel activation using different mechanical stimulation conditions. With good spatial resolution and depth of penetration, ultrasound is often proposed as the tool of choice for such therapeutic applications. Despite the identification of many ion channels as mechanosensitive in recent years, only a limited number of MSCs have been reported to be activated by ultrasound with substantial evidence. Furthermore, although many therapeutic implications using ultrasound have been explored, few offered insights into the molecular basis and the biological effects induced by ultrasound in relieving pain and accelerate tissue healing. In this review, we examined the literature, in particular studies that provided evidence of cellular responses to ultrasound, with and without the target ion channels. The ultrasound activation conditions were then summarized for these ion channels, and these conditions were related to their mode of activation based on the current biological concepts. The overall goal is to bridge the results relating to the activation of MSCs that is specific for ultrasound with the current knowledge in molecular structure and the available physiological evidence that may have facilitated such phenomena. We discussed how collating the information revealed by available scientific investigations helps in the design of a more effective stimulus device for the proposed translational purposes. Traditionally, studies on the effects of ultrasound have focused largely on its mechanical and physical interaction with the targeted tissue through thermal-based therapies as well as non-thermal mechanisms including ultrasonic cavitation; gas body activation; the direct action of the compressional, tensile and shear stresses; radiation force; and acoustic streaming. However, the current review explores and attempts to establish whether the application of low-intensity ultrasound may be associated with the activation of specific MSCs, which in turn triggers relevant cell signaling as its molecular mechanism in achieving the desired therapeutic effects. Non-invasive brain stimulation has recently become an area of intense research interest for rehabilitation, and the implication of low-intensity ultrasound is particularly critical given the need to minimize heat generation to preserve tissue integrity for such applications.

A preliminary study on the role of Piezo1 channels in myokine release from cultured mouse myotubes

It has long been known that regular physical exercise induces short and long term benefits reducing the risk of cardiovascular disease, diabetes, osteoporosis, cancer and improves sleep quality, cognitive level, mobility, autonomy in enderly. More recent is the evidence on the endocrine role of the contracting skeletal muscle. Exercise triggers the release of miokines, which act in autocrine, paracrine and endocrine ways controlling the activity of muscles but also of other tissues and organs such as adipose tissue, liver, pancreas, bones, and brain. The mechanism of release is still unclear.Neuromuscular electrical stimulation reproduces the beneficial effects of physical activity producing physiological metabolic, cardiovascular, aerobic responses consistent with those induced by exercise. In vitro, Electrical Pulse Stimulations (EPS) of muscle cells elicit cell contraction and mimic miokine release in the external medium.Here we show that, in cultured mouse myotubes, EPS induce contractile activity and the release of the myokine IL-6. Gadolinium highly reduces EPS-induced IL-6 release, suggesting the involvement of mechanical activated ion channels. The chemical activation of mechanosensitive Piezo1 channels with the specific agonist Yoda1 stimulates IL-6 release similarly to EPS, suggesting the involvement of Piezo1 channels in the control of the myokine release. The expression of Piezo1 protein in myotubes was confirmed by the Western blot analysis.To the best of our knowledge, this is the first evidence of a Piezo1-mediated effect in myokine release and suggests a potential translational use of specific Piezo1 agonists for innovative therapeutic treatments reproducing/enhancing the benefits of exercise mediated by myokines.

Single ion channel recording in 3D culture of stem cells using patch-clamp technique

Tri-dimensional (3D) cell aggregates or spheroids are considered to be closer to physiological conditions than traditional 2D cell culture. Mesenchymal stem cells (MSCs) assembling in spheroids have increased the survival of transplanted cells. The organization of stem cells in 3D culture affects cell microenvironment and their mechanical properties. The regulation of the biological processes that maintain crucial physiological reactions of MSCs is closely related to the functioning of ion channels. The pattern of expression, role and regulatory mechanisms of ion channels could be significantly different in 3D compared to 2D culture, and, thus, needed to be properly analyzed on the level of ionic currents. Electrophysiological data on the features of ion channels functioning in 3D cell culture models are currently very limited in the literature. This gap of knowledge may be associated with technical difficulties that exist when researchers try to apply the standard patch clamp method for the registration of ion channels in cells aggregated in spheroids. In this regard, our study focuses on solving emerging technical difficulties and presents an example of their successful solution. Here, we developed a specific approach and have recorded the activity of mechanosensitive stretch-activated ion channels (SACs) in endometrial MSCs (eMSCs) assembled in spheroids. Moreover, we observed functional interplay of SACs with potassium channels of big conductance (BK) in the plasma membrane of eMSC spheroids consistently to revealed earlier in routine 2D cultured cells. Additionally, we observed a significant decrease in the frequency of SACs activation in spheroids that may indicate the differences in the level of functional expression of channels in 3D culture comparing to 2D culture of eMSCs.

Polycystin-2 (TRPP2): Ion channel properties and regulation.

Polycystin-2 (TRPP2, PKD2, PC2) is the product of the PKD2 gene, whose mutations cause Autosomal Dominant Polycystic Kidney Disease (ADPKD). PC2 belongs to the superfamily of TRP (Transient Receptor Potential) proteins that generally function as Ca2+-permeable nonselective cation channels implicated in Ca2+ signaling. PC2 localizes to various cell domains with distinct functions that likely depend on interactions with specific channel partners. Functions include receptor-operated, nonselective cation channel activity in the plasma membrane, intracellular Ca2+ release channel activity in the endoplasmic reticulum (ER), and mechanosensitive channel activity in the primary cilium of renal epithelial cells. Here we summarize our current understanding of the properties of PC2 and how other transmembrane and cytosolic proteins modulate this activity, providing functional diversity and selective regulatory mechanisms to its role in the control of cellular Ca2+ homeostasis.