Stretch-activated Channel Blocker Research Articles

Active force generation contributes to the complexity of spontaneous activity and to the response to stretch of murine cardiomyocyte cultures.

Cardiomyocyte cultures exhibit spontaneous electrical and contractile activity, as in a natural cardiac pacemaker. In such preparations, beat rate variability exhibits features similar to those of heart rate variability in vivo. Mechanical deformations and forces feed back on the electrical properties of cardiomyocytes, but it is not fully elucidated how this mechano‐electrical interplay affects beating variability in such preparations. Using stretchable microelectrode arrays, we assessed the effects of the myosin inhibitor blebbistatin and the non‐selective stretch‐activated channel blocker streptomycin on beating variability and on the response of neonatal or fetal murine ventricular cell cultures against deformation. Spontaneous electrical activity was recorded without stretch and upon predefined deformation protocols (5% uniaxial and 2% equibiaxial strain, applied repeatedly for 1 min every 3 min). Without stretch, spontaneous activity originated from the edge of the preparations, and its site of origin switched frequently in a complex manner across the cultures. Blebbistatin did not change mean beat rate, but it decreased the spatial complexity of spontaneous activity. In contrast, streptomycin did not exert any manifest effects. During the deformation protocols, beat rate increased transiently upon stretch but, paradoxically, also upon release. Blebbistatin attenuated the response to stretch, whereas this response was not affected by streptomycin. Therefore, our data support the notion that in a spontaneously firing network of cardiomyocytes, active force generation, rather than stretch‐activated channels, is involved mechanistically in the complexity of the spatiotemporal patterns of spontaneous activity and in the stretch‐induced acceleration of beating. Key points Monolayer cultures of cardiac cells exhibit spontaneous electrical and contractile activity, as in a natural cardiac pacemaker. Beating variability in these preparations recapitulates the power‐law behaviour of heart rate variability in vivo. However, the effects of mechano‐electrical feedback on beating variability are not yet fully understood.Using stretchable microelectrode arrays, we examined the effects of the contraction uncoupler blebbistatin and the non‐specific stretch‐activated channel blocker streptomycin on beating variability and on stretch‐induced changes of beat rate.Without stretch, blebbistatin decreased the spatial complexity of beating variability, whereas streptomycin had no effects.Both stretch and release increased beat rate transiently; blebbistatin attenuated the increase of beat rate upon stretch, whereas streptomycin had no effects.Active force generation contributes to the complexity of spatiotemporal patterns of beating variability and to the increase of beat rate upon mechanical deformation. Our study contributes to the understanding of how mechano‐electrical feedback influences heart rate variability.

Effects of HOCl oxidation on excitation–contraction coupling: Implications for the pathophysiology of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a fatal X-linked genetic disease characterized by progressive loss of skeletal muscle. The mechanisms underlying the DMD pathology likely involve the complex interaction between reactive oxygen species (ROS) impaired Ca2+ handling and chronic inflammation, characterized by the presence of immune cells such as neutrophils. Hypochlorous acid (HOCl) is a highly reactive form of ROS produced endogenously via the actions of myeloperoxidase, an enzyme secreted by neutrophils. Myeloperoxidase activity is significantly elevated in dystrophic muscle. This study aimed to determine the effect of HOCl exposure on excitation-contraction coupling and its potential contribution to the dystrophic pathology. Isolated extensor digitorum longus (EDL) muscles and single fibers from C57 (wild type) and mdx (dystrophic) mice were used to investigate the effects of HOCl on whole muscle function, intracellular Ca2+ handling, and myofilament force production. HOCl exposure significantly decreased maximum specific force in isolated EDL muscles by 26% and 49%, respectively, in C57 and mdx mice (P < 0.0001). In single interosseous fibers, HOCl exposure significantly increased resting intracellular Ca2+ concentration by ∼17-19% (P < 0.05) and decreased the amplitude of electrically induced Ca2+ transients by ∼45% and 50%, respectively, in C57 and mdx fibers (C57, P < 0.05; mdx, P < 0.01). These effects of HOCl on resting Ca2+ could be blocked via application of tetracaine (ryanodine receptor blocker) or Gd3+ (stretch-activated channel blocker; C57, P < 0.01; mdx, P < 0.01 for both). The effect of HOCl on Ca2+ transient amplitude was significantly reduced by Gd3+ (C57, P < 0.05; mdx, P < 0.01). In chemically skinned EDL fibers, HOCl exposure decreased maximum Ca2+-activated force by ∼40% in both C57 and mdx fibers (P < 0.001). These results indicate that HOCl potently affects excitation-contraction coupling via impaired Ca2+ handling and myofilament force production. Hence, HOCl potentially links the chronic inflammation, oxidative stress, and impaired Ca2+ handling that underlies the dystrophic pathology.

A Yoda1-Based Approach to Investigate Piezo1 Channels in Red Blood Cells Using Automated Patch Clamp Technology

Piezo1 is a mechanosensitive ion channel supposed to regulate the volume and maintain the structural integrity in Red Blood Cells (RBCs), as gain-of-function mutations in this channel are associated to the RBC disease Hereditary Xerocytosis (Zarychanski et al. Blood 2012; Bae et al. Proceedings of the National Academy of Sciences 2013). Piezo1 is activated by several mechanical forces, including stretching, poking and shear stress and allows Ca2+ and other cations to enter the cell generating an electrical response. In 2015, it has been discovered that Piezo1 is sensitive to a small molecule, Yoda1 (Syeda et al. Elife 2015), which keeps the channel open and affects its inactivation kinetics. This finding has created new possibilities to elucidate Piezo1 gating mechanism and explore its functional significance in physiological and pathophysiological conditions.Here, we present a patient with a novel PIEZO1 mutation (R2110W) and a patch clamp-based high-throughput screening assay for Piezo1 activity. We established a protocol to detect functional Piezo1 mutations upon chemical stimulation by Yoda1, yet were not able to stimulate the channel via mechanical force, i.e. pressure steps and shear-stress.The assay was first developed on Neuro2A (N2A), a neuroblastoma cell-line endogenously expressing Piezo1 channels (kindly provided by Max-Delbrück Center, Berlin), due to larger abundance of Piezo1 channels in these cells. Initial experiments were performed on the Patchliner (Nanion Technologies GmbH, Munich), a medium-throughput automated patch clamp system able to record up to 8 cells at a time. Currents were elicited using a voltage ramp ranging from -100 to +80 mV for 300 ms, the holding potential was set to -60 mV. A significantly increased whole-cell current was observed upon 10 µM Yoda1 application in half of the recorded cells and the resulting Yoda1-induced currents were inhibited by 30 µM gadolinium chloride, a non-specific blocker of stretch-activated channels. The assay was then implemented on the SyncroPatch 384PE (Nanion Technologies GmbH, Munich), capable of recording up to 384 cells in parallel under identical experimental conditions, thus allowing for reliable statistical analysis. Yoda1 responding cells were selected based on strict quality control (QC) criteria, i.e. the seal resistance stability over time. In one example NPC-384 chip 140 out of 384 N2A cells (37%) passed the QC criteria and 85 cells (60% of the valid cells) were considered as Yoda1 responders. Finally, we investigated Piezo1 electrophysiological properties in healthy and patient RBCs carrying the novel PIEZO1 R2110W mutation. Similar to N2A cells, RBCs currents were analyzed and divided into Yoda1 responders and non-responders according to our QC criteria. The increase in whole-cell currents induced by Yoda1 application was significantly higher in patient compared to control RBCs, which was also reflected by a higher number of Yoda1 responders compared to control. Residue R2110W is structurally located in a gating sensitive area of the channel protein suggesting a gain-of-function. This would be in line with previously described mutations in PIEZO1 (Albuisson et al. Nature Communications 2013) and the mild form of anaemia observed in the patient. Furthermore, we excluded any involvement of Gardos channels in the Yoda1-induced currents by comparing measurements in the presence and absence of the specific Gardos channel inhibitor TRAM-34.Altogether, our work demonstrates that high-throughput patch clamping can provide a robust assay to study functional Piezo1 impairments in primary RBCs without expressing the mutated channel protein in a heterologous expression system. Our approach may be used to detect other channelopathies not only in RBCs and may serve as routine screening assay for diseases related to ion channel dysfunctions in general, complementary to gene sequencing. DisclosuresNo relevant conflicts of interest to declare.

Hyperosmolarity evokes histamine release from ileum mucosa by stimulating a cholinergic pathway

Changes in extracellular osmolarity lead to alteration in cellular volume. In the study, we examined the effects of hyperosmolarity on short-circuit currents (Isc) in the rat ileum using the Ussing chamber technique. Mucosal exposure to 20 mM glucose evoked a decrease of ISC in the rat ileum, which was antagonized by the stretch-activated channel blocker GdCl3, TTX and atropine, respectively. In contrast, it was not blocked by phlorizin, a Na+-glucose cotransporter SGLT1 inhibitor. Furthermore, the unabsorbed substances, such as sucrose, lactulose or urea, also induced a decrease of ISC in rat ileum. ELISA results revealed that 20 mM glucose stimulated the release of histamine from rat ileum mucosa, which was attenuated by TTX. In addition, the glucose-induced ISC was depressed by pyrilamine, a histamine H1 receptor blocker (H1 antagonist) whereas it was not affected by ranitidine (H2 antagonist), clobenpropit (H3 antagonists) or JNJ7777120 (H4 antagonist), respectively. The ion substitution experiments suggest that the changes of Na+ and HCO3− ion flux underlie the glucose-induced ISC. In conclusion, osmotic stimulus decreased the basal ISC of rat ileum by evoking histamine release from ileum mucosa. The changes of Na+ and HCO3− ion transport are involved in the glucose-evoked decrease of basal ISC.

Drug therapy for atrial fibrillation: quo vadis?

Atrial fibrillation has always been the most common sustained cardiac arrhythmia, and its incidence is increasing worldwide. Despite evolving ablation techniques, the vastness of the number of cases entrenches drug therapy as the mainstay of treatment for the majority of cases both now and in the foreseeable future. Drug therapy for atrial fibrillation includes drugs for ventricular rate control, anti-coagulation, and cardioversion/maintenance of sinus rhythm (rhythm control). This review summarizes the available data on new drugs in each of these 3 areas. In the area of rhythm control, it is clear that primary prevention of atrial fibrillation is achieved by a number of drugs in common clinical usage in hypertension, heart failure, and vascular disease, viz. blockers of the renin-angiotensin system and statins. Primary prevention is also promising with novel therapies such as anti-inflammatory therapy, pirfenidone, and Ω-3 poly-unsaturated fatty acids. Secondary prevention with anti-arrhythmic drugs producing multiple channel blockade is proven to be efficacious, and atrial-selective anti-arrhythmic drugs are an attractive development and will avoid ventricular pro-arrhythmia. A number of new drugs with novel mechanisms of action have mostly not yet undergone clinical trials, but are discussed here, and include gap junction modulators, stretch-activated channel blockers, sodium-calcium exchange inhibitors and new ion channel blockers.

Efficacy of Precordial Percussion Pacing Assessed in a Cardiac Standstill Microminipig Model.

Potential cardiovascular benefits of precordial percussion pacing (PPP) during cardiac standstill are unknown.Methods and Results:A cardiac standstill model in amicrominipigwas created by inducing complete atrioventricular block with a catheter ablation technique (n=7). Next, the efficacy of cardiopulmonary resuscitation by standard chest compressions (S-CPR), PPP and ventricular electrical pacing in this model were analyzed in series (n=4). To assess the mechanism of PPP, a non-selective, stretch-activated channel blocker, amiloride, was administered during PPP (n=3). Peak systolic and diastolic arterial pressures during S-CPR, PPP and ventricular electrical pacing were statistically similar. However, the duration of developed arterial pressure with PPP was comparable to that with ventricular electrical pacing, and significantly greater than that with S-CPR. Amiloride decreased the induction rate of ventricular electrical activity by PPP in a dose-related manner. Each animal survived without any neurological deficit at 24, 48 h and 1 week, even with up to 2 h of continuous PPP. In amicrominipigmodel of cardiac standstill, PPP can become a novel means to significantly improve physiological outcomes after cardiac standstill or symptomatic bradyarrhythmias in the absence of cardiac pacing. Activation of the non-selective stretch-activated channels may mediate some of the mechanophysiological effects of PPP. Further study of PPP by itself and together with S-CPR is warranted using cardiac arrest models of atrioventricular block and asystole.

Dysregulation of Intracellular Ca2+ in Dystrophic Cortical and Hippocampal Neurons.

Duchenne muscular dystrophy (DMD) is an inherited X-linked disorder characterized by skeletal muscle wasting, cardiomyopathy, as well as cognitive impairment. Lack of dystrophin in striated muscle produces dyshomeostasis of resting intracellular Ca2+ ([Ca2+]i), Na+ ([Na+]i), and oxidative stress. Here, we test the hypothesis that similar to striated muscle cells, an absence of dystrophin in neurons from mdx mice (a mouse model for DMD) is also associated with dysfunction of [Ca2+]i homeostasis and oxidative stress. [Ca2+]i and [Na+]i in pyramidal cortical and hippocampal neurons from 3 and 6months mdx mice were elevated compared to WT in an age-dependent manner. Removal of extracellular Ca2+ reduced [Ca2+]i in both WT and mdx neurons, but the decrease was greater and age-dependent in the latter. GsMTx-4 (a blocker of stretch-activated cation channels) significantly decreased [Ca2+]i and [Na+]i in an age-dependent manner in all mdx neurons. Blockade of ryanodine receptors (RyR) or inositol triphosphate receptors (IP3R) reduced [Ca2+]i in mdx. Mdx neurons showed elevated and age-dependent reactive oxygen species (ROS) production and an increase in neuronal damage. In addition, mdx mice showed a spatial learning deficit compared to WT. GsMTx-4 intraperitoneal injection reduced neural [Ca2+]i and improved learning deficit in mdx mice. In summary, mdx neurons show an age-dependent dysregulation in [Ca2+]i and [Na+]i which is mediated by plasmalemmal cation influx and by intracellular Ca2+ release through the RyR and IP3R. Also, mdx neurons have elevated ROS production and more extensive cell damage. Finally, a reduction of [Ca2+]i improved cognitive function in mdx mice.

Enhancement of Automaticity by Mechanical Stretch of the Isolated Guinea Pig Pulmonary Vein Myocardium.

The effects of mechanical stretch on the automaticity of the guinea pig isolated pulmonary vein preparations were studied in microelectrode experiments. The application of cumulative mechanical stretch to the preparations resulted in increases in the firing rate of spontaneous electrical activity in the myocardial layer. This effect was significantly inhibited by stretch-activated channel blockers such as gadolinium or streptomycin. These results suggest that acute mechanical stretch enhances the automaticity of the guinea pig pulmonary vein myocardium through the opening of the stretch-activated channels.

Beat-to-Beat Variability in Preload Unmasks Latent Risk of Torsade de Pointes in Anesthetized Chronic Atrioventricular Block Dogs.

Beat-to-beat variability in ventricular repolarization (BVR) associates with increased arrhythmic risk. Proarrhythmic remodeling in the dog with chronic AV-block (CAVB) compromises repolarization reserve and associates with increased BVR, which further increases upon dofetilide infusion and correlates with Torsade de Pointes (TdP) arrhythmias. It was hypothesized that these pro-arrhythmia-associated increases in BVR are induced by beat-to-beat variability in preload. Left ventricular monophasic action potential duration (LVMAPD) was recorded in acute (AAVB) and CAVB dogs, before and after dofetilide infusion. BVR was quantified as short-term variability of LVMAPD. The PQ-interval was controlled by pacing: either a constant or an alternating preload pattern was established, verified by PV-loop. The effect of the stretch-activated channel blocker, streptomycin, on BVR was evaluated in a second CAVB group. At alternating preload only, BVR was increased after proarrhythmic remodeling (0.45±0.14 ms AAVB vs. 2.2±1.1 ms CAVB, P<0.01). At CAVB, but not at AAVB, dofetilide induced significant proarrhythmia. Preload variability augmented the dofetilide-induced BVR increase at CAVB (+1.5±0.8 ms vs. +0.9±0.9 ms, P=0.058). In the second group, the increase in baseline BVR by alternating preload (0.3±0.03 ms to 1.0±0.8 ms, P<0.01) was abolished by streptomycin (0.5±0.2 ms, P<0.05). In CAVB dogs, the inverse relation between BVR and repolarization reserve originates from an augmented sensitivity of ventricular repolarization to beat-to-beat preload changes. Stretch-activated channels appear to be involved in the mechanism of BVR. (Circ J 2016; 80: 1336-1345).

Stretch-induced increase in cardiac contractility is independent of myocyte Ca2+ while block of stretch channels by streptomycin improves contractility after ischemic stunning.

Stretching the cardiac left ventricle (LV) enhances contractility but its effect on myoplasmic [Ca2+] is controversial. We measured LV pressure (LVP) and [Ca2+] as a function of intra-LV stretch in guinea pig intact hearts before and after 15 min global stunning ± perfusion with streptomycin (STM), a stretch-activated channel blocker. LV wall [Ca2+] was measured by indo-1 fluorescence and LVP by a saline-filled latex balloon inflated in 50 μL steps to stretch the LV. We implemented a mathematical model to interpret cross-bridge dynamics and myofilament Ca2+ responsiveness from the instantaneous relationship between [Ca2+] and LVP ± stretching. We found that: (1) stretch enhanced LVP but not [Ca2+] before and after stunning in either control (CON) and STM groups, (2) after stunning [Ca2+] increased in both groups although higher in STM versus CON (56% vs. 39%), (3) STM-enhanced LVP after stunning compared to CON (98% vs. 76% of prestunning values), and (4) stretch-induced effects on LVP were independent of [Ca2+] before or after stunning in both groups. Mathematical modeling suggested: (1) cooperativity in cross-bridge kinetics and myofilament Ca2+ handling is reduced after stunning in the unstretched heart, (2) stunning results in depressed myofilament Ca2+ sensitivity in the presence of attached cross-bridges regardless of stretch, and (3) the initial mechanism responsible for increased contractility during stretch may be enhanced formation of cross-bridges. Thus stretch-induced enhancement of contractility is not due to increased [Ca2+], whereas enhanced contractility after stunning in STM versus CON hearts results from improved Ca2+ handling and/or enhanced actinomyosin cross-bridge cycling.

Effects of the Selective Stretch-Activated Channel Blocker GsMtx4 on Stretch-Induced Changes in Refractoriness in Isolated Rat Hearts and on Ventricular Premature Beats and Arrhythmias after Coronary Occlusion in Swine.

Mechanical factors may contribute to ischemic ventricular arrhythmias. GsMtx4 peptide, a selective stretch-activated channel blocker, inhibits stretch-induced atrial arrhythmias. We aimed to assess whether GsMtx4 protects against ventricular ectopy and arrhythmias following coronary occlusion in swine. First, the effects of 170-nM GsMtx4 on the changes in the effective refractory period (ERP) induced by left ventricular (LV) dilatation were assessed in 8 isolated rat hearts. Then, 44 anesthetized, open-chest pigs subjected to 50-min left anterior descending artery occlusion and 2-h reperfusion were blindly allocated to GsMtx4 (57 μg/kg iv. bolus and 3.8 μg/kg/min infusion, calculated to attain the above concentration in plasma) or saline, starting 5-min before occlusion and continuing until after reflow. In rat hearts, LV distension induced progressive reductions in ERP (35±2, 32±2, and 29±2 ms at 0, 20, and 40 mmHg of LV end-diastolic pressure, respectively, P<0.001) that were prevented by GsMTx4 (33±2, 33±2, and 32±2 ms, respectively, P=0.002 for the interaction with LV end-diastolic pressure). Pigs receiving GsMtx4 had similar number of ventricular premature beats during the ischemic period as control pigs (110±28 vs. 103±21, respectively, P=0.842). There were not significant differences among treated and untreated animals in the incidence of ventricular fibrillation (13.6 vs. 22.7%, respectively, P=0.696) or tachycardia (36.4 vs. 50.0%, P=0.361) or in the number of ventricular tachycardia episodes during the occlusion period (1.8±0.7 vs. 5.5±2.6, P=0.323). Thus, GsMtx4 administered under these conditions does not suppress ventricular ectopy following coronary occlusion in swine. Whether it might protect against malignant arrhythmias should be tested in studies powered for these outcomes.

Activation of Ca2+-Dependent Cation Current by Fluid Shear Force in Atrial Myocytes

Atrial myocytes are subjected to fluid shear force (FSF) during each contraction and relaxation. Ionic currents regulated by shear force and their molecular integrity in cardiac myocytes have not been well-understood. We examined whether FSF activates specific current in atrial myocytes and underlying mechanisms for FSF-sensitive ionic current using whole-cell patch-clamp technique. A FSF of ∼16 dyne/cm2 was applied to entire single atrial myocyte using automated micro-puffing apparatus. A FSF-sensitive current (IFSF) was detected in lowly Ca2+-buffered (0.5 mM EGTA) atrial myocytes, but not in highly Ca2+-buffered (≥4 mM EGTA or 10 mM BAPTA) myocytes. The IFSF showed an outward rectification with a reversal potential of about -6 mV. The IFSF was inhibited by high concentrations (20-50 μM) of ryanodine and by replacement of external and internal cation with impermeant NMDG+, suggesting that IFSF is a Ca2+ release-dependent cation current. Application of either transient receptor potential melastatin subfamily 4 (TRPM4) inhibitor 9-phenanthrol or TRPM4-specific antibodies removed most of inward IFSF and ∼80% of outward IFSF. However, stretch-activated cation channel blocker GsMTx-4 did not affect IFSF. Interestingly, IFSF was strongly inhibited by inositol 1,4,5-trisphosphate receptor (IP3R) blockers, 2-APB (2 μM) or xestospongin C. In addition, in atrial myocytes isolated from type 2 IP3R (IP3R2) knock-out mouse, IFSF was not detected, although 9-phenanthrol-sensitive IFSF was recorded in wild-type myocytes. Co-immunostaining of TRPM4 and IP3R2 in rat atrial myocytes revealed peripheral localization of these proteins with some co-localizations. These results suggest that fluid shear stimuli may activate TRPM4 channels in atrial myocytes via Ca2+ releases triggered by the activation of nearby IP3R2.

Local membrane deformation and micro-injury lead to qualitatively different responses in osteoblasts

Micro-damage of bone tissue is known to regulate bone turnover. However, it is unknown if individual bone cells can differentiate between membrane deformation and micro-injury. We generated osteoblasts from mouse bone marrow or bone morphogenetic protein 2-transfected C2C12 cells. Single cells were mechanically stimulated by indentation with the atomic force microscopy probe with variable force load either resulting in membrane deformation only, or leading to membrane penetration and micro-injury. Changes in the cytosolic free calcium concentration ([Ca 2+] i) in fluo4-AM loaded cells were analyzed. When deformation only was induced, it resulted in an immediate elevation of [Ca 2+] i which was localized to the probe periphery. Multiple consecutive local Ca 2+ responses were induced by sequential application of low level forces, with characteristic recovery time of ~2 s. The duration of [Ca 2+] i elevations was directly proportional to the tip-cell contact time. In contrast, cell micro-injury resulted in transient global elevations of [Ca 2+] i, the magnitude of which was independent of the tip-cell contact time. Sequential micro-injury of the same cell did not induce Ca 2+ response within 30 s of the first stimulation. Both local and global Ca 2+elevations were blocked in Ca 2+-free media or in the presence of stretch-activated channel blocker Gd 3+. In addition, amount of Ca 2+ released during global responses was significantly reduced in the presence of PLC inhibitor Et-18-OCH 3. Thus, we found qualitative differences in calcium responses to mechanical forces inducing only membrane deformation or deformation leading to micro-injury.

Local membrane deformation and micro-injury lead to qualitatively different responses in osteoblasts.

Micro-damage of bone tissue is known to regulate bone turnover. However, it is unknown if individual bone cells can differentiate between membrane deformation and micro-injury. We generated osteoblasts from mouse bone marrow or bone morphogenetic protein 2-transfected C2C12 cells. Single cells were mechanically stimulated by indentation with the atomic force microscopy probe with variable force load either resulting in membrane deformation only, or leading to membrane penetration and micro-injury. Changes in the cytosolic free calcium concentration ([Ca (2+)] i) in fluo4-AM loaded cells were analyzed. When deformation only was induced, it resulted in an immediate elevation of [Ca (2+)] i which was localized to the probe periphery. Multiple consecutive local Ca (2+) responses were induced by sequential application of low level forces, with characteristic recovery time of ~2 s. The duration of [Ca (2+)] i elevations was directly proportional to the tip-cell contact time. In contrast, cell micro-injury resulted in transient global elevations of [Ca (2+)] i, the magnitude of which was independent of the tip-cell contact time. Sequential micro-injury of the same cell did not induce Ca (2+) response within 30 s of the first stimulation. Both local and global Ca (2+)elevations were blocked in Ca (2+)-free media or in the presence of stretch-activated channel blocker Gd (3+). In addition, amount of Ca (2+) released during global responses was significantly reduced in the presence of PLC inhibitor Et-18-OCH 3. Thus, we found qualitative differences in calcium responses to mechanical forces inducing only membrane deformation or deformation leading to micro-injury.

Mechanical control of cell biology. Effects of cyclic mechanical stretch on cardiomyocyte cellular organization

ObjectivesThe aim of our study was to elucidate how cyclic mechanical stretch is sensed by cardiomyocytes and in which way it affects cytoskeletal organization. MethodsNeonatal rat cardiomyocytes, cultured on flexible membranes, were subjected to cyclic mechanical stretch (1 Hz, 10% elongation) for 24 h using either round or rectangular loading posts for equibi-axial or uni-axial stretch, respectively, using the FlexCell stretch system. Cells were treated either with vehicle, the focal adhesion kinase (FAK) inhibitor PF-573,228 (200 nM), or the stretch-activated ion channel blocker gadolinium (Gd3+; 100 μM). ResultsCyclic mechanical stretch (36 mm diameter silicone membrane, equibi-axial stretch, 10% elongation, 1 Hz) induced elongation of the cardiomyocytes together with accentuation of Cx43 at the cell poles, and with an orientation of the cell axis between the radial axis and the circumferential axis (mean deviation: 11° from the circumference). Moreover, stretch resulted in ca. 1.4 fold increased Cx43 expression. FAK was found to be phosphorylated at the edges of the cells. In order to find out, how cardiomyocytes might sense stretch, we investigated possible effects of Gd3+and PF-573,228. Gd3+ had no effect on elongation or polarization and did not affect stretch-induced Cx43 expression. Interestingly, the FAK inhibitor completely antagonized the stretch-induced elongation, orientation and Cx43-polarization. However, the stretch-induced Cx43 expression was insensitive to this treatment. In order to clarify our result that the cells in equibi-axial stretch did not exactly organize to the circumference or to the radial axis, we decided to use a uni-axial stretch protocol. In uni-axially stretched cells, we found that the cardiomyocytes also showed elongation, Cx43 polarization, and orientation near to the stretch axis, but not exactly in the stretch axis but ca. 25° oblique to it. Furthermore, we investigated the tubular system, the Golgi apparatus, the SR and the nucleus. After 24 h stretch the microtubules were localized nearly (but not completely) parallel to the stretch axis (i.e. in longitudinal cell axis). Moreover, the localization of nucleus and the Golgi was also changed: while under static conditions, the Golgi was distributed more or less around the nucleus, after stretch the Golgi was accentuated at one site of the nucleus facing a cell pole with the nucleus facing the opposite cell pole. The plus motor protein kinesin accentuated at the cell poles and at the cell periphery, while the minus motor protein dynein was found near to the Golgi apparatus. ConclusionsThe stretch signal sensing is mediated via FAK and leads to intracellular re-organization and orientation. The oblique orientation of the cell with regard to the direction of stretch may define a directed force vector which could allow the cell to orientate.

Possible contribution of pannexin-1 to ATP release in human upper airway epithelia.

Pannexins are a family of transmembrane nonselective channel proteins that participate in the release of ATP into extracellular space. Previous studies have suggested that pannexin‐1 (Panx1) may constitute a local autocrine/paracrine system via transmitter ATP in association with the purinergic P2X7 receptor. In this study, we investigate the expressions of Panx1 and P2X7 in human nasal mucosa, together with hypotonic stress‐induced ATP release from this tissue. Twenty men and one woman ranging in age from 10 to 82 years with an average age of 44.2 ± 4.4 years participated in the study. Inferior turbinates were collected from patients with chronic hypertrophic rhinitis during endoscopic endonasal surgery. The expressions of Panx1 and P2X7 were examined by fluorescence immunohistochemistry and quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR). We also examined hypotonic stress‐induced ATP release from the turbinate mucosa and the effects of channel blockers in an ex vivo experiment. Substantial expressions of both proteins were observed in human nasal mucosa. The immunoreactivity for Panx1 was stronger than that for P2X7. The presence of the transcripts of Panx1 and P2X7 was also shown by qRT‐PCR. Ten and 100 μmol/L carbenoxolone (a Panx1 channel blocker) significantly inhibited the ATP release from the nasal mucosa, but flufenamic acid (a connexin channel blocker) and gadolinium (a stretch‐activated channel blocker) did not. These results indicate the coexistence of Panx1 and P2X7 in, and Panx1‐dependent ATP release from, the human nasal mucosa, suggesting the possible participation of these molecules in the physiological functions of the upper airway.

The effect of pressure on potassium currents in the inner hair cells isolated from guinea-pig cochlea.

An increase in hydrostatic pressure in the endolymphatic system causes hydrops-related inner ear diseases such as Meniere's disease or low tone sensorineural hearing loss. In the present study, we investigated the effects of pressure exerted on potassium currents in acutely isolated inner hair cells of the guinea-pig cochlea using whole-cell voltage-clamp techniques. By applying negative or positive pressure via the patch pipette using a syringe, intracellular hydropressure was changed between -40 cm H2O to +20 cm H2O. Negative pressure potentiated the amplitude of potassium currents, whereas positive pressure suppressed the amplitude of potassium currents. Gadolinium, a blocker of stretch-activated cation channels, did not influence pressure-dependent changes in potassium currents; however, cinnarizine blocked pressure-dependent changes in potassium currents. The current changes were not dependent on the sign of the pressure change, that is, similar increases in negative pressures (between -10 cm H2O and -40 cm H2O) and similar decreases in positive pressures (between +10 cm H2O and +20 cm H2O) were observed.

Fluid Pressure-Activated Non-Selective Cation Current and Cl- Current in Rat Atrial Myocytes

When intact atrial muscle is exposed to turbulant flow or high fluid pressure during valve diseases, it produces arrhythmias. Here we characterized a novel fluid pressure (FP)-gated ionic current (IFP) in single rat atrial myocytes using a whole-cell patch clamp. A flow of pressurized (∼16 dyn/cm2) fluid was applied onto single rat atrial myocytes using a microperfusion method. The application of FP with a normal bath solution elicited a transient inward current (∼1 pA/pF at −80 mV). The magnitude of IFP was increased in a pressure-dependent manner. The removal of extracellular Ca2+ largely enhanced the IFP and eliminated the current adaptation. Under physiological ionic gradients, the IFP displayed an inwardly- and outwardly-rectifying current-voltage relationship with a reversal potential (Erev) of approximately −52 mV. The Cl− channel blockers, DIDS and 9-AC, suppressed inward and outward IFP by about 50% and 70-80%, respectively. In symmetrical Cl− solutions, the Erev was shifted rightward (≌−18 mV) and the outwardly rectifying IFP was attenuated. In the symmetrical Cl− conditions, removal of extracellular Na+ largely reduced inward IFP, and produced a left shift of Erev (≌−64 mV). In addition, the elimination of internal K+ shifted Erev to ≌+8.4 mV and decreased outward IFP. Although low concentrations of extracellular Ca2+ blocked IFP with a negative shift of Erev, high concentrations of extracellular Ca2+ produced a right shift of Erev. Gadolinium ion (Gd3+), the stretch-activated channel blocker, partially blocked the inward IFP. In current-clamped cells, FP of the same magnitude elicited spontaneous membrane depolarization with repetitive action potentials and prolonged action potential durations. These results indicate that FP may activate an outwardly rectifying Cl− channel and a Gd3+- and Ca2+-sensitive non-selective cation channel that carries Na+, K+, and Ca2+.

Neuroprotective effect of gadolinium: a stretch-activated calcium channel blocker in mouse model of ischemia–reperfusion injury

The present study was designed to investigate the potential of gadolinium, a stretch-activated calcium channel blocker in ischemic reperfusion (I/R)-induced brain injury in mice. Bilateral carotid artery occlusion of 12min followed by reperfusion for 24h was given to induce cerebral injury in male Swiss mice. Cerebral infarct size was measured using triphenyltetrazolium chloride staining. Memory was assessed using Morris water maze test and motor incoordination was evaluated using rota-rod, lateral push, and inclined beam walking tests. In addition, total calcium, thiobarbituric acid reactive substance (TBARS), reduced glutathione (GSH), and acetylcholinesterase (AChE) activity were also estimated in brain tissue. I/R injury produced a significant increase in cerebral infarct size. A significant loss of memory along with impairment of motor performance was also noted. Furthermore, I/R injury also produced a significant increase in levels of TBARS, total calcium, AChE activity, and a decrease in GSH levels. Pretreatment of gadolinium significantly attenuated I/R-induced infarct size, behavioral and biochemical changes. On the basis of the present findings, we can suggest that opening of stretch-activated calcium channel may play a critical role in ischemic reperfusion-induced brain injury and that gadolinium has neuroprotective potential in I/R-induced injury.

The Stretch-Activated Channel Blocker Gd3+Reduces Palytoxin Toxicity in Primary Cultures of Skeletal Muscle Cells

Palytoxin (PLTX) is one of the most toxic seafood contaminants ever isolated. Reports of human food-borne poisoning ascribed to PLTX suggest skeletal muscle as a primary target site. Primary cultures of mouse skeletal muscle cells were used to study the relationship between Ca(2+) response triggered by PLTX and the development of myotoxic insult. Ca(2+) imaging experiments revealed that PLTX causes a transitory intracellular Ca(2+) response (transient phase) followed by a slower and more sustained Ca(2+) increase (long-lasting phase). The transient phase is due to Ca(2+) release from intracellular stores and entry through voltage-dependent channels and the Na(+)/Ca(2+) exchanger (reverse mode). The long-lasting phase is due to a massive and prolonged Ca(2+) influx from the extracellular compartment. Sulforhodamine B assay revealed that the long-lasting phase is the one responsible for the toxicity in skeletal muscle cells. Our data analyzed, for the first time, pathways of PLTX-induced Ca(2+) entry and their correlation with PLTX-induced toxicity in skeletal muscle cells. The cellular morphology changes induced by PLTX and the sensitivity to gadolinium suggest a role for stretch-activated channels.