Role Of Stretch-activated Ion Channels Research Articles

The Role of Stretch-Activated Channels in Mediating mTORC1 Signaling in Isolated Rat Soleus Muscle in Response to Mechanical Stimulation after Functional Unloading

The aim of the present work was to identify the role of stretch-activated ion channels (SAC) in the transmission of mechanical signals to the key mTORC1/p70S6k anabolic pathways in the soleus muscle in rats after seven days of functional unloading of the hindlimbs. Functional unloading of the hindlimb muscles was by antiorthostatic suspension (hindlimb suspension, HS). The rats were divided into four groups: 1) group C (animal-house control; 2) C + Gd (controls whose muscles were incubated with the SAC inhibitor GdCl3; 3) HS (functional unloading for seven days); and 4) HS + Gd (a group with seven days of antiorthostatic suspension followed by incubation of muscles with GdCl3). After completion of antiorthostatic suspension, isolated soleus muscles were subjected to a series of eccentric contractions (EC). Muscles from intact animals responded to EC with increases in the phosphorylation of key markers of mTORC1 signaling: p70S6k and rpS6. The extent of mechanically induced increases in the levels of phosphorylation of these protein markers were significantly reduced to essentially identical extents in the “pure” suspension (HS) group and the HS group given the SAC blocker (HS + Gd) as compared with the increase in muscles from intact control group. These results lead to the conclusion that decreases in the mechanically dependent responses of the mTORC1 signal pathway in the soleus muscle in rats after seven days of functional unloading may be linked with inactivation of stretch-activated ion channels.

An Anabolic Signaling Response of Rat Soleus Muscle to Eccentric Contractions Following Hindlimb Unloading: A Potential Role of Stretch-Activated Ion Channels.

Mechanisms that convert a mechanical signal into a biochemical response in an atrophied skeletal muscle remain poorly understood. The aims of the study were to evaluate a temporal response of anabolic signaling and protein synthesis (PS) to eccentric contractions (EC) in rat soleus during hindlimb unloading (HU); and to assess a possible role of stretch-activated ion channels (SAC) in the propagation of a mechanical signal to mTORC1 following HU. Following HU, an isolated soleus was subjected to EC. Upon completion of EC, muscles were collected for western blot analyses to determine the content/phosphorylation of the key anabolic markers. We found that a degree of EC-induced p70S6K phosphorylation and the rate of PS in the soleus of 3- and 7-day unloaded rats was significantly less than that in control. A decrease in EC-induced phosphorylation of p70S6K, RPS6 and PS in the 7-day unloaded soleus treated with SAC inhibitor did not differ from that of the 7-day unloaded soleus without SAC blockade. The results of the study suggest that (i) HU results in a blunted anabolic response to a bout of EC, (ii) attenuation of mTORC1-signaling and PS in response to EC in unloaded soleus may be associated with inactivation of SAC.

Acute recovery from disuse atrophy: the role of stretch-activated ion channels in the activation of anabolic signaling in skeletal muscle.

The aim of the study was to 1) measure time-course alternations in the rate of protein synthesis (PS) and phosphorylation status of the key anabolic markers, and 2) find out the role of stretch-activated ion channels (SACs) in the activation of anabolic signaling in the rat soleus during an acute reloading following disuse atrophy. Wistar rats were subjected to 14-day hindlimb suspension (HS) followed by 6, 12, and 24 h of reloading. To examine the role of SAC in the reloading-induced activation of anabolic signaling, the rats were treated with gadolinium (Gd3+), a SAC blocker. The content of signaling proteins was determined by Western blot. c-Myc mRNA expression was assessed by RT-PCR. After 24-h reloading, the PS rate was elevated by 44% versus control. After 6-h reloading, the p-70-kDa ribosomal protein S6 kinase (p70S6k) and translation initiation factor 4E-binding protein 1 (4E-BP1) did not differ from control; however, 12-h reloading resulted in an upregulation of both p70s6k and 4E-BP1 phosphorylation versus control. The phosphorylation of AKT (Ser473) and glycogen synthase kinase-3β (Ser9) was reduced after HS and then completely restored by 12-h reloading. c-Myc was significantly upregulated during the entire reloading. Gd3+ treatment during reloading (12 h) prevented a full phosphorylation of p70S6k, rpS6, 4E-BP1, as well as PS activation. The results of the study suggest that 1) enhanced PS during the acute recovery from HS may be associated with the activation of ribosome biogenesis as well as mammalian target of rapamycin complex 1 (mTORC1)-dependent signaling pathways, and 2) functional SACs are necessary for complete activation of mTORC1 signaling in rat soleus during acute recovery from HS.

PO-064 A potential role of stretch-activated channels in anabolic mechanotransduction in the atrophied rat soleus muscle

Objective Prolonged immobilization or unloading of skeletal muscle causes muscle disuse atrophy, which is characterized by a reduction in muscle cross-sectional area and compromised contractile function. To date, the mechanisms of anabolic mechanotransduction in the atrophied mammalian skeletal muscle remain poorly understood. The aim of the present study was to assess a possible role of stretch-activated ion channels (SAC) in the propagation of a mechanical signal to anabolic signaling and protein synthesis (PS) in an isolated rat soleus muscle following mechanical unloading.
 Methods The mechanical unloading was performed via hindlimb suspension (HS). Twenty-eight male Wistar rats weighing were randomly assigned to the following 4 groups (n=7/group): 1) vivarium control (C), 2) control + SAC inhibitor (gadolinium) (C+Gd3+), 3) 7-day HS (HS), 4) 7-day HS + SAC inhibitor (HS+Gd3+). Following unloading, an isolated rat soleus was placed in an organ culture medium and subjected to a bout of eccentric contractions (EC). Upon completion of the EC, muscles were collected for Western blot analyses to determine the content of the key anabolic markers. The rate of PS was measured by SUnSET technique.
 Results EC-induced increase in PS was significantly less in the HS and HS+ Gd3+ groups vs. the C group. There was no statistically significant difference between the HS and HS+ Gd3+ groups in terms of EC-induced increase in muscle PS. A decrease in EC-induced phosphorylation of p70S6K, 4E-BP1, RPS6 and GSK-3beta in the 7-day unloaded soleus treated with SAC inhibitor did not differ from that of the 7-day unloaded soleus without SAC blockade. Thus, the inhibition of SAC with gadolinium did not lead to further decline in EC-induced phosphorylation of the key anabolic markers and muscle PS.
 Conclusions The results of the study suggest that attenuation of mTORC1-signaling and PS in response to EC in unloaded soleus muscle may be associated with inactivation of SAC. The study was supported by the RFBR grant # 16-34-60055.

The role of stretch-activated ion channels in acute respiratory distress syndrome: finally a new target?

Mechanical ventilation (MV) and oxygen therapy (hyperoxia; HO) comprise the cornerstones of life-saving interventions for patients with acute respiratory distress syndrome (ARDS). Unfortunately, the side effects of MV and HO include exacerbation of lung injury by barotrauma, volutrauma, and propagation of lung inflammation. Despite significant improvements in ventilator technologies and a heightened awareness of oxygen toxicity, besides low tidal volume ventilation few if any medical interventions have improved ARDS outcomes over the past two decades. We are lacking a comprehensive understanding of mechanotransduction processes in the healthy lung and know little about the interactions between simultaneously activated stretch-, HO-, and cytokine-induced signaling cascades in ARDS. Nevertheless, as we are unraveling these mechanisms we are gathering increasing evidence for the importance of stretch-activated ion channels (SACs) in the activation of lung-resident and inflammatory cells. In addition to the discovery of new SAC families in the lung, e.g., two-pore domain potassium channels, we are increasingly assigning mechanosensing properties to already known Na(+), Ca(2+), K(+), and Cl(-) channels. Better insights into the mechanotransduction mechanisms of SACs will improve our understanding of the pathways leading to ventilator-induced lung injury and lead to much needed novel therapeutic approaches against ARDS by specifically targeting SACs. This review 1) summarizes the reasons why the time has come to seriously consider SACs as new therapeutic targets against ARDS, 2) critically analyzes the physiological and experimental factors that currently limit our knowledge about SACs, and 3) outlines the most important questions future research studies need to address.

Piezo1 in Smooth Muscle Cells Is Involved in Hypertension-Dependent Arterial Remodeling.

The mechanically activated non-selective cation channel Piezo1 is a determinant of vascular architecture during early development. Piezo1-deficient embryos die at midgestation with disorganized blood vessels. However, the role of stretch-activated ion channels (SACs) in arterial smooth muscle cells in the adult remains unknown. Here, we show that Piezo1 is highly expressed in myocytes of small-diameter arteries and that smooth-muscle-specific Piezo1 deletion fully impairs SAC activity. While Piezo1 is dispensable for the arterial myogenic tone, it is involved in the structural remodeling of small arteries. Increased Piezo1 opening has a trophic effect on resistance arteries, influencing both diameter and wall thickness in hypertension. Piezo1 mediates a rise in cytosolic calcium and stimulates activity of transglutaminases, cross-linking enzymes required for the remodeling of small arteries. In conclusion, we have established the connection between an early mechanosensitive process, involving Piezo1 in smooth muscle cells, and a clinically relevant arterial remodeling.

Inhibition of stretch-activated ion channels on endothelial cells disrupts nitric oxide-mediated arterial outward remodeling

Outward arterial remodeling is a physiological response to accommodate chronically elevated blood flow and requires endothelial cells (ECs) and expression of endothelial nitric oxide synthase (eNOS). ECs may sense elevated flow via stretch-activated ion channels (SACs). We evaluated the role of SACs in regulation of flow-induced arterial expansion and eNOS expression by ECs. A high-flow environment was created in the common carotid arteries (CCAs) of mice via contralateral common carotid artery (CCA) ligation. Either streptomycin for SAC blockade or saline for placebo was delivered to the mice. CCAs were harvested for morphometric analysis 7 days post procedure. Cultured ECs were exposed to flow with wall shear stresses (WSSs) of 1.5–10 Pa for 24 h in presence or absence of streptomycin. Immunofluorescent staining was used for eNOS quantification. In vivo, CCA expansion in streptomycin-treated mice (n = 7) was significantly less than in the placebo-treated group (n = 8) (p = 0.015). In vitro, streptomycin exposure significantly inhibited eNOS expression at WSS >2.5 Pa (p = 0.001) while not affecting eNOS expression at baseline WSS (1.5–2.5 Pa). Blockade of SACs with streptomycin impairs outward arterial remodeling and eNOS expression at high WSSs. Activation of SACs under elevated WSS may contribute to vessel expansion by upregulating eNOS in ECs.

Stretch-Activated Calcium Channels Are Essential For Functional Adaptations Following Repeated Loading In Skeletal Muscle

Although subjecting a muscle to unaccustomed eccentric exercise often results in cellular damage, adhering to a long-term eccentric exercise program results in the repeated bout effect (RBE). Although the role of stretch-activated ion channels (SAC) in muscle adaptation has not been fully realized, evidence suggests that blocking them may reduce membrane disruption. Transient changes in intracellular calcium during muscle contraction have been shown to modulate gene expression providing a possible link between eccentric exercise and the RBE. PURPOSE: Test the hypothesis that SAC function is necessary for functional adaptation following chronic exercise. METHODS: Dorsiflexors of 2 groups of NZW rabbits were subjected to chronic repetitive eccentric exercise bouts (3x/week) for 4 weeks. Muscle function and damage indices were measured before and after the exercise protocol to gauge the adaptive response. Group 1 (streptomycin) received daily intramuscular injections of streptomycin (300mg/kg body weight) for duration of the study. Group 2 (control) underwent exercise only. RESULTS: Both groups were susceptible to eccentric exercise induced damage on day 1 as measured by a reduction in peak torque following the first exercise bout (−57.3±5.6%, −51.5±1.9%, p>.05). Following 4 weeks of eccentric exercise, the control group generated a greater peak isometric torque compared to the streptomycin group (+34.5±3.7%, +1.4±8.8%, p<.05), and optimized function to a longer muscle length (torque-joint angle shift: +4.3±0.7o, −12.8±7.0 o, p<.05). Also, the control group exhibited a greater resistance to exercise-induced damage on the final day as measured by torque reduction (−25.8±1.0%) compared to the streptomycin group (−33.2±2.7%, p<.05). CONCLUSION: SAC blocking did not affect peak torque production on day 1, but attenuated adaptive responses during repeated eccentric loading. Following long-term eccentric exercise, streptomycin treated muscles failed to improve force generation and experienced mal-adaptation by producing peak isometric torque at shorter muscle length. We conclude that SAC are essential for beneficial functional adaptations to exercise in skeletal muscle. This work was Supported by the ACSM Research Endowment Award, and The Ohio State University Pomerene Chair in Sports Medicine.

Increased coronary perfusion augments cardiac contractility in the rat through stretch-activated ion channels.

The role of stretch-activated ion channels (SACs) in coronary perfusion-induced increase in cardiac contractility was investigated in isolated isometrically contracting perfused papillary muscles from Wistar rats. A brief increase in perfusion pressure (3-4 s, perfusion pulse, n = 7), 10 repetitive perfusion pulses (n = 4), or a sustained increase in perfusion pressure (150-200 s, perfusion step, n = 7) increase developed force by 2.7 +/- 1.1, 7.7 +/- 2.2, and 8.3 +/- 2.5 mN/mm(2) (means +/- SE, P < 0.05), respectively. The increase in developed force after a perfusion pulse is transient, whereas developed force during a perfusion step remains increased by 5.1 +/- 2.5 mN/mm(2) (P < 0.05) in the steady state. Inhibition of SACs by addition of gadolinium (10 micromol/l) or streptomycin (40 and 100 micromol/l) blunts the perfusion-induced increase in developed force. Incubation with 100 micromol/l N(omega)-nitro-L-arginine [nitric oxide (NO) synthase inhibition], 10 micromol/l sodium nitroprusside (NO donation) and 0.1 micromol/l verapamil (L-type Ca(2+) channel blockade) are without effect on the perfusion-induced increase of developed force. We conclude that brief, repetitive, or sustained increases in coronary perfusion augment cardiac contractility through activation of stretch-activated ion channels, whereas endothelial NO release and L-type Ca(2+) channels are not involved.

Modification of stretch-induced shortening of repolarization by streptomycin in the isolated rabbit heart.

The exact mechanism of mechano-electrical feedback and stretch-induced arrhythmias is unknown, but the role of stretch-activated ion channels and specific calcium channels has been proposed. The aim of the present study was to test the hypothesis that stretch-activated ion channels and not calcium channels contribute to stretch-related alterations of repolarization and that these effects can be neutralized by stretch-activated channel block. We studied the interaction of acute ventricular dilatation and the stretch-activated channel blocker streptomycin and the specific calcium channel blocker verapamil in an isolated retrogradely perfused rabbit heart model in which the left ventricular size is modified by abruptly changing the volume of a fluid-filled balloon placed in the left ventricle. Acute ventricular dilatation led to a rate-dependent decrease in repolarization. The mean effective refractory period (ERP) and monophasic action potential duration (MAP90) for cycle lengths between 300 and 1,000 ms decreased from 174.2+/-9 ms and 178.9+/-7 ms to 161.6+/-11 ms and 169.7+/-5 ms, respectively. Streptomycin (80 microM) inhibited this stretch-related shortening of repolarization (ERP: 175.4+/-8 ms; MAP90: 179.7+/-8 ms, p < 0.05) but had almost no effect on already dilated ventricles. Counteraction of the observed electrophysiologic changes could only be achieved by increasing the streptomycin concentration to 200 microM. Streptomycin nearly completely suppressed stretch-related ectopic ventricular complexes. In contrast, verapamil (1 microM) had no effect on stretch-related changes in repolarization and stretch-induced arrhythmias. The present study indirectly implicates stretch-activated ion channels in the genesis of stretch-related changes in repolarization and arrhythmias. The electrophysiologic changes after ventricular dilatation to a degree that increases left ventricular pressure in a clinically relevant range can be influenced by the stretch-activated channel blocker streptomycin but not by specific calcium channel block. This may have clinically important implications for the development of new antiarrhythmic drugs.

Flow Stimulates Nitric Oxide Release in Guinea Pig Heart: Role of Stretch-Activated Ion Channels

Blood flow regulates vessel tone triggering the release of nitric oxide; however, the mechanism involved in this phenomenon is unknown. We investigated whether coronary flow induces nitric oxide release in the isolated perfused guinea pig heart and the role of the stretch-activated ion channels in the effect of flow. We used gadolinium (3 μM) in order to block these channels, and estimated nitric oxide release by an oxyhemoglobin method. The results have shown a flow-dependent stimulation of nitric oxide release (fivefold increase at perfusion flow of 25 ml/min). Gadolinium inhibited this effect in a dose-dependent fashion. Acetylcholine was able to stimulate nitric oxide release in presence of gadolinium. We concluded that coronary flow stimulates nitric oxide release in the guinea pig heart. Stretch-activated ion channels mediate this effect. Acetylcholine and flow stimulate nitric oxide release by different mechanisms of action.

Angiotensin and Endothelin

Changes in intracellular pH (pHi) can produce marked effects on cardiac function, and, therefore, it is important that the cell possess mechanisms by which pHi is regulated, especially after intracellular acidosis associated with myocardial ischemia. The Na+/H+ exchanger (NHE) and the Na+/HCO3− symport represent the 2 major pathways by which alkalinization occurs in cardiac cells. The NHE not only regulates pHi but also cell volume and intracellular signaling in response to a variety of stimuli. The NHE in mammalian myocardium1 has received considerable attention in the past decade, because it has been linked to cardiac growth and reperfusion injury.2 3 The NHE is activated by mechanical stretch in cultured cardiac myocytes and is thought to be a primary factor in influencing the anabolic state of the ventricular myocardium in response to pressure overload.4 5 Activation of the NHE occurs via phosphorylation of the cytoplasmic domain, which appears to be mediated by protein kinase C.6 Mechanical stretch of neonatal cardiac myocytes has also been associated with activation of second messengers, such as inositol triphosphate, protein kinase C, Raf-1 kinase, and mitogen-activated protein (MAP) kinase, all of which can contribute to reexpression of a number of fetal genes associated with cardiac hypertrophy, including β-myosin heavy chain, skeletal α-actin, and atrial natriuretic peptide.7 In studies using neonatal rat cardiac myocytes cultured on elastic membranes, stretch-mediated increases in MAP kinase activity and protein synthesis were attenuated by treatment of cells with the NHE antagonist Hoe 694,2 8 suggesting that the NHE has an important role in mediating growth in the pressure-overloaded myocardium. Deformation of the cardiac myocyte as it occurs in vivo, or in vitro under experimental or pathological conditions, presents a mechanical stress for cellular structures. This …

Ca2+ uptake in GH3 cells during hypotonic swelling: the sensory role of stretch-activated ion channels.

Hypotonic cell swelling triggers an increase in intracellular Ca2+ concentration that is deemed responsible for the subsequent regulated volume decrease in many cells. To understand the mechanisms underlying this increase, we have studied the Ca2+ sources that contribute to hypotonic cell swelling-induced Ca2+ increase (HICI) in GH3 cells. Fura 2 fluorescence of cell populations revealed that extracellular, but not intracellular, stores of Ca2+ were required. HICI was abolished by nifedipine, a blocker of L-type Ca2+ channels, and Gd3+, a nonspecific blocker of stretch-activated channels (SACs), suggesting two components for the Ca2+ membrane pathway: L-type Ca2+ channels and SACs. Using HICI as an assay, we found that venom from the spider Grammostola spatulata could block HICI without blocking L-type Ca2+ channels. The venom did, however, block SAC activity. This suggests that Ca(2+)-permeable SACs, rather than L-type Ca2+ channels, are the sensing elements for HICI. These results support the model for volume regulation in which SACs, activated by an increase of the membrane tension during hypotonic cell swelling, trigger HICI, leading to a volume decrease.

Calcium imaging of mechanically induced fluxes in tissue-cultured chick heart: role of stretch-activated ion channels.

Heart rate and contractility are sensitive to stretch. To better understand the origin of these effects, we have studied the effect of mechanical stimuli on a model system of tissue-cultured heart cells. Gently prodding cells with a pipette produced a Ca2+ influx that often led to waves of calcium-induced calcium release (CICR) spreading from the site of stimulation. Ca2+ release could also be produced by pulling on neighboring cells. The response was blocked by removing extracellular Ca2+ or by adding 20 microM Gd3+ to normal saline. The mechanical sensitivity probably arose from stretch-activated ion channels (SACs) based on several lines of evidence. Chick heart cells contain nonselective cation SACs that pass Ca2+ as well as Na+ and K+. Both the SACs and the fluorescence response are blocked by 20 microM Gd3+. Removal of Ca2+ from the extracellular medium blocked the fluorescent response. Cultures without SACs (grown in the absence of embryo extract) had no mechanically induced fluxes. These data contradict the recent claim that SAC activity is a patch-clamp artifact (C.E. Morris and R. Horn, Science Wash. DC 256: 1246-1249, 1991). The SACs had a density of approximately 1/micron 2 and were expected to pass less than 20 fA of Ca2+ current under physiological conditions. The change in intracellular concentration of Ca2+ ([Ca2+]i) resulting from activation of SACs may be too small to induce CICR unless the channels pass current into a restricted space (N. LeBlanc and J.R. Hume, Science Wash. DC 248: 372, 1990).(ABSTRACT TRUNCATED AT 250 WORDS)