Fluidization and Resolidification of the Human Bladder Smooth Muscle Cell in Response to Transient Stretch

Background

Cells resident in certain hollow organs are subjected routinely to large transient stretches, including every adherent cell resident in lungs, heart, great vessels, gut, and bladder. We have shown recently that in response to a transient stretch the adherent eukaryotic cell promptly fluidizes and then gradually resolidifies, but mechanism is not yet understood.

Principal Findings

In the isolated human bladder smooth muscle cell, here we applied a 10% transient stretch while measuring cell traction forces, elastic modulus, F-actin imaging and the F-actin/G-actin ratio. Immediately after a transient stretch, F-actin levels and cell stiffness were lower by about 50%, and traction forces were lower by about 70%, both indicative of prompt fluidization. Within 5min, F-actin levels recovered completely, cell stiffness recovered by about 90%, and traction forces recovered by about 60%, all indicative of resolidification. The extent of the fluidization response was uninfluenced by a variety of signaling inhibitors, and, surprisingly, was localized to the unstretch phase of the stretch-unstretch maneuver in a manner suggestive of cytoskeletal catch bonds. When we applied an “unstretch-restretch” (transient compression), rather than a “stretch-unstretch” (transient stretch), the cell did not fluidize and the actin network did not depolymerize.

Conclusions

Taken together, these results implicate extremely rapid actin disassembly in the fluidization response, and slow actin reassembly in the resolidification response. In the bladder smooth muscle cell, the fluidization response to transient stretch occurs not through signaling pathways, but rather through release of increased tensile forces that drive acute disassociation of actin.     

On the Inhibition of Muscle Contraction Caused by Exposure to Hypertonic Solutions

The evidence supporting a site of inhibition of excitation contraction (E-C) coupling near the plasma membrane (the "glycerol effect," the K⁺-potentiating effect) for muscle in hypertonic solution was reinvestigated. It was found, using whole frog sartorii, that there was a rehydration of muscle soaked in glycerol Ringer after 30 min and a large swelling (to 140% after 1 hr soaking) upon return of the muscle to normal Ringer, suggesting that significant amounts of glycerol enter the fibers during this time. While contrary to the original report of the glycerol effect, this finding was consistent with other studies involving the use of single fibers. Also reexamined was the potentiating effect of K⁺ on the hypertonic inhibition of muscle contraction. It was found that muscles exposed to this KCl pretreatment swell so that they are less dehydrated in hypertonic solutions, thus accounting for the observed potentiation. After being treated instead with a K₂-tartrate Ringer solution, muscles did not swell and, as determined with twitch recordings, did not display any potentiation in hypertonic solutions—even though the [K₊] was higher than an osmotically equivalent KCl solution. The evidence was thus consistent with alternative hypotheses in which inhibition of contraction occurs at a later stage in E-C coupling or involves the contractile process itself.     

Tension in Isolated Frog Muscle Fibers Induced by Hypertonic Solutions

The effect of hypertonic solutions on the tension of isolated twitch muscle fibers of the frog has been investigated. Increased tonicity up to about 1.7 times normal (1.7 T) caused a very small, graded, maintained tension increase. Above about 1.7 T a large, transient contracture response was superimposed on the small tension. The contracture response was graded with tonicity and reached a maximum at 2.5 T of 108 ± 25 mN·mm² a third of the maximum tetanic tension in isotonic solution. Contracture tension developed with a delay which decreased with increased tonicity. The contracture threshold was lower and the delay shorter in small fibers than in large. Contractures were obtained equally well in depolarized as in polarized fibers. They were completely suppressed by 0.1–0.5 mM tetracaine. The possible mechanism responsible for the tension-inducing effect of hypertonic solutions is discussed in terms of the close similarity between the properties of these contractures and those caused by caffeine, and it is suggested that the effect is due to a release of calcium from internal stores.     

The Postnatal Functional Development of Muscle Stretch Receptors in the Rat

The response to a 5-sec stretch of the triceps muscle was studied in dorsal root filaments L₅ of 72 infant rats (1-19 days old) under urethane anesthesia.

More than 50% of all units in 1-day-old rats responded by repetitive firing until the end of the 5-sec stretch (slowly adapting or SA receptors), while the rest ceased to fire earlier (relatively rapidly adapting or l/2 SA receptors), or gave an “on” response only. The number of units exhibiting an SA response increased with age and attained 80% in 5-day-old rats. By the 10th day of life, almost 90% of endings behaved as SA receptors. During development, the maximal discharge frequencies at the peak of stretch increased markedly, and their values in 18-day-old rats were comparable to those in adult rats. The phasic component of the response to stretch, although less well defined in the younger animals, was already present even in 1 -day-old rats. Adaptation of the static response during maintained stretch was relatively steep in all the age groups studied.

The results indicate that, in the rat, large numbers of muscle stretch receptors are capable of responding to sustained stretch as SA receptors, even at an age when their morphological and ultra-structural maturation is not yet fully accomplished.     

Caffeine- and Potassium-Induced Contractures of Frog Striated Muscle Fibers in Hypertonic Solutions

The effect of hypertonic solutions on the caffeine- and KCl-induced contractures of isolated fibers of frog skeletal muscle was tested. Hypertonic solutions, twice the normal osmotic strength, prepared by adding NaCl or sucrose, potentiate the caffeine-induced contractures. The fibers may develop tensions of 3.6 kg/cm² of fiber transverse section. The same hypertonic medium reduced the peak tension of KCl-induced contractures. Thus the hypertonic condition does not affect the contractile mechanism itself. These findings give further support to the view that the differential effect of hypertonic solution is on the excitation-contraction coupling mechanism. Extracellular calcium is not essentially required for the first few of a series of caffeine-induced contractures either in hypertonic or in isotonic solutions.     

Volume and Twitch Tension Changes in Single Muscle Fibers in Hypertonic Solutions

Single muscle fibers were exposed to solutions made hypertonic (approximately 460 milliosmols/kg water) by addition of either NaCl, glycerol, urea, acetamide, ethylene glycol, or propylene glycol. The changes in either the fiber twitch tension or the volume were measured. In the case of NaCl both fiber volume and twitch tension fall rapidly to 64 and 27% of the respective initial value. These two values were maintained for the duration of the exposure. In the case of the other substances, the fiber volume and twitch tension also decreased but in these cases the effect was transient and the fibers recovered their initial volume and twitch tension. The rate of recovery in the different hypertonic media increased in the order: glycerol < urea < ethylene glycol < propylene glycol < acetamide. In the cases of the last three substances, the initial twitch value was recovered in less than 5 min and even surpassed. However, on returning to normal Ringer the fibers' ability to twitch or to develop potassium contractures was lost. The return of the fibers to normal Ringer after exposure to these hypertonic solutions causes a transient swelling of the fibers. However, when fibers were swelled by exposure to hypotonic media, they did not lose their ability to twitch on return to the normal Ringer.     

Autocrine Phosphorylation of p70S6k in Response to Acute Stretch in Myotubes

Phosphorylation of 70-KDa S6 kinase (p70^S6k) is correlated with in vivo skeletal muscle hypertrophy. Experiments tested whether mechanical stretch is sufficient to increase p70^S6k phosphorylation in skeletal myotubes. Immediately following stretch, there was a small increase in p70^S6k phosphorylation (63.2 ± 8.5%) with maximal phosphorylation at 3 h (129.5 ± 22.2%) and it remained elevated through 24 h (46.0 ± 17.2%). To test whether an autocrine mechanism is involved, unstretched myotubes were incubated with medium from the stretch group for 10 min. Conditioned medium resulted in the phosphorylation of p70^S6k in unstretched myotubes (92.8 ± 28.9%) to levels comparable to the 3-h stretch group. These data indicate that p70^S6k is phosphorylated in stretched myotubes via a mechanism that most likely involves an autocrine signaling pathway.     

Hypertrophic Gene Expression Induced by Chronic Stretch of Excised Mouse Heart Muscle

Altered mechanical stress and strain in cardiac myocytes induce modifications in gene expression that affects cardiac remodeling and myocyte contractile function. To study the mechanisms of mechanotransduction in cardiomyocytes, probing alterations in mechanics and gene expression has been an effective strategy. However, previous studies are self-limited due to the general use of isolated neonatal rodent myocytes or intact animals. The main goal of this study was to develop a novel tissue culture chamber system for mouse myocardium that facilitates loading of cardiac tissue, while measuring tissue stress and deformation within a physiological environment. Intact mouse right ventricular papillary muscles were cultured in controlled conditions with superfusate at 95% O₂/ 5% CO₂, and 34°C, such that cell to extracellular matrix adhesions as well as cell to cell adhesions were undisturbed and both passive and active mechanical properties were maintained without significant changes. The system was able to measure the induction of hypertrophic markers (BNP, ANP) in tissue after 2 hrs and 5 hrs of stretch. ANP induction was highly correlated with the diastolic load of the muscle but not with developed systolic load. Load induced ANP expression was blunted in muscles from muscle-LIM protein knockout mice, in which defective mechanotransduction pathways have been predicted.     

Muscle Atrophy in Response to Cytotoxic Chemotherapy Is Dependent on Intact Glucocorticoid Signaling in Skeletal Muscle

Cancer cachexia is a syndrome of weight loss that results from the selective depletion of skeletal muscle mass and contributes significantly to cancer morbidity and mortality. The driver of skeletal muscle atrophy in cancer cachexia is systemic inflammation arising from both the cancer and cancer treatment. While the importance of tumor derived inflammation is well described, the mechanism by which cytotoxic chemotherapy contributes to cancer cachexia is relatively unexplored. We found that the administration of chemotherapy to mice produces a rapid inflammatory response. This drives activation of the hypothalamic-pituitary-adrenal axis, which increases the circulating level of corticosterone, the predominant endogenous glucocorticoid in rodents. Additionally, chemotherapy administration results in a significant loss of skeletal muscle mass 18 hours after administration with a concurrent induction of genes involved with the ubiquitin proteasome and autophagy lysosome systems. However, in mice lacking glucocorticoid receptor expression in skeletal muscle, chemotherapy-induced muscle atrophy is completely blocked. This demonstrates that cytotoxic chemotherapy elicits significant muscle atrophy driven by the production of endogenous glucocorticoids. Further, it argues that pharmacotherapy targeting the glucocorticoid receptor, given in concert with chemotherapy, is a viable therapeutic strategy in the treatment of cancer cachexia.     

Lamin A/C Regulates Endothelial Glucocorticoid Receptor Nuclear Translocation in Response to Cyclic Stretch

The glucocorticoid receptor (GR) has multiple phosphorylation sites that can be activated by MAPKs, which have been previously shown to be activated in response to cyclic stretch in endothelial cells. It is possible therefore that physiological and/or pathological degree of cyclic stretch may also initiate phosphorylation-induced changes in GR subcellular localization as we previously showed with shear stress. However, little is known about the effects of cyclic stretch on glucocorticoid receptor (GR) activity in endothelial cells. We used control and lamin shRNA BAECs and subjected them to ligand (dexamethasone) treatment, physiological stretch (10% at 1 Hz), or pathological stretch (20% at 1 Hz or 10% at 2 Hz), in order to evaluate GR nuclear translocation in endothelial cells with and without lamin A/C as well as potential upstream protein regulators of GR subcellular movement during cyclic stretch. Upon exposure to pathological degrees of stretching, control shRNA BAECs showed greater nuclear concentration of GR at each time point compared to when they were stretched at physiological parameters. The response of GR in lamin-deficient cells to cyclic stretching was relatively non-existent compared to that observed in control shRNA cells. Our results suggest that in cells with lamin A/C, cyclic stretch activates GR through the JNK pathway, and ERK has some inhibitory role on GR nuclear translocation. DUSP proteins become upregulated in response to stretch as a result of GR activation (DUSP1) or by stretch-induced MAPK signaling. In lamin-deficient cells, only the combination of cyclic stretch and p38 inhibition was able to induce marginal nuclear translocation. Increased MAPK phosphorylation due to lamin A/C absence could drive DUSP expression as a negative feedback mechanism. Upregulation of the cytoplasmic DUSP6 suggests a significant role of ERK in reducing GR sensitivity to mechanical strain.     

The Response of Duck Erythrocytes to Hypertonic Media : Further evidence for a volume-controlling mechanism

The addition of a hypertonic bathing medium to duck erythrocytes results in an initial instantaneous phase of osmotic shrinkage and, when the [K]_o of the hypertonic solution is larger than "normal," in a second, more prolonged phase, the volume regulatory phase. During the latter, which also requires extracellular Na, the cells swell until they approach their initial isotonic volume. The increase in cell volume during the volume regulatory phase is accomplished by a gain in the cell content of K, Cl, and H₂O. There is also a smaller increase in the Na content of the cell. Potassium is accumulated against an electrochemical gradient and is therefore actively transported into the cell. This accumulation is associated with an increase, although dissimilar, in both K influx and efflux. Changes in cell size during the volume regulatory phase are not altered by 10^-4 M ouabain, although this concentration of ouabain does change the cellular cation content. The response is independent of any effect of norepinephrine. The changes in cell size during the volume regulatory phase are discussed as the product of a volume controlling mechanism identical in principle to the one reported in the previous paper which controls cell volume in hypotonic media. Similarly, this mechanism can regulate cell size, when the Na-K exchange, ouabain-inhibitable pump mechanism is blocked.     

An Embryonic Myosin Isoform Enables Stretch Activation and Cyclical Power in Drosophila Jump Muscle

The mechanism behind stretch activation (SA), a mechanical property that increases muscle force and oscillatory power generation, is not known. We used Drosophila transgenic techniques and our new muscle preparation, the jump muscle, to determine if myosin heavy chain isoforms influence the magnitude and rate of SA force generation. We found that Drosophila jump muscles show very low SA force and cannot produce positive power under oscillatory conditions at pCa 5.0. However, we transformed the jump muscle to be moderately stretch-activatable by replacing its myosin isoform with an embryonic isoform (EMB). Expressing EMB, jump muscle SA force increased by 163% and it generated net positive power. The rate of SA force development decreased by 58% with EMB expression. Power generation is Pi dependent as >4 mM Pi was required for positive power from EMB. Pi increased EMB SA force, but not wild-type SA force. Our data suggest that when muscle expressing EMB is stretched, EMB is more easily driven backward to a weakly bound state than wild-type jump muscle. This increases the number of myosin heads available to rapidly bind to actin and contribute to SA force generation. We conclude that myosin heavy chain isoforms influence both SA kinetics and SA force, which can determine if a muscle is capable of generating oscillatory power at a fixed calcium concentration.     

剪应力和周向应变联合作用下血管内皮细胞内自由Ca^2＋响应

机械力调节血管内皮细胞功能。Ca2 在机械力信号转导中扮演了重要的角色。本文研究剪应力和周向应变联合作用下血管内皮细胞内自由Ca2 浓度的变化规律,结果表明,在生理周向应变条件(小于15%)下,同时暴露于剪应力和周向应变的细胞内自由Ca2 浓度变化更依赖于剪应力大小而非周向应变的大小,Ca2 浓度升高主要是胞外Ca2 内流引起的。     

Genetic Response of Rat Supraspinatus Tendon and Muscle to Exercise

Inflammation is a complex, biologic event that aims to protect and repair tissue. Previous studies suggest that inflammation is critical to induce a healing response following acute injury; however, whether similar inflammatory responses occur as a result of beneficial, non-injurious loading is unknown. The objective of this study was to screen for alterations in a subset of inflammatory and extracellular matrix genes to identify the responses of rat supraspinatus tendon and muscle to a known, non-injurious loading condition. We sought to define how a subset of genes representative of specific inflammation and matrix turnover pathways is altered in supraspinatus tendon and muscle 1) acutely following a single loading bout and 2) chronically following repeated loading bouts. In this study, Sprague-Dawley rats in the acute group ran a single bout of non-injurious exercise on a flat treadmill (10 m/min, 1 hour) and were sacrificed 12 or 24 hours after. Rats in the chronic group ran 5 days/wk for 1 or 8 weeks. A control group maintained normal cage activity. Supraspinatus muscle and tendon were harvested for RNA extractions, and a custom Panomics QuantiGene 2.0 multiplex assay was used to detect 48 target and 3 housekeeping genes. Muscle/tendon and acute/chronic groups had distinct gene expression. Components of the arachidonic acid cascade and matrix metalloproteinases and their inhibitors were altered with acute and chronic exercise. Collagen expression increased. Using a previously validated model of non-injurious exercise, we have shown that supraspinatus tendon and muscle respond to acute and chronic exercise by regulating inflammatory- and matrix turnover-related genes, suggesting that these pathways are involved in the beneficial adaptations to exercise.     

人类小腿肌肉对运动训练生理响应的电学特性研究

目的 运动训练已被证明能够改善许多慢性肌肉功能疾病,被用于治疗衰老型肌萎缩。本文采用电阻抗成像(electrical impedance tomography,EIT)研究人类小腿肌肉对运动训练生理响应的电学特性,旨在使用EIT方法可视化运动训练对人类小腿响应肌肉隔室内肌肉纤维体积增加的效果。方法 实验对象被要求在连续5个实验日进行左、右腿单侧提踵训练,应用EIT检测每日运动训练前和运动训练后小腿肌肉的电导率分布。为了定量分析运动训练对响应肌肉隔室的作用,使用配对样本t检验分析EIT重建图像的空间平均电导率<σ>。结果 运动训练后,由小腿腓肠肌组成的M₁肌肉隔室空间平均电导率<σ>_M1显著增加。此外,连续5个实验日的EIT测量结果显示,运动训练前的空间平均电导率<σ^pre>_M1呈上升趋势。所有实验对象在实验日1早晨进行实验前的腿部瘦体重与<σ>_M1呈线性关系,即<σ>_M1随腿部瘦体重增加而增加;运...     

Nitrergic Proprioceptive Afferents Originating from Quadriceps Femoris Muscle are Related to Monosynaptic Ia-Motoneuron Stretch Reflex Circuit in the Dog

1. The aim of the present study was to examine the occurrence of the neuronal nitric oxide synthase immunoreactivity in the stretch reflex circuit pertaining to the quadriceps femoris muscle in the dog.2. Immunohistochemical processing for neuronal nitric oxide synthase and histochemical staining for nicotinamide adenine dinucleotide phosphate diaphorase were used to demonstrate the presence of neuronal nitric oxide synthase in the proprioceptive afferents issuing in the quadriceps femoris muscle. The retrograde tracer Fluorogold injected into the quadriceps femoris muscle was used to detect the proprioceptive afferents and their entry into the L5 and L6 dorsal root ganglia.3. A noticeable number of medium-sized intensely nitric oxide synthase immunolabelled somata (1000–2000 μm² square area) was found in control animals in the dorsolateral part of L5 and L6 dorsal root ganglia along with large-caliber intraganglionic nitric oxide synthase immunolabelled fibers, presumed to be Ia axons. Before entering the dorsal funiculus the large-caliber nitric oxide synthase immunolabelled fibers of the L5 and L6 dorsal roots formed a massive medial bundle, which upon entering the dorsal root entry zone reached the dorsolateral part of the dorsal funiculus and were distributed here in a funnel-shaped fashion. The largest nitric oxide synthase immunolabelled fibers, 8.0–9.2 μm in diameter, remained close to the dorsal horn, while medium-sized fibers were seen dispersed across the medial portion of the dorsal funiculus. Single, considerably tapered nitric oxide synthase immunolabelled fibers, 2.2–4.6 μm in diameter, were seen to proceed in ventrolateral direction until they reached the mediobasal portion of the dorsal horn and the medial part of lamina VII. In lamina IX, only short fragments of nitric oxide synthase immunoreactive fibers and their terminal ramifications could be seen. Nitric oxide synthase immunolabelled terminals varying greatly in size were identified in control material at the base of the dorsal horn, in the vicinity of motoneurons ventrally and ventrolaterally in L5 and L6 segments and in Clarke’s column of L3 and L4 segments. Injections of the retrograde tracer Fluorogold into the quadriceps femoris muscle and cut femoral nerve, combined with nitric oxide synthase immunohistochemistry of the L5 and L6 dorsal root ganglia, confirmed the existence of a number of medium-sized nitric oxide synthase immunoreactive and Fluorogold-fluorescent somata presumed to be proprioceptive Ia neurons (1000–2000 μm² square area) in the dorsolateral part of both dorsal root ganglia. L5 and L6 dorsal rhizotomy caused a marked depletion of nitric oxide synthase immunoreactivity in the medial bundle of the L5 and L6 dorsal roots and in the dorsal funiculus of L5 and L6 segments.4. The analysis of control material and the degeneration of the large- and medium-caliber nitric oxide synthase immunoreactive Ia fibers in the dorsal funiculus of L5 and L6 segments confirmed the presence of nitric oxide synthase in the afferent limb of the monosynaptic Ia-motoneuron stretch reflex circuit related to the quadriceps femoris muscle. Abbreviations ABC, avidin–biotin complex; bNOS, neuronal nitric oxide synthase; bNOS-IR, neuronal nitric oxide synthase immunoreactive; bNOS-IRB_s, neuronal nitric oxide synthase immunoreactive boutons; cNOS, catalytic nitric oxide synthase; DAB, diaminobenzidine; DF, dorsal funiculus; DH, dorsal horn; DREZ-one, dorsal root entry zone; DRG_s, dorsal root ganglia; eNOS, endothelial nitric oxide synthase; FG, Fluorogold; FN, femoral nerve; mNOS, macrophage nitric oxide synthase; NADPHd, nicotinamide adenine dinucleotide phosphate diaphorase; NBT, nitroblue tetrazolium; NO, nitric oxide; NOS, nitric oxide synthase; NOS-IR, nitric oxide synthase immunoreactive; PBS, phosphate-buffered saline; VGLUT1 and VGLUT2, vesicular glutamate transporters     

An Embryonic Myosin Isoform Enables Stretch Activation and Cyclical Power in Drosophila Jump Muscle

The mechanism behind stretch activation (SA), a mechanical property that increases muscle force and oscillatory power generation, is not known. We used Drosophila transgenic techniques and our new muscle preparation, the jump muscle, to determine if myosin heavy chain isoforms influence the magnitude and rate of SA force generation. We found that Drosophila jump muscles show very low SA force and cannot produce positive power under oscillatory conditions at pCa 5.0. However, we transformed the jump muscle to be moderately stretch-activatable by replacing its myosin isoform with an embryonic isoform (EMB). Expressing EMB, jump muscle SA force increased by 163% and it generated net positive power. The rate of SA force development decreased by 58% with EMB expression. Power generation is Pi dependent as >4 mM Pi was required for positive power from EMB. Pi increased EMB SA force, but not wild-type SA force. Our data suggest that when muscle expressing EMB is stretched, EMB is more easily driven backward to a weakly bound state than wild-type jump muscle. This increases the number of myosin heads available to rapidly bind to actin and contribute to SA force generation. We conclude that myosin heavy chain isoforms influence both SA kinetics and SA force, which can determine if a muscle is capable of generating oscillatory power at a fixed calcium concentration.     

Azelnidipine Inhibits Cultured Rat Aortic Smooth Muscle Cell Death Induced by Cyclic Mechanical Stretch

Acute aortic dissection is the most common life-threatening vascular disease, with sudden onset of severe pain and a high fatality rate. Clarifying the detailed mechanism for aortic dissection is of great significance for establishing effective pharmacotherapy for this high mortality disease. In the present study, we evaluated the influence of biomechanical stretch, which mimics an acute rise in blood pressure using an experimental apparatus of stretching loads in vitro, on rat aortic smooth muscle cell (RASMC) death. Then, we examined the effects of azelnidipine and mitogen-activated protein kinase inhibitors on mechanical stretch-induced RASMC death. The major findings of the present study are as follows: (1) cyclic mechanical stretch on RASMC caused cell death in a time-dependent manner up to 4 h; (2) cyclic mechanical stretch on RASMC induced c-Jun N-terminal kinase (JNK) and p38 activation with peaks at 10 min; (3) azelnidipine inhibited RASMC death in a concentration-dependent manner as well as inhibited JNK and p38 activation by mechanical stretch; and (4) SP600125 (a JNK inhibitor) and SB203580 (a p38 inhibitor) protected against stretch-induced RASMC death; (5) Antioxidants, diphenylene iodonium and tempol failed to inhibit stretch-induced RASMC death. On the basis of the above findings, we propose a possible mechanism where an acute rise in blood pressure increases biomechanical stress on the arterial walls, which induces RASMC death, and thus, may lead to aortic dissection. Azelnidipine may be used as a pharmacotherapeutic agent for prevention of aortic dissection independent of its blood pressure lowering effect.