Multiscale modeling of calcium dynamics in ventricular myocytes with realistic transverse tubules

Spatial-temporal Ca(2+) dynamics due to Ca(2+) release, buffering, and reuptaking plays a central role in studying excitation-contraction (E-C) coupling in both normal and diseased cardiac myocytes. In this paper, we employ two numerical methods, namely, the meshless method and the finite element method, to model such Ca(2+) behaviors by solving a nonlinear system of reaction-diffusion partial differential equations at two scales. In particular, a subcellular model containing several realistic transverse tubules (or t-tubules) is investigated and assumed to reside at different locations relative to the cell membrane. To this end, the Ca(2+) concentration calculated from the whole-cell modeling is adopted as part of the boundary constraint in the subcellular model. The preliminary simulations show that Ca(2+) concentration changes in ventricular myocytes are mainly influenced by calcium release from t-tubules.     

Spontaneous Calcium Transients in Cultured Cortical Networks During Development

Spontaneous neuronal activity plays an important role in the development of the brain. Developmental changes in the spontaneous activity pattern of neuronal networks in vitro have been extensively studied by using the microelectrode array (MEA) recording system. However, little is known about the transition of spontaneous intracellular calcium dynamics, and the relationship between calcium transients and electrical activity during development. In the present paper, we carry out simultaneous recording of spontaneous electrical activity and intracellular calcium transients of rat cortical networks cultured on MEA. In one-week cultures, periodic synchronized bursts are observed and are followed by synchronized calcium transients. In three-week cultures, synchronized calcium transients are rarely observed despite the presence of highly complicated synchronized activity. Between these two states, in two-week cultures, slow, radial propagation of calcium waves independent of electrical activity is observed. Pharmacological treatments with the purinergic receptor antagonist suramin and gap junction blocker 18-$beta$ glycyrrhetinic acid reveal that the spontaneous radial calcium waves are mediated by the astrocytic network, and suggest that the astrocytic calcium waves can influence the electrical firing patterns of networks by locally affecting neuronal signaling. These results indicate that the various dynamics of intracellular calcium transients regulate the network maturation processes.      

Multiscale FEM modeling of vascular tone: from membrane currents to vessel mechanics

Regulation of vascular tone is a complex process that remains poorly understood. Here, we present our recent efforts for the development of physiologically realistic models of arterial segments for the analysis of vasoreactivity in health and disease. Multiscale modeling integrates intracellular and cell membrane components into whole-cell models of calcium and membrane potential dynamics. Single-cell models of vascular cells are combined into a multicellular model of the vascular wall, and vessel wall biomechanics are integrated with calcium dynamics in the smooth muscle layer. At each scale, continuum models using finite element method can account for spatial heterogeneity in calcium signaling and for nonuniform deformations of a vessel segment. The outlined approach can be used to investigate cellular mechanisms underlying altered vasoreactivity in hypertension.     

Mechanism of Ca2+ increase in myoblasts derived from chicken embryos

The mechanism of intracellular calcium ions (Ca(2+)) increase in chicken myoblasts was studied using histological, immunohistochemical, immunoblotting and Ca(2+) imaging techniques. Mononuclear myoblasts at embryonic day 12 (E12) contained myofibrils in the peripheral cytoplasm, and the sarcoplasmic reticulum was observed in the cytoplasm. Several Ca(2+)-related receptors, namely acetylcholine (ACh) receptors, dihydropyridine receptors (DHPRs) and ryanodine receptors (RyRs), were detected in the tissue as early as E12. Western blotting analyses detected one band corresponding to RyR subtype 3 (RyR3) at E12 and two bands corresponding to RyR1 and RyR3 after E13. Ca(2+) imaging of mononuclear myoblasts in vitro revealed an intense Ca(2+)-increase response to ACh stimulation, and this effect was abolished after EGTA addition to the culture medium. Nifedipine treatment also led to a lack of Ca(2+) increase in response to ACh stimulation, while ryanodine treatment led to a weak Ca(2+)-increase response. On the other hand, multinuclear myoblasts showed a Ca(2+)-increase response to ACh stimulation in the presence of not only EGTA but also nifedipine, although ryanodine treatment led to a lack of Ca(2+) increase. These results suggest that the mechanism of Ca(2+) increase in mononuclear myoblasts involves extracellular Ca(2+) entry through DHPRs, which is amplified by Ca(2+) release from the cytoplasmic Ca(2+) store, while multinuclear myoblasts mainly depend on Ca(2+) release from the cytoplasmic Ca(2+) store.     

2-Aminoethoxydiphenyl borate (2-APB) inhibits capacitative calcium entry independently of the function of inositol 1,4,5-trisphosphate receptors

Capacitative calcium entry (CCE), the mechanism that replenishes intracellular calcium stores after depletion, is essential to intracellular calcium signaling. CCE is mediated by the channels in the plasma membrane generally referred to as "store operated channels (SOCs)". However, the molecular identity of the SOCs has never been determined, and the mechanism of the activation of SOCs remains to be elucidated. Recent studies have demonstrated that 2-aminoethoxydiphenyl borate (2-APB), which has been found to be an antagonist of inositol 1,4,5-trisphosphate receptors (IP3Rs), inhibits CCE, suggesting that IP3Rs channel activity is essential to the generation of CCE. However, CCE has also been reported to occur normally in IP3R-deficient cells. In order to resolve this discrepancy, we investigated the effect of 2-APB on CCE in IP3Rs-deficient cells. In response to store depletion with thapsigargin or N,N,N',N'-tetrakis (2-pyridylmethyl) ethylene diamine (TPEN), CCE was generated in IP3Rs-deficient cells the same as in wild-type cells, however, 2-APB abolished CCE in IP3Rs-deficient cells, despite the fact that this cell line does not possess functional IP3Rs. We also examined the effect of 2-APB on several types of TRP Ca2+ channels, which exhibit properties similar to those of SOCs. 2-APB had a different inhibitory effect on spontaneous and thapsigargin-induced Ba2+ influx in cells that transiently expressed individual TRP subtypes. These results suggest that the channel activity of IP3Rs is not essential to the generation of CCE in this cell line and that 2-APB inhibits CCE independently of the function of IP3Rs.     

Application of real-time confocal laser scanning microscopy to observe living cells in tissue specimens

Fluorescence microscopy imaging has developed into an important tool for the study of cell structure and function in cell biology. This non-invasive technique permits the characterization, localization and qualitative quantification of free ions, messengers, pH, voltage and other molecules in living cells. The regulation of cytosolic Ca2+ homeostasis is essential for cells. However, most investigations have used cultured or isolated cells as an experimental model and, consequently, provide only limited insight into the mechanisms that operate in tissue in situ. More useful information could be obtained by studying intact tissue specimens. The calcium dynamics of some tissue specimens, such as arteriole smooth muscle cells, supra cervical ganglia and peripheral nerve bundles, were analysed in this study. Real-time confocal microscopy revealed that individual cells exhibited different [Ca2+]i dynamics and the responses to transmitters/modulators were heterogeneous. It is important that the confocal microscopes have good detection performances, due to the reduction of stray light. We conclude that real-time confocal microscopy is a useful tool for investigating structural and functional changes of cells in living tissues, although suitable tissue-preparation is important for these measurements.     

植物细胞钙离子检测与成像技术研究进展

钙离子是一种重要的第二信使,参与调节植物多种生理和发育过程.胞内钙离子呈现复杂的时空动态变化,只有实时捕获钙离子在植株整体水平以及亚细胞水平的变化,才能深入理解钙信号的重要功能.过去几十年中,人们在钙离子检测与成像技术方面进行了大量探索,本文总结了近年来植物科学研究中常用的钙离子检测与成像技术,着重介绍了化学荧光探针和基于荧光蛋白钙离子探针的原理、特点、浓度计算方法、在细胞质以及细胞器钙离子检测与成像中的应用等,并总结了化学荧光探针的装载方法.     

低功率激光对细胞质膜通透性及细胞功能的影响

目的:探索低功率激光对细胞质膜通透性及细胞功能的影响。方法:以波长为632.8nm,功率密度为5.4mW/cm~2的氦氖激光照射人外周血淋巴细胞15、30、60分钟,并采用钙荧光指示剂Fura—2/Am定量测试法检测淋巴细胞内游离钙浓度和质膜Ca~(2+)—Mg~(2+)—ATP酶活性变化。结果:照射后淋巴细胞内游离钙浓度明显低于正常(P<0.05);同时细胞膜Ca~(2+)—Mg~(2+)—ATP酶活性增加(P<0.05);而且照射后细胞内游离钙浓度降低与质膜上Ca~(2+)—Mg~(2+)—ATP酶的激活呈负相关。结论:低功率激光照射激活细胞膜Ca~(2+)—Mg~(2+)—ATP酶活性,使细胞膜对钙通透性发生变化,且影响到细胞内Ca~(2+)贮存,造成细胞膜通透性和细胞功能的改变。     

Ultrafast Near‐Infrared Light‐Triggered Intracellular Uncaging to Probe Cell Signaling

The possibility of regulating cell signaling with high spatial and temporal resolution within individual cells and complex cellular networks has important implications in biomedicine. This article demonstrates a general strategy that uses near‐infrared tissue‐penetrating laser pulses to uncage biomolecules from plasmonic gold‐coated liposomes, i.e., plasmonic liposomes, to activate cell signaling in a nonthermal, ultrafast, and highly controllable fashion. Near‐infrared picosecond laser pulse induces transient nanobubbles around plasmonic liposomes. The mechanical force generated from the collapse of nanobubbles rapidly ejects encapsulated compound within 0.1 ms. This article shows that single pulse irradiation triggers the rapid intracellular uncaging of calcein from plasmonic liposomes inside endolysosomes. The uncaged calcein then evenly distributes over the entire cytosol and nucleus. Furthermore, this article demonstrates the ability to trigger calcium signaling in both an immortalized cell line and primary dorsal root ganglion neurons by intracellular uncaging of inositol triphosphate (IP₃), an endogenous cell calcium signaling second messenger. Compared with other uncaging techniques, this ultrafast near‐infrared light‐driven molecular uncaging method is easily adaptable to deliver a wide range of bioactive molecules with an ultrafast optical switch, enabling new possibilities to investigate signaling pathways within individual cells and cellular networks.     

基于荧光显微镜的显微视觉图像增强处理

针对显微视觉景深较小，经常出现图像模糊、不清晰等问题，文中提出采取一种图像增强的处理方法。通过对显微采集原始图像进行灰度变换、直方图均衡化等处理方法从而快速准确地获得目标的图像特征。基于图像增强、目标细胞定位处理对荧光图像中信息进行分析，找寻目标位置，提高细胞定位精确度。根据图像清晰度评价函数显示，经由处理后的图像边界和细节部分清晰，有助于医学应用。     

A preliminary model of gastrointestinal electromechanical coupling

Gastrointestinal (GI) motility is coordinated by several cooperating mechanisms, including electrical slow wave activity, the enteric nervous system (ENS), and other factors. Slow waves generated in interstitial cells of Cajal (ICC) depolarize smooth muscle cells (SMC), generating basic GI contractions. This unique electrical coupling presents an added layer of complexity to GI electromechanical models, and a current barrier to further progress is the lack of a framework for ICC-SMC-contraction coupling. In this study, an initial framework for the electromechanical coupling was developed in a 2-D model. At each solution step, the slow wave propagation was solved first and [Ca(2+)](i) in the SMC model was related to a Ca(2+)-tension-extension relationship to simulate active contraction. With identification of more GI-specific constitutive laws and material parameters, the ICC-SMC-contraction approach may underpin future GI electromechanical models of health and disease states.     

Fabrication of a pneumatically driven single-cell trap

This paper describes the creation of a pneumatically driven single-cell trap and its ability to trap a live single cell. The cell-trap device consists of a pneumatic vibrator array and a trap chamber used to trap single cells. The entire structure was manufactured with polydimethylsiloxane (PDMS). External compressed air was used to actuate the pneumatic vibrators. The magnitude and frequency of the pneumatic force were controlled by programmable electromagnetic valves. The live cell in the trap chamber was manipulated using the velocity field of the cell media induced by deformation of the vibrator diaphragm. The cell was successfully trapped in the central equilibrium region of the device, at which point the axial velocity fields generated by the vibrators were minimized. The intracellular calcium response of a single cell induced by mechanical stimuli during cell trapping was measured to verify the effectiveness of the pneumatic cell trap as a stress-free trapping method. The results showed that the internal signaling in the cell was sensitive to the extracellular physical environment. Since the measured intensity of the intracellular calcium of the single cell was very minor, the pneumatic cell trap described in this paper was categorized as a harmless trapping method.     

PKA信号通路与ryanodine受体介导的心肌肌浆网钙泄漏关系的研究

目的：研究β肾上腺素激活的PKA信号通路与成年大鼠心肌细胞RyR2介导的肌浆网Ca^2＋泄漏之间的关系,探讨治疗心力衰竭的新方法。方法：NiCl2（5mmol/L）和毒胡萝卜素（TG,100nmol/L）分别阻断成年大鼠心室肌细胞的钠钙交换体和钙泵,胞外灌流异丙肾上腺素（ISO,1μmol/L）。采用激光扫描共聚焦显微镜记录灌流前后细胞内钙火花及[Ca2＋]i的变化,并使用Ca^2＋通道阻断剂钌红（RR,5μmol/L）对β肾上腺素刺激作用进行逆转,进而阻断RyR2介导的肌浆网内钙泄漏。结果：ISO胞外灌流后,心肌细胞钙火花发生率和[Ca^2＋]i与对照组相比明显升高（n=10,P〈0.05）;加入RR后,心肌细胞内钙火花发生率与对照组比较显著降低（n=20,P〈0.05）,[Ca^2＋]i两组没有明显差别（n=20,P〉0.05）。结论：ISO激活PKA信号通路后可诱导心肌细胞RyR2介导的钙泄漏,RR可有效地逆转这种钙泄漏。     

Electroaddressing of Cell Populations by Co‐Deposition with Calcium Alginate Hydrogels

Electroaddressing of biological components at specific device addresses is attractive because it enlists the capabilities of electronics to provide spatiotemporally controlled electrical signals. Here, the electrodeposition of calcium alginate hydrogels at specific electrode addresses is reported. The method employs the low pH generated at the anode to locally solubilize calcium ions from insoluble calcium carbonate. The solubilized Ca²⁺ can then bind alginate to induce this polysaccharide to undergo a localized sol‐gel transition. Calcium alginate gel formation is shown to be spatially controlled in the normal and lateral dimensions. The deposition method is sufficiently benign that it can be used to entrap the bacteria E. coli. The entrapped cells are able to grow and respond to chemical inducers in their environment. Also, the entrapped cells can be liberated from the gel network by adding sodium citrate that can compete with alginate for Ca²⁺ binding. The capabilities of calcium alginate electrodeposition is illustrated by entrapping reporter cells that can recognize the quorum sensing autoinducer 2 (AI‐2) signaling molecule. These reporter cells were observed to recognize and respond to AI‐2 generated from an external bacterial population. Thus, calcium alginate electrodeposition provides a programmable method for the spatiotemporally controllable assembly of cell populations for cell‐based biosensing and for studying cell‐cell signaling.     

Reducing overhead in movement based dynamic location management scheme for cellular networks

One of the key issues in cellular mobile communication is to find the current location of mobile terminal (MT) to deliver the services, which is called location management (LM). Much research has been done on dynamic LM that reduced the LM cost up to a large extent. In movement based dynamic LM scheme, the location area is defined in the form of ring of cells for individual user. Whenever an MT visits a cell outside of its current location area (LA), it triggers location update (LU). For this purpose, network must inform the mobile terminal about ID of all the cells present in its current location area. In this paper, a simple way of cell-ID assignment is proposed under which, network sends only the ID of center cell of LA ring to MT and then MT can compute IDs of all other cells in its location area. This saves a significant amount of wireless bandwidth by minimizing the signaling traffic at VLR level and thus reduces the mobility management overhead.     

Performance bounds of a multiple-step paging strategy in future universal mobile telecommunication systems

In future mobile telecommunications, due to the huge number of users and the specific functions that support mobility, a significant amount of signaling load will have to be carried by the finite capacity of the radio link. Hence, methods aiming at radio link signaling load reduction are welcome. In this paper we propose and analyze a method that saves paging signaling load by exploiting information related to the terminal location during the most recent interaction between the terminal and the network. The penalty paid is extra processing power and extra paging delays. An analytical model is developed so as to describe the performance versus traffic intensity and mobility conditions. The performance of the proposed paging scheme is investigated, and it is shown that the method operates well even in the worst case, which is the high user mobility conditions. Possible extensions of the method, which exploit information related to the mobility degree of each individual user or information characterizing the mobility conditions in a certain location area, are also proposed.This paper has been partially funded by CEC through the RACE 2066 Mobile Network (MONET) project. The paper does not present the views of the project as a whole, but those of the authors.     

Ultrashort electrical pulses open a new gateway into biological cells

An electrical model for biological cells predicts that for pulses with durations shorter than the charging time of the outer membrane, there is an increasing probability of electric field interactions with intracellular structures. Experimental studies in which human cells were exposed to pulsed electric fields of up to 300-kV/cm amplitude, with durations as short as 10 ns, have confirmed this hypothesis. The observed effects include the breaching of intracellular granule membranes without permanent damage to the cell membrane, abrupt rises in intracellular free calcium levels, and enhanced expression of genes. At increased electric fields, the application of submicrosecond pulses induces apoptosis (programmed cell death) in biological cells, an effect that has been shown to reduce the growth of tumors. Possible applications of the intracellular electroeffect are enhancing gene delivery to the nucleus, controlling cell functions that depend on calcium release (causing cell immobilization), and treating tumors.     

Photophysics of Conjugated Oligoelectrolytes Relevant to Two-Photon Fluorescence-Lifetime Imaging Microscopy

Conjugated oligoelectrolytes (COEs) comprise a class of cell-membrane intercalating molecules that serve as effective optical reporters. However, little is known about the photophysical properties of COEs in biological environments such as buffers, cell membranes, and intracellular organelles, which is critical to optimize performance. Herein, how COE self-assembly depends on the dielectric environment (polarity and ion content) is explored based on the representative molecule 6-ring phenylenevinylene (PV) conjugated oligoelectrolyte (COE-S6), and its optical properties within mammalian cells are subsequently studied. Two-photon fluorescence lifetime imaging microscopy (FLIM), confocal laser scanning microscopy, and optical properties in solutions are brought together to obtain information about the location, accumulation, and characteristics of the local surroundings. FLIM imaging lifetime phasor plots, decays, and fluorescence spectra on stained mammalian cells provide evidence of successful COE-S6 internalization via endocytosis. The fluorescence lifetime of COE-S6 is identical when in A549 mammalian cells and in giant unilamellar vesicle model membranes, thereby providing a correlation between living system and artificial constructs.     

Stimulation of isolated ventricular myocytes within an open architecture microarray

This paper is concerned with the physiological responses of single heart cells within microfluidic chambers, in response to stimulation by integrated microelectrodes. To enable these investigations, which included the measurement of action potential duration, intracellular Ca2+ and cell shortening, a series of microfluidic chambers (50 microm wide, 180 microm long, 400 microm high, 500 microm pitch) and connecting channels (200 microm wide, 5000 microm long, 50 microm high, 500 microm pitch) were replica-moulded into the silicone elastomer, polydimethylsiloxane (PDMS). The structures were formed against a master of posts and lines, photolithograhically patterned into the high aspect ratio photoresist SU-8. The chambers within the slab of PDMS were aligned against pairs of stimulating gold microelectrodes (50 microm long, 20 microm wide, 0.1-10 microm thick, 180 microm apart) patterned on a microscope coverslip base, thus defining cavities of approximately 4 nL volume. The assembly was filled with physiological saline and single isolated rabbit ventricular myocytes were introduced by micropipetting, thus creating limited volumes of saline above individual myocytes that could be varied between 4 nL and > or = 4 microL. The application of transient current pulses to the cells via the electrodes caused transient contractions with constant amplitude (recorded as changes in sarcomere length), confirming that excitation contraction coupling (EC coupling) remained functional in these limited volumes. Continuous monitoring of the intracellular Ca2+ (using calcium sensitive dyes) showed, that in the absence of bath perfusion, the amplitude of the transients remained constant for approximately 3 min in the 4-nL volume and approximately 20 min for the 4 microL volume. Beyond this time, the cells became unexcitable until the bath was renewed. The action potential duration (APD) was recorded at stimulation frequencies of 1 Hz and 0.5 Hz using potential sensitive dyes and was prolonged at the higher pacing rate. These studies show the prolonged electrical stimulation of isolated adult cardiac myocytes in microchambers with unimpaired EC coupling as verified on optical records of the action potential, Ca2+ transients and cell shortening. The open architecture provided free (pipetting) access for drug dispensation without cross talk between neighboring microwells, and multiplexed optical detection can be realized to study EC coupling on arrays of cells under both control and experimental conditions.     

牛磺酸在体外对缺血/再灌注新生大鼠心肌细胞内游离钙的影响

目的:观察低氧、复氧对心肌细胞内Ca2+浓度([Ca2+]i)的影响,以及牛磺酸模拟心肌缺血/再灌注(I/R)过程中减轻钙超载的作用。方法:采用SD大鼠乳鼠进行心肌细胞培养,建立模拟I/R模型。实验分4组:①正常对照组;②模拟缺血/缺氧组:细胞低氧180min;③模拟I/R组:细胞低氧180min,复氧180min;③牛磺酸+I/R组:先加入终浓度为20mmol.L-1的牛磺酸,再行低氧180min,复氧180min。以Fluo-4/AM荧光指示剂负载,应用激光共聚焦显微镜技术检测心肌细胞[Ca2+]i变化。结果:对照组心肌细胞[Ca2+]i荧光强度(23.71±2.37)U和荧光光密度较低;低氧180min后复氧即刻,[Ca2+]i荧光强度开始增加(57.52±8.31),复氧180min后[Ca2+]i荧光强度(71.13±4.74)U和光密度显著增高(P<0.01vs对照组)。而牛磺酸组细胞内荧光强度和光密度较模拟I/R组显著降低[(42.42±4.17)U vs(71.13±4.74)U,P<0.01]。结论:心肌细胞缺血/缺氧导致Ca2+超载;模拟I/R Ca2+超载加剧,而牛磺酸有明显减轻心肌细胞模拟I/R时Ca2+超载的作用。