Electrophysiological properties of the membrane of smooth muscle cells from bovine lymphatic vessels

Using single sucrose gap technique, studies have been made on electrophysiological properties of the membrane in myocytes of the lymphatic vessels in the ox Bos taurus. It was shown that electrical stimulation does not induce tetanic contraction in the myocytes. The results obtained indicate strong similarity between electrophysiological properties of the myocytes in the lymphatic vessels and those of the myocardial cells in homoiotherms. Refractory state which follows the action potential, accounts for a possibility of rhythmic activity in the myocytes of the lymphatic vessels. Both single and rhythmic stimulation produce in the myocytes the "all-or-none" response. The main factor determining the level of excitability in the myocytes is the intravascular pressure. The latter exerts its influence on contractile activity via changes in the electrical activity (the membrane potential, duration of the plateau phase and the number of fast peak potentials on this plateau).     

Optogenetic versus Electrical Stimulation of Human Cardiomyocytes: Modeling Insights

Optogenetics provides an alternative to electrical stimulation to manipulate membrane voltage, and trigger or modify action potentials (APs) in excitable cells. We compare biophysically and energetically the cellular responses to direct electrical current injection versus optical stimulation mediated by genetically expressed light-sensitive ion channels, e.g., Channelrhodopsin-2 (ChR2). Using a computational model of ChR2(H134R mutant), we show that both stimulation modalities produce similar-in-morphology APs in human cardiomyocytes, and that electrical and optical excitability vary with cell type in a similar fashion. However, whereas the strength-duration curves for electrical excitation in ventricular and atrial cardiomyocytes closely follow the theoretical exponential relationship for an equivalent RC circuit, the respective optical strength-duration curves significantly deviate, exhibiting higher nonlinearity. We trace the origin of this deviation to the waveform of the excitatory current—a nonrectangular self-terminating inward current produced in optical stimulation due to ChR2 kinetics and voltage-dependent rectification. Using a unifying charge measure to compare energy needed for electrical and optical stimulation, we reveal that direct electrical current injection (rectangular pulse) is more efficient at short pulses, whereas voltage-mediated negative feedback leads to self-termination of ChR2 current and renders optical stimulation more efficient for long low-intensity pulses. This applies to cardiomyocytes but not to neuronal cells (with much shorter APs). Furthermore, we demonstrate the cell-specific use of ChR2 current as a unique modulator of intrinsic activity, allowing for optical control of AP duration in atrial and, to a lesser degree, in ventricular myocytes. For self-oscillatory cells, such as Purkinje, constant light at extremely low irradiance can be used for fine control of oscillatory frequency, whereas constant electrical stimulation is not feasible due to electrochemical limitations. Our analysis offers insights for designing future new energy-efficient stimulation strategies in heart or brain.     

Simulation and mechanistic investigation of the arrhythmogenic role of the late sodium current in human heart failure

Heart failure constitutes a major public health problem worldwide. The electrophysiological remodeling of failing hearts sets the stage for malignant arrhythmias, in which the role of the late Na(+) current (I(NaL)) is relevant and is currently under investigation. In this study we examined the role of I(NaL) in the electrophysiological phenotype of ventricular myocytes, and its proarrhythmic effects in the failing heart. A model for cellular heart failure was proposed using a modified version of Grandi et al. model for human ventricular action potential that incorporates the formulation of I(NaL). A sensitivity analysis of the model was performed and simulations of the pathological electrical activity of the cell were conducted. The proposed model for the human I(NaL) and the electrophysiological remodeling of myocytes from failing hearts accurately reproduce experimental observations. The sensitivity analysis of the modulation of electrophysiological parameters of myocytes from failing hearts due to ion channels remodeling, revealed a role for I(NaL) in the prolongation of action potential duration (APD), triangulation of the shape of the AP, and changes in Ca(2+) transient. A mechanistic investigation of intracellular Na(+) accumulation and APD shortening with increasing frequency of stimulation of failing myocytes revealed a role for the Na(+)/K(+) pump, the Na(+)/Ca(2+) exchanger and I(NaL). The results of the simulations also showed that in failing myocytes, the enhancement of I(NaL) increased the reverse rate-dependent APD prolongation and the probability of initiating early afterdepolarizations. The electrophysiological remodeling of failing hearts and especially the enhancement of the I(NaL) prolong APD and alter Ca(2+) transient facilitating the development of early afterdepolarizations. An enhanced I(NaL) appears to be an important contributor to the electrophysiological phenotype and to the dysregulation of [Ca(2+)](i) homeostasis of failing myocytes.     

Endothelium-dependent effect of bradykinin and ATP on rabbit ventricular myocytes.

We have investigated the effects of bradykinin (BK) and ATP on Ca2+ transient induced by electrical-field stimulation in freshly isolated rabbit ventricular myocytes, in the presence or absence of rabbit aortic endothelial cells. BK and ATP induced an increase in intracellular Ca2+ concentration ([Ca2+]i) in the endothelial cells, but had no significant effect on Ca2+ transient in electrical-field stimulated ventricular myocytes. In the presence of cultured endothelial cells, the amplitude of Ca2+ transient induced by electrical stimulation in ventricular myocytes was decreased. BK and ATP further reduced the amplitude of Ca2+ transient induced by electrical stimulation in ventricular myocytes. These data show that BK and ATP have endothelium-dependent depressing effects on ventricular myocytes and indicate that substances released from endothelial cells in response to BK and ATP stimulation can modulate ventricular myocytes excitation-contraction coupling. However, identification of the cardioactive mediators produced by the endothelial cells requires further study.     

Recovery from central apnea: effect of stimulus duration and end-tidal CO2 partial pressure

The present study was designed to investigate the effect of stimulus duration and chemosensory input on the recovery of central respiratory activity from apnea induced by superior laryngeal nerve (SLN) electrical stimulation. Newborn piglets less than 8 days of age were anesthetized, paralyzed, and mechanically ventilated at differing levels of end-tidal CO2 partial pressure (PCO2). The vagi were cut bilaterally in the neck. Integrated phrenic nerve activity was used as the index of respiratory activity. SLN stimulation caused apnea that persisted after stimulus cessation. The length of apnea following stimulus cessation was directly related to stimulus duration and inversely related to end-tidal PCO2. After apnea, respiratory activity returned gradually to the initial control level. The recovery pattern was well described by a linear regression function using the natural logarithm of time as the independent variable. Prolonging stimulus duration progressively inhibited the amount of initial respiratory activity following apnea. On the other hand, the rate of respiratory recovery was independent of stimulus duration and, except at low end-tidal PCO2 following long (30 s) stimuli, was independent of the end-tidal PCO2 level. These results demonstrate that a long-acting central mechanism regulates recovery from apnea induced by SLN stimulation.     

The myocytes of the lymphatic vessels]

Histo- and ultrastructure of myocytes of the lymphatic vessels has been studied in some farm animals. Architectonics, amount and interconnection of myocytes in the lymphangion wall have been investigated. Communicational and gap myo-myocytic contacts revealed give a possibility to suppose that there exists a direct connection between myocytes. This is important for conducting electrical stimulation from one cell to another. For the myocytes abundance of myofilaments, and in some--an essential accumulation of mitochondria are specific; they are morphological manifestation of contractile function of myocytes.     

Transient finite element modeling of functional electrical stimulation

Transcutaneous functional electrical stimulation is commonly used for strengthening muscle. However, transient effects during stimulation are not yet well explored. The effect of an amplitude change of the stimulation can be described by static model, but there is no differency for different pulse duration. The aim of this study is to present the finite element (FE) model of a transient electrical stimulation on the forearm. Discrete FE equations were derived by using a standard Galerkin procedure. Different tissue conductive and dielectric properties are fitted using least square method and trial and error analysis from experimental measurement. This study showed that FE modeling of electrical stimulation can give the spatial-temporal distribution of applied current in the forearm. Three different cases were modeled with the same geometry but with different input of the current pulse, in order to fit the tissue properties by using transient FE analysis. All three cases were compared with experimental measurements of intramuscular voltage on one volunteer.     

压力负荷性心衰小鼠左心室跨壁L-型钙电流的变化

为了观察正常和心衰时心内膜下和心外膜下心肌细胞L-型钙电流(ICa-L)的差别,我们采用主动脉弓狭窄的方法建立小鼠压力超负荷性心衰模型,采用全细胞膜片钳技术记录了正常、主动脉狭窄(band)及假手术对照(sham)组动物左心室游离壁内、外膜下心肌细胞的动作电位时程(action potential duration,APD)和ICa-L。结果显示:(1)与sham组同龄的正常小鼠左心室心内膜下细胞动作电位复极达90％的时程(APD90)为(38．2±6．44)ms,较心外膜下细胞的APD90(15.67±5.31)ms明显延长,二者的比值约为2．5:1;内膜下细胞和外膜下细胞ICa-L密度没有差异,峰电流密度分别为(-2.7±0.49)pA/pF和(-2.54±0．53)pA/pF;(2)Band组内、外膜下细胞的动作电位复极达50％的时程(APD50)、APD90均较sham组显著延长,尤以内膜下细胞延长突出,分别较sham组延长了400％和360％,内、外膜下细胞APD90的比值约为4.2:1;(3)与sham组相比, band组内膜下细胞ICa-L密度显著减小,在+10 mV～+40 mV的4个电压下分别降低了20．2％、21．4％、21.6％和25.7％(P< 0．01),但其激活电位、峰电位和翻转电位没有改变;band组外膜下细胞的ICa-L密度与同期sham组相比无明显变化;band组钙通道激活、失活及复活的动力学特征与sham组相比没有改变。以上结果提示,生理状态下小鼠左心室内、外膜下细胞ICa-L密度不存在明显差别,提示ICa-L与APD跨壁异质性的产生无关;心衰时左心室内、外膜下细胞APD明显延长,以内膜下细胞延长尤为突出,内膜下细胞ICa-L密度明显减少,而外膜下细胞ICa-L密度无明显改变,这种ICa-L的非同步变化在心衰时可能起到对抗APD延长、减少复极离散度的有益作用。     

Myomaker mediates fusion of fast myocytes in zebrafish embryos

Myomaker (also called Tmem8c), a new membrane activator of myocyte fusion was recently discovered in mice. Using whole mount in situ hybridization on zebrafish embryos at different stages of embryonic development, we show that myomaker is transiently expressed in fast myocytes forming the bulk of zebrafish myotome. Zebrafish embryos injected with morpholino targeted against myomaker were alive after yolk resorption and appeared morphologically normal, but they were unable to swim, even under effect of a tactile stimulation. Confocal observations showed a marked phenotype characterized by the persistence of mononucleated muscle cells in the fast myotome at developmental stages where these cells normally fuse to form multinucleated myotubes. This indicates that myomaker is essential for myocyte fusion in zebrafish. Thus, there is an evolutionary conservation of myomaker expression and function among Teleostomi.     

Ca disorder caused by rapid electrical field stimulation can be modulated by CaMKIIδ expression in primary rat atrial myocytes

Abnormalities in intracellular Ca²⁺ handing are believed to contribute to arrhythmogenesis during atrial fibrillation (AF). Ca²⁺/calmodulin-dependent protein kinaseII δ (CaMKIIδ) overexpression was detected in atrial myocytes from patients and animal models with persistent AF. In the present study, we found that rapid electrical field stimulation applied to primary atrial myocytes altered the CaMKIIδ activity, not expression level, resulting in Ca²⁺ disorder. By lentivirus mediated delivery of CaMKIIδ gene or siRNA into atrial myocytes, cells with different CaMKIIδ expression were generated. Changes of CaMKIIδ expression altered the sarcoplasmic reticulum (SR) Ca²⁺ release and L-type Ca²⁺ channels current (I_Ca) in both steady and electrical stimulating state. These results revealed the important role of CaMKIIδ in Ca²⁺ disorder caused by electrical field stimulation. It also provided a potential method to improve Ca²⁺ disorder in AF by modulating CaMKIIδ expression level.     

Inducible polymorphic ventricular tachyarrhythmias in a transgenic mouse model with a long Q-T phenotype

We created a mouse model with a prolonged Q-T interval and spontaneous arrhythmias by overexpressing the NH(2) terminus and first transmembrane segment (Kv1.1N206Tag) of a delayed rectifier potassium channel (LQT(+/-) mouse). Analyses were performed using whole cell recordings of cardiac myocytes, surface electrocardiography, and programmed electrical stimulation. Action potential duration (APD) was prolonged to the same extent and was more highly variable in myocytes derived from LQT(+/-) and LQT(+/+) mice than in myocytes derived from wild-type (WT) FVB mice. Under ketamine anesthesia, the Q-T interval of both LQT(+/+) and LQT(+/-) mice was comparably prolonged versus that of WT mice. Stimulation of the right ventricle using an intracardiac catheter induced polymorphic ventricular tachyarrhythmias in 50% of the LQT(+/-) mice and 36% of the LQT(+/+) mice, whereas polymorphic ventricular tachyarrhythmias were not inducible in WT mice. The analyses of LQT(+/-) and LQT(+/+) mice indicate that prolongation of the Q-T interval in LQT mice is associated with prolonged APD, increased dispersion of APD among cardiocytes, and inducibility of polymorphic ventricular tachycardia, providing the substrate for spontaneous arrhythmias in these animals.     

Temporal and spatial properties of cellular Ca2+ flux in trout ventricular myocytes

Confocal microscopy was used to investigate the temporal and spatial properties of Ca(2+) transients and Ca(2+) sparks in ventricular myocytes of the rainbow trout (Oncorhynchus mykiss). Confocal imaging confirmed the absence of T tubules and the long ( approximately 160 microm), thin ( approximately 8 microm) morphology of trout myocytes. Line scan imaging of Ca(2+) transients evoked by electrical stimulation in cells loaded with fluo 4 revealed spatial inhomogeneities in the temporal properties of Ca(2+) transients across the width of the myocytes. The Ca(2+) wavefront initiated faster, rose faster, and reached larger peak amplitudes in the periphery of the myocyte compared with the center. These differences were exacerbated by stimulation with the L-type Ca(2+) channel agonist (-)BAY K 8644 or by sarcoplasmic reticulum (SR) inhibition with ryanodine and thapsigargin. Results reveal that the shape of the trout myocyte allows for rapid diffusion of Ca(2+) from the cell periphery to the cell center, with SR Ca(2+) release contributing to the cytosolic Ca(2+) rise in a time-dependent manner. Spontaneous Ca(2+) sparks were exceedingly rare in trout myocytes under control conditions (1 sparking cell from 238 cells examined). This is in marked contrast to the rat where a total of 56 spontaneous Ca(2+) sparks were observed in 9 of 11 myocytes examined. Ca(2+) sparklike events were observed in a very small number of trout myocytes (15 sparks from 9 of 378 cells examined) after stimulation with either (-)BAY K 8644 or high Ca(2+) (6 mM). Reducing temperature to 15 degrees C in intact myocytes or permeabilizing myocytes to adjust intracellular conditions to favor Ca(2+) spark detection was without significant effects. Possible reasons for the rarity of Ca(2+) sparks in a cardiac myocyte with an active SR are discussed.     

Calcium handling in zebrafish ventricular myocytes

The zebrafish is an important model for the study of vertebrate cardiac development with a rich array of genetic mutations and biological reagents for functional interrogation. The similarity of the zebrafish (Danio rerio) cardiac action potential with that of humans further enhances the relevance of this model. In spite of this, little is known about excitation-contraction coupling in the zebrafish heart. To address this issue, adult zebrafish cardiomyocytes were isolated by enzymatic perfusion of the cannulated ventricle and were subjected to amphotericin-perforated patch-clamp technique, confocal calcium imaging, and/or measurements of cell shortening. Simultaneous recordings of the voltage dependence of the L-type calcium current (I(Ca,L)) amplitude and cell shortening showed a typical bell-shaped current-voltage (I-V) relationship for I(Ca,L) with a maximum at +10 mV, whereas calcium transients and cell shortening showed a monophasic increase with membrane depolarization that reached a plateau at membrane potentials above +20 mV. Values of I(Ca,L) were 53, 100, and 17% of maximum at -20, +10, and +40 mV, while the corresponding calcium transient amplitudes were 64, 92, and 98% and cell shortening values were 62, 95, and 96% of maximum, respectively, suggesting that I(Ca,L) is the major contributor to the activation of contraction at voltages below +10 mV, whereas the contribution of reverse-mode Na/Ca exchange becomes increasingly more important at membrane potentials above +10 mV. Comparison of the recovery of I(Ca,L) from acute and steady-state inactivation showed that reduction of I(Ca,L) upon elevation of the stimulation frequency is primarily due to calcium-dependent I(Ca,L) inactivation. In conclusion, we demonstrate that a large yield of healthy atrial and ventricular myocytes can be obtained by enzymatic perfusion of the cannulated zebrafish heart. Moreover, zebrafish ventricular myocytes differed from that of large mammals by having larger I(Ca,L) density and a monophasically increasing contraction-voltage relationship, suggesting that caution should be taken upon extrapolation of the functional impact of mutations on calcium handling and contraction in zebrafish cardiomyocytes.     

虚拟视觉刺激引起幼年斑马鱼捕食行为放弃的研究

目的 当动物重复某种行为以逃避危险或获取奖励而无法成功时，会产生放弃。放弃是一种常见且基本的行为，在小鼠等模式动物中已经被广泛研究，但是其部分神经机制仍未被阐明。幼年斑马鱼适合进行全脑光学成像，是神经科学领域的重要模式生物。已经有研究者通过持续电击等消极刺激诱发斑马鱼放弃行为，然而奖励刺激能否引起斑马鱼放弃尚无报道。本文对奖励刺激引起的斑马鱼放弃行为进行了探究。方法 通过给予斑马鱼虚拟的食物视觉刺激，检验斑马鱼对虚拟食物的捕食情况，比较斑马鱼捕食频率和单次捕食时长随时间的变化。结果 虚拟的食物视觉刺激可以引起斑马鱼的捕食行为，接受25 min虚拟刺激后，8日龄以上斑马鱼的捕食频率和单次捕食时长均出现显著下降。结论 此研究丰富了斑马鱼放弃行为的研究范式，实验结果表明，缺失真实奖励的虚拟食物刺激可以诱导斑马鱼放弃捕食行为，这将进一步加深对动物放弃行为的理解，推动对其神经机制的研究。     

Functional and electrophysiologic effects of polyunsaturated fatty acids on exictable tissues: heart and brain.

It has been shown in animals and probably in humans, that n-3 polyunsaturated fatty acids (PUFAs) are antiarrhythmic. The free PUFAs stabilize the electrical activity of isolated cardiac myocytes by inhibiting sarcolemmal ion channels, so that a stronger electrical stimulus is required to elicit an action potential and the relative refractory period is markedly prolonged. This appears at present to be the probable major antiarrhythmic mechanism of the PUFAs. They similarly inhibit the Na+ and Ca2+ currents in rat hippocampal neurons which results in an increase in the electrical threshold for generalized seizures using the cortical stimulation model in rats.     

Involvement of Ca2+ waves in excitation-contraction coupling of rat atrial cardiomyocytes

Two-dimensional and line-scan analyses of the early phase Ca2+ transients in rat cardiomyocytes were performed with a rapid-scanning laser confocal microscope and fluo-3 to elucidate the mechanism of activation of Ca2+ release from the sarcoplasmic reticulum in atrial myocytes which lack a well developed T-tubular network. On electrical stimulation of ventricular myocytes, Ca2+ concentration began to rise earliest at the Z-line level and became uniform throughout the cytoplasm within about 10 msec. In contrast, on stimulation of atrial myocytes, the earliest rise in Ca2+ occurred at the cell periphery and then spread to the cell interior; cytoplasmic Ca2+ became uniform after more than 30msec. The velocity of the propagation of rise in Ca2+ was 112 +/- 5.1 microm/sec (n = 10), which was similar to that of spontaneous Ca2+ waves observed in atrial and ventricular myocytes. No difference in frequency, amplitude and kinetics of spontaneous Ca2+ sparks was observed between the subsarcolemmal and central regions of atrial myocytes. Ryanodine concentration-dependently decreased the contractile force of isolated rat atrial and ventricular tissue preparations; the sensitivity was higher in atrial myocytes. The present study visualized the involvement of a propagated Ca2+-induced-Ca+ release mechanism in atrial but not ventricular myocytes. This difference may underlie some of the atrioventricular difference in response to physiological and pharmacological stimuli.     

Neuromuscular electrical stimulation for skeletal muscle function

Lack of neural innervation due to neurological damage renders muscle unable to produce force. Use of electrical stimulation is a medium in which investigators have tried to find a way to restore movement and the ability to perform activities of daily living. Different methods of applying electrical current to modify neuromuscular activity are electrical stimulation (ES), neuromuscular electrical stimulation (NMES), transcutaneous electrical nerve stimulation (TENS), and functional electrical stimulation (FES). This review covers the aspects of electrical stimulation used for rehabilitation and functional purposes. Discussed are the various parameters of electrical stimulation, including frequency, pulse width/duration, duty cycle, intensity/amplitude, ramp time, pulse pattern, program duration, program frequency, and muscle group activated, and how they affect fatigue in the stimulated muscle.     

Agrotis segetum雄蛾触角叶性信息素反应神经元对电刺激触角神经的反应

为探讨电刺激Agrotis segetum雄蛾触角神经是否可以作为MGC中神经元的识别手段,采用细胞内电生理记录方法,共记录34个对性信息素有反应的MGC神经元,并测试了其中12个神经元对性信息素刺激的反应,22个神经元对性信息素刺激和电刺激的反应。结果表明,MGC神经元对性信息素及电刺激的反应模式基本一致,为一种双相反应模式。两种刺激方式均能诱导出兴奋反应,电刺激得到的兴奋反应比由信息素刺激引起的要短;MGC神经元对两种刺激的超极化反应（抑制反应）幅度影响没有显著性差别,在电刺激实验的22个神经元上,超极化反应幅度和抑制时间都与神经元本身放电频率有一定的相关性。超极化反应是在LN参与下一定的神经回路对刺激所产生的反应而形成的。这提示两种刺激所作用的神经回路应是一致的,但从整个实验过程记录到的神经元情况来看,还须进一步结合形态学实验来验证电刺激触角神经作为MGC神经元的识别手段。     

Mathematical models for fatigue minimization during functional electrical stimulation

We previously reported the development of a force- and fatigue-model system that predicted accurately forces during repetitive fatiguing activation of human skeletal muscles using brief duration (six-pulse) stimulation trains. The model system was tested in the present study using force responses produced by longer duration stimulation trains, containing up to 50 pulses. Our results showed that our model successfully predicted the peak forces produced when the muscle was repetitively activated with stimulation trains of frequencies ranging from 20 to 40 Hz, train durations ranging from 0.5 to 1 s, and varied pulse patterns. The predicted peak forces throughout each protocol matched the experimental peak forces with r² values above 0.9 and predicted successfully the forces at the end of each protocol with <15% error for all protocols tested. The success of our model system further supports its potential use for the design of optimal stimulation patterns for individual users during functional electrical stimulation.     

Analytical modeling of the hysteresis phenomenon in guinea pig ventricular myocytes

In the present study, we have demonstrated hysteresis phenomena in the excitability of single, enzymatically dissociated guinea pig ventricular myocytes. Membrane potentials were recorded with patch pipettes in the whole-cell current clamp configuration. Repetitive stimulation with depolarizing current pulses of constant cycle length and duration but varying strength led to predictable excitation (1:l) and non-excitation (1:0) patterns depending on current strength. In addition, transition between patterns depended on the direction of current intensity change and stable hysteresis loops were obtained in stimulus:response pattern vs. current intensity plots in 14 cells. Increase of pulse duration and decrease of stimulation rate contributed to a reduction in hysteresis loop areas. Changes in amplitude and shape of the subthreshold responses during the transitions from one stable pattern to the other, suggested that activity led to an increase in membrane resistance, particularly in the voltage domain between resting potential, and threshold. Therefore, we modelled the dynamic behaviour of the single cells as a function of diastolic membrane resistance, using previously published analytical solutions. Numerical iteration of the analytical model equations closely reproduced the experimental hysteresis loops in both qualitative and quantitative ways. In particular, the effect of stimulation frequency on the model was similar to the experimental findings. The overall study suggests that the excitability pattern of guinea pig ventricular myocytes accounts for hysteresis and bistabilities when current intensity is allowed to fluctuate around threshold levels.