A model of calcium dynamics in cardiac myocytes based on the kinetics of ryanodine-sensitive calcium channels.

The ryanodine-sensitive calcium channels are pivotal to signal transduction and cell function in many cell types, including cardiac myocytes. In this paper a kinetic model is proposed for these channels. In the model there are two Ca regulatory sites on the channel protein, one positive and the other negative. Cytoplasmic Ca binds to these regulatory sites independently It is assumed that the binding of Ca to the positive site is a much faster process than binding to the negative site. At steady state, the channel opening as a function of the Ca concentration is a bell-shaped curve. The model predicts the adaptation of channels to constant Ca stimulus. When this model is applied to cardiac myocytes, it predicts excitability with respect to Ca perturbations, smoothly graded responses, and Ca oscillations in certain pathological circumstances. In a spatially distributed system, traveling Ca waves in individual myocytes exist under certain conditions. This model can also be applied to other systems where the ryanodine-sensitive channels have been identified.     

心肌细胞的钙致钙释放

心肌细胞兴奋－收缩偶联由胞内钙变中介和调控。去极化进进入细胞的少量钙通过钙下释放（ＣＩＣＲ）过程发肌质多（ＳＲ）释放更多的钙,使胞浆钙浓度升高,导致收缩近年来证明,ＳＲ钙放呈梯级特征,提出了局部控制模型,以解释这种现象。钙火花的发现,直观地证硒钙释放单位的存在,进一步支持了局部控制模型。此外,钙释放通道的适应现象,可能是ＣＩＣＲ这一正反馈过程的负调节机制。     

Autonomic regulation of calcium and potassium channels is oppositely modulated by microtubules in cardiac myocytes

We recently showed that colchicine treatment of rat ventricular myocytes increases the L-type Ca2+ current (I(Ca)) and intracellular Ca2+ concentration ([Ca2+](i)) transients and interferes with adrenergic signaling. These actions were ascribed to adenylyl cyclase (AC) stimulation after G(s) activation by alpha,beta-tubulin. Colchicine depolymerizes microtubules into alpha,beta-tubulin dimers. This study analyzed muscarinic signals in myocytes with intact or depolymerized microtubules. Myocytes were loaded with the Ca2+ indicator fluo 3 and were field stimulated at 1 Hz or voltage clamped. In untreated cells, carbachol (CCh; 1 microM) induced ACh-activated K(+) current [I(K(ACh))], which happens via betagamma-subunits from the activation of G(i). Carbachol also reduced [Ca2+](i) transients and contractions. Once G(i) is activated by muscarinic agonist, the alpha(i)-subunit is released from the betagamma-subunits, but it is silent, and its inhibition of the AC/cAMP cascade, manifested by I(Ca) reduction, is not seen unless AC has been previously activated. In colchicine-treated cells, CCh caused greater reductions of [Ca2+](i) transients and contractions than in untreated cells. The alpha(i)-subunit became effective in signaling through the AC/cAMP cascade and reduced I(Ca) without changing its voltage-dependence. Isoproterenol (Iso) regained its efficacy and reversed I(Ca) inhibition by CCh. Stimulation of I(Ca) by forskolin persisted in colchicine-treated cells when Iso was ineffective. The effect of CCh on I(K(ACh)) was occluded in colchicine-treated cells. Colchicine treatment, per se, may increase I(K(ACh)) by betagamma-subunits released from G(s) to mask this effect of CCh. Microtubules suppress I(Ca) regulation by alpha(i); their disruption releases restraints that unmask muscarinic inhibition of I(Ca). Summarily, colchicine treatment reverses regulation of ventricular excitation-contraction coupling by autonomic agents.     

Inactivation of calcium channels in vascular smooth muscle myocytes

Many of the structural domains involved in Ca²⁺ channel (CACN) inactivation are also involved in determining their sensitivity to antagonist inhibition. We hypothesize that differences in inactivation properties and their structural determinants may suggest candidate domains as targets for the development of novel, selective antagonists. The characteristics of Ca²⁺ current (I_Ca) inactivation, steady-state inactivation (SSIN), and recovery from inactivation were studied in freshly dispersed smooth muscle cells from rabbit portal vein (RPV) using whole-cell, voltage-clamp methods. The time course of inactivation could be represented by two time constants. Increasing I_Ca by increasing [Ca²⁺]_o or with more negative holding potentials decreased both time constants. With Sr²⁺, Ba²⁺, or Na⁺ as the charge carrier, I_Ca inactivation was also represented by two time constants, both of which were larger than those found with Ca²⁺. With Ca²⁺, Sr²⁺, or Ba²⁺ as the charge carrier, both time constants had minimum values near the voltage associated with maximum current. When Na⁺ (140 mM) was the charge carrier, voltages for I_max (−20 mV) or τ_min (o mV) did not correspond. SSIN of I_Ca had a half-maximum voltage of −32±4 mV for Ca²⁺, −43 mV±5 mV for Sr²⁺, −41±5 mV for Ba²⁺, and −68±6 mV for Na⁺. The slope factor for SSIN per e-fold voltage change was 6.5±0.2 mV for Ca²⁺, 6.8±0.3 for Sr²⁺, and 6.6±0.2 for Ba²⁺, representing four equivalent charges. When Na⁺ or Li⁺ was the charge carrier, the slope factor was 13.5±0.7 mV, representing two equivalent charges. For I_Ca in rat left ventricular (rLV) myocytes, there was no difference in the slope factor of SSIN for Ca²⁺ and Na⁺. The rate of recovery of I_Ca from inactivation varied inversely with recovery voltage and was independent of the charge carrier. These results suggest that inactivation of I_Ca in PV myocytes possess an intrinsic voltage dependence that is modified by Ca²⁺. For RPV but not rLV I_Ca, the charge of the permeating ion confers the voltage-dependency of SSIN.     

Criticality in intracellular calcium signaling in cardiac myocytes

Calcium (Ca) is a ubiquitous second messenger that regulates many biological functions. The elementary events of local Ca signaling are Ca sparks, which occur randomly in time and space, and integrate to produce global signaling events such as intra- and intercellular Ca waves and whole-cell Ca oscillations. Despite extensive experimental characterization in many systems, the transition from local random to global synchronous events is still poorly understood. Here we show that criticality, a ubiquitous dynamical phenomenon in nature, is responsible for the transition from local to global Ca signaling. We demonstrate this first in a computational model of Ca signaling in a cardiac myocyte and then experimentally in mouse ventricular myocytes, complemented by a theoretical agent-based model to delineate the underlying dynamics. We show that the interaction between the Ca release units via Ca-induced Ca release causes self-organization of Ca spark clusters. When the coupling between Ca release units is weak, the cluster-size distribution is exponential. As the interactions become strong, the cluster-size distribution changes to a power-law distribution, which is characteristic of criticality in thermodynamic and complex nonlinear systems, and facilitates the formation and propagation of Ca waves and whole-cell Ca oscillations. Our findings illustrate how criticality is harnessed by a biological cell to regulate Ca signaling via self-organization of random subcellular events into cellular-scale oscillations, and provide a general theoretical framework for the transition from local Ca signaling to global Ca signaling in biological cells.     

Effects of antisense oligonucleotides to the cardiac Na+/Ca2+ exchanger on calcium dynamics in cultured cardiac myocytes.

The present study was designed to explore the role of the Na+/Ca2+ exchanger on spontaneous beating of cultured cardiac myocytes. Antisense oligonucleotides (AS) based on the sequence of the cardiac Na+/Ca2+ exchanger were used to decrease expression of this Ca2+ transporting protein in cardiac myocytes. An application of AS (10 microM) caused an increase in beating rate of myocytes within 6-24 h. After 24 h of exposure, AS increased the beating rate from an average rate of 77 beats/min in control and sense-treated myocytes to 103 beats/min. Moreover, myocytes treated for 24 h with 10 microM AS exhibited an increase in diastolic [Ca2+]i levels. The antisense treatment also led to a approximately 20% decrease in expression of Na+/Ca2+ exchanger proteins within 6-24 h. Changes in mRNA levels following AS treatment could not be detected within 3- to 24-h periods. The results of these studies suggest that the Na+/Ca2+ exchanger plays a potentiating role in spontaneous the beating process by regulating [Ca2+]i dynamics and that even a small reduction in the levels of the exchanger protein has marked effects on the handling of [Ca2+]i during the cardiac cycle.     

In silico simulations reveal that RYR distribution affects the dynamics of calcium release in cardiac myocytes

The dyads of cardiac myocytes contain ryanodine receptors (RYRs) that generate calcium sparks upon activation. To test how geometric factors of RYR distribution contribute to the formation of calcium sparks, which cannot be addressed experimentally, we performed in silico simulations on a large set of models of calcium release sites (CRSs). Our models covered the observed range of RYR number, density, and spatial arrangement. The calcium release function of CRSs was modeled by RYR openings, with an open probability dependent on concentrations of free Ca²⁺ and Mg²⁺ ions, in a rapidly buffered system, with a constant open RYR calcium current. We found that simulations of spontaneous sparks by repeatedly opening one of the RYRs in a CRS produced three different types of calcium release events (CREs) in any of the models. Transformation of simulated CREs into fluorescence signals yielded calcium sparks with characteristics close to the observed ones. CRE occurrence varied broadly with the spatial distribution of RYRs in the CRS but did not consistently correlate with RYR number, surface density, or calcium current. However, it correlated with RYR coupling strength, defined as the weighted product of RYR vicinity and calcium current, so that CRE characteristics of all models followed the same state-response function. This finding revealed the synergy between structure and function of CRSs in shaping dyad function. Lastly, rearrangements of RYRs simulating hypothetical experiments on splitting and compaction of a dyad revealed an increased propensity to generate spontaneous sparks and an overall increase in calcium release in smaller and more compact dyads, thus underlying the importance and physiological role of RYR arrangement in cardiac myocytes.     

Effect of platelet release products on cytosolic calcium in cardiac myocytes

The effect of platelet release products on cytosolic calcium [( Ca++]i) was examined by monitoring the fluorescence of chick embryonic heart cells loaded with the fluorescent calcium indicator indo-1 AM. Cell free filtrate of platelet release products was obtained from rabbit platelets activated with thrombin or collagen. This filtrate caused a rapid increase in both systolic and diastolic [Ca++]i in a dose-dependent manner. The effect was not blocked by pretreating the platelets with aspirin or a thromboxane synthetase inhibitor. It was not mimicked by a thromboxane analog, or by several substances known to be released from platelets including ADP, serotonin, or platelet activating factor. Apyrase or ATP-gamma S had no effect on the activity. The responsible product was heat-sensitive, trypsin-sensitive, and partitioned into the aqueous phase of a chloroform suspension. It has a low molecular weight (less than 3kD) and is sensitive to 2-mercaptoethanol. Protease inhibitor appears to prolong the activity. These results suggest that trypsin-sensitive peptide(s) released from activated platelets can increase [Ca++]i in cardiac cells.     

应激对大鼠心室肌细胞L-型钙离子通道的影响

目的 :研究应激对心室肌细胞L 型钙离子通道电流及激活、失活门控动力学特性的影响。方法 :制备离体心室肌细胞并用去甲肾上腺素 (NE)诱导建立应激心肌细胞模型 ,应用流式细胞术 (FCM )和Fura 2荧光分光光度法测定应激心室肌细胞的凋亡率和心肌细胞内游离钙浓度变化。利用膜片钳全细胞钳制记录法记录应激心室肌细胞L 型钙离子通道电流I V曲线图及稳态激活、失活曲线图。结果 :FCM检测发现 1 0 - 4mol/L的NE模拟应激可导致心室肌细胞凋亡率明显上升 ,对照组 :0 .36 % ,应激组 :2 .1 7% (P <0 .0 1 )。 1 0 - 4mol/L的NE干预使Ica L峰电流幅值显著性上升 ,激活曲线分析发现应激后曲线明显左移 ,半数最大激活膜电位 (V1 /2 )为 (- 1 4 .59± 0 .2 4 )mV ,较之于对照组 :(- 0 .69± 0 .36)mV ,差异显著 ,而稳态失活曲线特性未发生有意义的改变。实验组心肌细胞内游离钙浓度较对照组升高 1 6 .7%。结论 :应激可导致L 型钙离子通道电流增加 ,通道更易于激活 ,通道的这种异常活动可能导致钙超载 ,从而引起心肌细胞凋亡 ,介导应激性心肌损伤的发生     

A mathematical model of spontaneous calcium release in cardiac myocytes

In cardiac myocytes, calcium (Ca) can be released from the sarcoplasmic reticulum independently of Ca influx from voltage-dependent membrane channels. This efflux of Ca, referred to as spontaneous Ca release (SCR), is due to Ryanodine receptor fluctuations, which can induce spontaneous Ca sparks, which propagate to form Ca waves. This release of Ca can then induce delayed after-depolarizations (DADs), which can lead to arrhythmogenic-triggered activity in the heart. However, despite its importance, to date there is no mathematical model of SCR that accounts for experimentally observed features of subcellular Ca. In this article, we present an experimentally based model of SCR that reproduces the timing distribution of spontaneous Ca sparks and key features of the propagation of Ca waves emanating from these spontaneous sparks. We have coupled this model to an ionic model for the rabbit ventricular action potential to simulate SCR within several thousand cells in cardiac tissue. We implement this model to study the formation of an ectopic beat on a cable of cells that exhibit SCR-induced DADs.     

心肌细胞发育过程中胞浆内钙稳态的调控

Ca^2＋信号是细胞和各器官生长发育、行使其生理功能的基础,维持心肌细胞的钙稳态是保持正常心脏功能的先决条件。作为在胚胎发育过程中最早出现并行使功能的器官,胚胎期心脏的形态结构发生了明显的变化,泵血功能不断增强,以适应不断增强的机体的生理需求。从胚胎到成年,心肌细胞的功能有非常大的改变,各钙离子通道的表达也发生明显变化。因此,发育早期心肌细胞的钙稳态调控与成熟心肌细胞有明显的不同,在发育过程中引起细胞收缩的Ca^2＋来源也有明显的变化。随着分子和细胞生物学研究的发展,以及胚胎干细胞体外分化模型的应用,人们对心肌细胞发育过程中钙稳态的调控有了进一步的认识。本文综述了早期心肌细胞发育过程中胞浆内钙稳态的变化,总结了早期心肌细胞钙稳态调控机制的最新研究进展。     

牛磺酸对大鼠心肌细胞内钙浓度的影响

牛磺酸 (Ｔａｕｒｉｎｅ ,Ｔａｕ)是可兴奋组织中含量最为丰富的游离氨基酸 ,是细胞自稳态的重要调节物质。在多种心血管疾病的临床与实验研究中具有明显的细胞保护作用。其作用机制与调节心肌细胞的钙浓度有关。用同位素示踪技术已证实Ｔａｕ在细胞内“高钙”状态下能抑制钙的跨膜内流。本文采用Ｆｕｒａ 2荧光技术测定Ｔａｕ对成年大鼠分离心肌细胞在静息、高钾去极化以及缺氧 /复氧条件下游离 [Ｃａ ]ｉ,旨在进一步探讨Ｔａｕ的作用机制。1　材料与方法(1)动物实验　雄性Ｗｉｓｔａｒ大鼠 (军事医学科学院四所提供 )。 2 0 %乌拉坦ｉｐ…     

FRET detection of calmodulin binding to the cardiac RyR2 calcium release channel

Calmodulin (CaM) binding to the type 2 ryanodine receptor (RyR2) regulates Ca release from the cardiac sarcoplasmic reticulum (SR). However, the structural basis of CaM regulation of the RyR2 is poorly defined, and the presence of other potential CaM binding partners in cardiac myocytes complicates resolution of CaM's regulatory interactions with RyR2. Here, we show that a fluorescence-resonance-energy-transfer (FRET)-based approach can effectively resolve RyR2 CaM binding, both in isolated SR membrane vesicles and in permeabilized ventricular myocytes. A small FRET donor was targeted to the RyR2 cytoplasmic assembly via fluorescent labeling of the FKBP12.6 subunit. Acceptor fluorophore was attached at discrete positions within either the N- or the C-lobe of CaM. FRET between FKBP12.6 and CaM bound to SR vesicles indicated CaM binding at a single high-affinity site within 60 Å of FKBP12.6. Micromolar Ca increased the apparent affinity of CaM binding and slowed CaM dissociation, but did not significantly affect maximal FRET efficiency at saturating CaM. FRET was strongest when the acceptor was attached at either of two positions within CaM's N-lobe versus sites in CaM's C-lobe, providing CaM orientation information. In permeabilized ventricular myocytes, FKBP12.6 and CaM colocalized to Z-lines, and the efficiency of energy transfer to both the N- and C-lobes of CaM was comparable to that observed in SR vesicle experiments. Results also indicate that both the location and orientation of CaM binding on the RyR2 are very similar to the skeletal muscle RyR1 isoform. Specific binding of CaM to functional RyR2 channels in the cardiac myocyte environment can be monitored using FKBP biosensors and FRET.     

Activation of calcium release assessed by calcium release-induced inactivation of calcium current in rat cardiac myocytes

In mammalian cardiac myocytes, calcium released into the dyadic space rapidly inactivates calcium current (I_Ca). We used this Ca²⁺ release-dependent inactivation (RDI) of I_Ca as a local probe of sarcoplasmic reticulum Ca²⁺ release activation. In whole cell patch-clamped rat ventricular myocytes, Ca²⁺ entry induced by short prepulses from —50 mV to positive voltages caused suppression of peak I_Ca during a test pulse. The negative correlation between peak I_Ca suppression and I_Ca inactivation during the test pulse indicated that RDI evoked by the prepulse affected only calcium channels in those dyads in which calcium release was activated. Ca²⁺ ions injected during the prepulse and during the subsequent tail current suppressed peak I_Ca in the test pulse to a different extent. Quantitative analysis indicated that equal Ca²⁺ charge was 3.5 times less effective in inducing release when entering during the prepulse than when entering during the tail. Tail Ca²⁺ charge injected by the first voltage-dependent calcium channel (DHPR) openings was three times less effective than that injected by DHPR reopenings. These findings suggest that calcium release activation can be profoundly influenced by the recent history of L-type Ca²⁺ channel activity due to potentiation of ryanodine receptors (RyRs) by previous calcium influx. This conclusion was confirmed at the level of single RyRs in planar lipid bilayers: using flash photolysis of the calcium cage NP-EGTA to generate two sequential calcium stimuli, we showed that RyR activation in response to the second stimulus was four times higher than that in response to the first stimulus. excitation-contraction coupling     

Effects of magnesium on inactivation of the voltage-gated calcium current in cardiac myocytes

The effects of changes in intracellular and extracellular free ionized [Mg2+] on inactivation of ICa and IBa in isolated ventricular myocytes of the frog were investigated using the whole-cell configuration of the patch-clamp technique. Intracellular [Mg2+] was varied by internal perfusion with solutions having different calculated free [Mg2+]. Increasing [Mg2+]i from 0.3 mM to 3.0 mM caused a 16% reduction in peak ICa amplitude and a 36% reduction in peak IBa amplitude, shifted the current-voltage relationship and the inactivation curve approximately 10 mV to the left, decreased relief from inactivation, and caused a dramatic increase in the rate of inactivation of IBa. The shifts in the current-voltage and inactivation curves were attributed to screening of internal surface charge by Mg2+. The increased rate of inactivation of IBa was due to an increase in both the steady-state level of inactivation as well as an increase in the rate of inactivation, as measured by two-pulse inactivation protocols. Increasing external [Mg2+] decreased IBa amplitude and shifted the current-voltage and inactivation curves to the right, but, in contrast to the effect of internal Mg2+, had little effect on the inactivation kinetics or the steady-state inactivation of IBa at potentials positive to 0 mV. These observations suggest that the Ca channel can be blocked quite rapidly by external Mg2+, whereas the block by [Mg2+]i is time and voltage dependent. We propose that inactivation of Ca channels can occur by both calcium-dependent and purely voltage-dependent mechanisms, and that a component of voltage-dependent inactivation can be modulated by changes in cytoplasmic Mg2+.