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。     

兴奋-收缩偶联中DHPR与RyR的相互作用

兴奋-收缩偶联（E—C coupling）依赖纽胞膜二氢吡啶受体（DHPR）／L型电压门控Ca^2＋通道和肌浆网兰诺定受体（RyR）／Ca^2＋释放通道的相互作用。在骨骼肌细胞中,DHPR与RyRl在结构上二机械偶联,不依赖细胞外Ca^2＋即可激活RyRl;在心肌细胞中,去极化激活DHPR,细胞外Ca^2＋内流,内流的Ca^2＋通过钙诱导钙释放（CICR）机制激活RyR2。最近的研究表明,DHPR与RyR之间的信号转导通常是双向的。DHPR与RyR机械和化学的双向偶联机制调节这两种Ca^2＋通道的效率、精确度和活性。     

β-肾上腺素能受体调控心肌细胞钙释放的分子机制

β-肾上腺素能受体（βAR）是最经典的G蛋白耦联受体。在心室肌细胞中,βAR可以提高细胞膜L-型钙通道（LCC）介导的钙内流的幅度和同步性,通过钙致钙释放机制触发肌质网（SR）更强的钙释放活动,从而起到调节心脏收缩能力的作用。然而,目前仍不清楚β-蛋白激酶A（PKA）信号通路如何直接调控肌质网钙释放通道ryanodine受体（RyR）的功能,该领域的研究结果存在很大争议。本文使用特殊的单通道钙成像技术,通过去极化方法激活单个LCC产生钙小星,记录触发的RyR钙火花。结果表明,在βAR激动剂异丙肾上腺素（1μM）作用20分钟内,单个LCC触发的钙火花幅度显著上升,且该效应不依赖于LCC单通道钙电流的变化;βAR激动下钙火花的钙释放电流幅度与肌质网钙储量的比值显著提高,表明βAR信号动员了更多的RyR通道参与同步钙释放活动;βAR激动下钙小星触发钙火花的耦联潜伏期时间缩短,成功率上升,表明βAR信号通路增强了LCC—RyR的分子间耦联效率。上述效应不依赖BAR引起的在肌质网钙储量上升,且能够被PKA抑制剂Rp-β-CPT-cAMP（100μM）和H89（10μM）消除。上述结果证明,βAR—PKA信号能够提高RvR对LCC单通道电流的响应速率和同步性。由此揭示的交感神经调节心脏功能的分子机制,将为进一步研究心脏疾病下βhR信号的异常变化奠定基础。     

钙火花研究进展与瞻望

钙离子是最广泛而又最重要的细胞内第二信使。自1993年以来,钙火花等一系列钙信号基本单元相继发现,揭示了细胞钙信号转导的数字-模拟二元特征：纳米-微米尺度上短暂的钙信号事件（数字系统）随机叠加于连续的全细胞钙信号（模拟系统）背景中。数字模式的微区域钙信号赋予细胞钙信号在时间、空间、幅度上多尺度多层次的精细结构。对钙火花激活机制、协同机制、终止机制等方面的研究,为钙释放通道阵列的门控及调节提出了新的见解和问题。钙火花等对于高域值钙依赖性过程（如肌细胞兴奋-收缩耦联、细胞兴奋性和神经细胞分泌）的激活和时空调控具有特别重要的生理和病理意义。钙信号“激-模二元性”的研究可望进一步揭示细胞钙信号的简单性与复杂性的统一。     

Ca2+对骨骼肌钙释放通道的调节

钙释放通道（calcium release channel）又称Ryanodine受体（RyR）,是细胞内质网膜上介导细胞内钙信号转导的离子通道。RyR1在骨骼肌细胞的兴奋-收缩偶联过程中起重要作用,是肌质网快速释放Ca^2＋的通道。许多调节因素,如一些内源性蛋白（FK结合蛋白、钙调素、钙结合蛋白）和一些离子（Ca^2＋、Mg^2＋）,通过不同的作用位点与RyR1结合,调控RyR1的结构与功能。研究表明,Ca^2＋是众多调节RyR1因素中的核心成分和前提条件,其对RyR1的结构与功能有重要的调控作用。     

心肌细胞膜与肌质网膜的结构功能耦联及其病理重塑机制

心脏的收缩功能依赖心肌细胞膜（包括横管）与肌质网的结构耦联以及其中L型钙通道与肌质网钙释放通道之间的钙致钙释放过程。在一些病理条件下,细胞膜与肌质网的耦联结构发生重塑,钙致钙释放机制受损,心肌细胞收缩力下降。其中,junctophilin-2等蛋白分子表达量减少是心力衰竭疾病中心肌细胞收缩能力下降的关键因素。     

内皮素-1通过L-型钙通道的Ca~(2+)内流和钙致钙释放诱导心肌细胞内[Ca~(2+)]的增加

本研究利用fura-2-AM荧光成像和膜片钳技术,发现内皮素-1(Endothelin-1,ET-1)可显著提高大鼠分离心肌细胞内钙离子水平([Ca2+]i),激活心肌细胞钙通道.ETA受体阻滞剂BQ123能够消除ET-1提高[Ca2+]i的效应,而ETB受体阻滞剂BQ788对该效应无影响.用ryanodine受体阻断剂ryanodine(10 μmol/L)预处理,可以使ET-1诱导的[Ca2+]i的增加抑制46.7%.蛋白激酶A(PKA)的抑制剂、蛋白激酶C(PKC)的抑制剂和血管紧张素Ⅱ一型受体(AT1 receptor)的抑制剂都能够抑制ET-1诱导的[Ca2+]i的增加.本研究发现ET-1能够提高全细胞L-型钙通道电流的幅度,增加L-型钙通道单通道的开放概率.并且BQ123完全阻止了ET-1诱导的L-型钙通道开放概率增加的效应.本研究证明了ET-1通过一系列机制调节钙超载,包括L-型钙通道的激活,钙致钙释放(CICR),ETA受体,PKC,PKA和血管紧张素Ⅱ一型受体也参与到了这个途径中.     

心肌细胞横管-肌质网耦联与钙信号调控

心肌细胞的兴奋沿横管传入细胞深处并激活L型钙通道,进而通过钙致钙释放机制激活肌质网ryanodine受体钙释放通道,由此产生的钙火花叠加成为细胞钙瞬变,引发心肌细胞同步化收缩.β1型肾上腺素受体介导的广域cAMP信号通过磷酸化L型钙通道、ryanodine受体、肌质网受磷蛋白,分别上调钙内流、钙释放和肌质网钙泵的钙回收...     

钙火花

“钙火花”是细胞钙信号转导的一个基本单位,由程和平、Cannell和Lederer于1993年在心肌细胞中首次发现并命名。当内质网“钙释放单位”（含50-300个簇状排列的钙释放通道分子）激活时,胞浆自由钙离子浓度局部瞬态增高,即产生钙火花。     

氨甲酰胆碱增加大鼠心室肌细胞收缩机制的初步研究

目的:本文研究非选择性M受体激动剂氨甲酰胆碱(Cch)对心肌细胞收缩的作用,并同时观察L-型钙流,探讨其机制.方法:以分离大鼠的单个心室肌细胞为对象,采用膜片钳和单细胞收缩测量技术,在35℃恒温,细胞外钙离子浓度1.8 mmol/L,0.2 Hz,1.0 Hz刺激下测量细胞收缩幅度和ICa(L)、INa/Ca.结果:①大鼠心肌细胞收缩与刺激频率呈负相关;②选择△L0.2 Hz/△L1.0 Hz≥1.25(n=6)的细胞给予1.0 Hz的刺激,100 μmol/L Cch增加大鼠心室肌细胞收缩28%.③ Cch作用能被非选择性M受体阻滞剂阿托品阻滞,选择性M1受体激动剂MCN-A-343 100 μmol/L对细胞收缩无影响.④ 100 μmol/L Cch对ICa(L)无影响,但增加从 10 mV复极到-40 mV所产生的晚期尾电流,提示INa/Ca加大.结论:Cch能加强大鼠心室肌细胞收缩,它的作用由M2受体介导,其机制可能是通过加强INa/Ca,增加肌质网(SR)内的钙内容和钙释放,导致细胞收缩加强,而非通过L-型钙流.     

Life and death of a cardiac calcium spark

Calcium sparks in cardiac myocytes are brief, localized calcium releases from the sarcoplasmic reticulum (SR) believed to be caused by locally regenerative calcium-induced calcium release (CICR) via couplons, clusters of ryanodine receptors (RyRs). How such regeneration is terminated is uncertain. We performed numerical simulations of an idealized stochastic model of spark production, assuming a RyR gating scheme with only two states (open and closed). Local depletion of calcium in the SR was inevitable during a spark, and this could terminate sparks by interrupting CICR, with or without assumed modulation of RyR gating by SR lumenal calcium. Spark termination by local SR depletion was not robust: under some conditions, sparks could be greatly and variably prolonged, terminating by stochastic attrition–a phenomenon we dub “spark metastability.” Spark fluorescence rise time was not a good surrogate for the duration of calcium release. Using a highly simplified, deterministic model of the dynamics of a couplon, we show that spark metastability depends on the kinetic relationship of RyR gating and junctional SR refilling rates. The conditions for spark metastability resemble those produced by known mutations of RyR2 and CASQ2 that cause life-threatening triggered arrhythmias, and spark metastability may be mitigated by altering the kinetics of the RyR in a manner similar to the effects of drugs known to prevent those arrhythmias. The model was unable to explain the distributions of spark amplitudes and rise times seen in chemically skinned cat atrial myocytes, suggesting that such sparks may be more complex events involving heterogeneity of couplons or local propagation among sub-clusters of RyRs.     

Shape, size, and distribution of Ca(2+) release units and couplons in skeletal and cardiac muscles.

Excitation contraction (e-c) coupling in skeletal and cardiac muscles involves an interaction between specialized junctional domains of the sarcoplasmic reticulum (SR) and of exterior membranes (either surface membrane or transverse (T) tubules). This interaction occurs at special structures named calcium release units (CRUs). CRUs contain two proteins essential to e-c coupling: dihydropyridine receptors (DHPRs), L-type Ca(2+) channels of exterior membranes; and ryanodine receptors (RyRs), the Ca(2+) release channels of the SR. Special CRUs in cardiac muscle are constituted by SR domains bearing RyRs that are not associated with exterior membranes (the corbular and extended junctional SR or EjSR). Functional groupings of RyRs and DHPRs within calcium release units have been named couplons, and the term is also loosely applied to the EjSR of cardiac muscle. Knowledge of the structure, geometry, and disposition of couplons is essential to understand the mechanism of Ca(2+) release during muscle activation. This paper presents a compilation of quantitative data on couplons in a variety of skeletal and cardiac muscles, which is useful in modeling calcium release events, both macroscopic and microscopic ("sparks").     

Putting out the fire: what terminates calcium-induced calcium release in cardiac muscle?

The majority of contractile calcium in cardiac muscle is released from stores in the sarcoplasmic reticulum (SR), by a process of calcium-induced calcium release (CICR) through ryanodine receptors. Because CICR is intrinsically self-reinforcing, the stability of and graded regulation of cardiac EC coupling appear paradoxical. It is now well established that this gradation results from the stochastic recruitment of varying numbers of elementary local release events, which may themselves be regenerative, and which can be directly observed as calcium sparks. Ryanodine receptors (RyRs) are clustered in dense lattices, and most calcium sparks are now believed to involve activation of multiple RyRs. This implies that local CICR is regenerative, requiring a mechanism to terminate it. It was initially assumed that this mechanism was inactivation of the RyR, but during the decade since the discovery of sparks, no sufficiently strong inactivation mechanism has been demonstrated in vitro and all empirically determined gating schemes for the RyR give unstable EC coupling in Monte Carlo simulations. We consider here possible release termination mechanisms. Stochastic attrition is the spontaneous decay of active clusters due to random channel closure; calculations show that it is much too slow unless assisted by another process. Calcium-dependent RyR inactivation involving third-party proteins remains a viable but speculative mechanism; current candidates include calmodulin and sorcin. Local depletion of SR release terminal calcium could terminate release, however calculations and measurements leave it uncertain whether a sufficient diffusion resistance exists within the SR to sustain such depletion. Depletion could be assisted by dependence of RyR activity on SR lumenal [Ca(2+)]. There is substantial evidence for such lumenal activation, but it is not clear if it is a strong enough effect to account for the robust termination of sparks. The existence of direct interactions among clustered RyRs might account for the discrepancy between the inactivation properties of isolated RyRs and intact clusters. Such coupled gating remains controversial. Determining the mechanism of release termination is the outstanding unsolved problem of cardiac EC coupling, and will probably require extensive genetic manipulation of the EC coupling apparatus in its native environment to unravel the solution.     

An integrative model of the cardiac ventricular myocyte incorporating local control of Ca2+ release

The local control theory of excitation-contraction (EC) coupling in cardiac muscle asserts that L-type Ca(2+) current tightly controls Ca(2+) release from the sarcoplasmic reticulum (SR) via local interaction of closely apposed L-type Ca(2+) channels (LCCs) and ryanodine receptors (RyRs). These local interactions give rise to smoothly graded Ca(2+)-induced Ca(2+) release (CICR), which exhibits high gain. In this study we present a biophysically detailed model of the normal canine ventricular myocyte that conforms to local control theory. The model formulation incorporates details of microscopic EC coupling properties in the form of Ca(2+) release units (CaRUs) in which individual sarcolemmal LCCs interact in a stochastic manner with nearby RyRs in localized regions where junctional SR membrane and transverse-tubular membrane are in close proximity. The CaRUs are embedded within and interact with the global systems of the myocyte describing ionic and membrane pump/exchanger currents, SR Ca(2+) uptake, and time-varying cytosolic ion concentrations to form a model of the cardiac action potential (AP). The model can reproduce both the detailed properties of EC coupling, such as variable gain and graded SR Ca(2+) release, and whole-cell phenomena, such as modulation of AP duration by SR Ca(2+) release. Simulations indicate that the local control paradigm predicts stable APs when the L-type Ca(2+) current is adjusted in accord with the balance between voltage- and Ca(2+)-dependent inactivation processes as measured experimentally, a scenario where common pool models become unstable. The local control myocyte model provides a means for studying the interrelationship between microscopic and macroscopic behaviors in a manner that would not be possible in experiments.     

A Human Ventricular Myocyte Model with a Refined Representation of Excitation-Contraction Coupling

Cardiac Ca²⁺-induced Ca²⁺ release (CICR) occurs by a regenerative activation of ryanodine receptors (RyRs) within each Ca²⁺-releasing unit, triggered by the activation of L-type Ca²⁺ channels (LCCs). CICR is then terminated, most probably by depletion of Ca²⁺ in the junctional sarcoplasmic reticulum (SR). Hinch et al. previously developed a tightly coupled LCC-RyR mathematical model, known as the Hinch model, that enables simulations to deal with a variety of functional states of whole-cell populations of a Ca²⁺-releasing unit using a personal computer. In this study, we developed a membrane excitation-contraction model of the human ventricular myocyte, which we call the human ventricular cell (HuVEC) model. This model is a hybrid of the most recent HuVEC models and the Hinch model. We modified the Hinch model to reproduce the regenerative activation and termination of CICR. In particular, we removed the inactivated RyR state and separated the single step of RyR activation by LCCs into triggering and regenerative steps. More importantly, we included the experimental measurement of a transient rise in Ca²⁺ concentrations ([Ca²⁺], 10–15 μM) during CICR in the vicinity of Ca²⁺-releasing sites, and thereby calculated the effects of the local Ca²⁺ gradient on CICR as well as membrane excitation. This HuVEC model successfully reconstructed both membrane excitation and key properties of CICR. The time course of CICR evoked by an action potential was accounted for by autonomous changes in an instantaneous equilibrium open probability of couplons. This autonomous time course was driven by a core feedback loop including the pivotal local [Ca²⁺], influenced by a time-dependent decay in the SR Ca²⁺ content during CICR.     

Suppression of Calcium Sparks in Rat Ventricular Myocytes and Direct Inhibition of Sheep Cardiac RyR Channels by EPA,DHA and Oleic Acid

The anti-arrhythmic effects of long-chain polyunsaturated fatty acids (PUFAs) may be related to their ability to alter calcium handling in cardiac myocytes. We investigated the effect of eicosapentanoic acid (EPA) and docosahexaenoic acid (DHA) on calcium sparks in rat cardiac myocytes and the effects of these PUFAs and the monounsaturated oleic acid on cardiac calcium release channels (RyRs). Visualization of subcellular calcium concentrations in single rat ventricular myocytes showed that intensity of calcium sparks was reduced in the presence of EPA and DHA (15 µM). It was also found that calcium sparks decayed more quickly in the presence of EPA but not DHA. Sarcoplasmic vesicles containing RyRs were prepared from sheep hearts and RyR activity was determined by either [³H]ryanodine binding or by single-channel recording. Bilayers were formed from phosphatidylethanolamine and phosphatidylcholine dissolved in either n-decane or n-tetradecane. EPA inhibited [³H]ryanodine binding to RyRs in SR vesicles with K _I = 40 µM. Poly- and mono-unsaturated free fatty acids inhibited RyR activity in lipid bilayers. EPA (cytosolic or luminal) inhibited RyRs with K _I =32 µM and Hill coefficient, n ₁ = 3.8. Inhibition was independent of the n-alkane solvent and whether RyRs were activated by ATP or Ca²⁺. DHA and oleic acid also inhibited RyRs, suggesting that free fatty acids generally inhibit RyRs at micromolar concentrations.     

RyRCa2+ leak limits cardiac Ca2+ window current overcoming the tonic effect of calmodulinin mice

Ca(2+) mediates the functional coupling between L-type Ca(2+) channel (LTCC) and sarcoplasmic reticulum (SR) Ca(2+) release channel (ryanodine receptor, RyR), participating in key pathophysiological processes. This crosstalk manifests as the orthograde Ca(2+)-induced Ca(2+)-release (CICR) mechanism triggered by Ca(2+) influx, but also as the retrograde Ca(2+)-dependent inactivation (CDI) of LTCC, which depends on both Ca(2+) permeating through the LTCC itself and on SR Ca(2+) release through the RyR. This latter effect has been suggested to rely on local rather than global Ca(2+) signaling, which might parallel the nanodomain control of CDI carried out through calmodulin (CaM). Analyzing the CICR in catecholaminergic polymorphic ventricular tachycardia (CPVT) mice as a model of RyR-generated Ca(2+) leak, we evidence here that increased occurrence of the discrete local SR Ca(2+) releases through the RyRs (Ca(2+) sparks) cause a depolarizing shift in activation and a hyperpolarizing shift in isochronic inactivation of cardiac LTCC current resulting in the reduction of window current. Both increasing fast [Ca(2+)](i) buffer capacity or depleting SR Ca(2+) store blunted these changes, which could be reproduced in WT cells by RyRCa(2+) leak induced with Ryanodol and CaM inhibition.Our results unveiled a new paradigm for CaM-dependent effect on LTCC gating and further the nanodomain Ca(2+) control of LTCC, emphasizing the importance of spatio-temporal relationships between Ca(2+) signals and CaM function.     

Control of Sarcoplasmic Reticulum Ca2+ Release by Stochastic RyR Gating within a 3D Model of the Cardiac Dyad and Importance of Induction Decay for CICR Termination

The factors responsible for the regulation of regenerative calcium-induced calcium release (CICR) during Ca²⁺ spark evolution remain unclear. Cardiac ryanodine receptor (RyR) gating in rats and sheep was recorded at physiological Ca²⁺, Mg²⁺, and ATP levels and incorporated into a 3D model of the cardiac dyad, which reproduced the time course of Ca²⁺ sparks, Ca²⁺ blinks, and Ca²⁺ spark restitution. The termination of CICR by induction decay in the model principally arose from the steep Ca²⁺ dependence of RyR closed time, with the measured sarcoplasmic reticulum (SR) lumen Ca²⁺ dependence of RyR gating making almost no contribution. The start of CICR termination was strongly dependent on the extent of local depletion of junctional SR Ca²⁺, as well as the time course of local Ca²⁺ gradients within the junctional space. Reducing the dimensions of the dyad junction reduced Ca²⁺ spark amplitude by reducing the strength of regenerative feedback within CICR. A refractory period for Ca²⁺ spark initiation and subsequent Ca²⁺ spark amplitude restitution arose from 1), the extent to which the regenerative phase of CICR can be supported by the partially depleted junctional SR, and 2), the availability of releasable Ca²⁺ in the junctional SR. The physical organization of RyRs within the junctional space had minimal effects on Ca²⁺ spark amplitude when more than nine RyRs were present. Spark amplitude had a nonlinear dependence on RyR single-channel Ca²⁺ flux, and was approximately halved by reducing the flux from 0.6 to 0.2 pA. Although rat and sheep RyRs had quite different Ca²⁺ sensitivities, Ca²⁺ spark amplitude was hardly affected. This suggests that moderate changes in RyR gating by second-messenger systems will principally alter the spatiotemporal properties of SR release, with smaller effects on the amount released.     

Termination of cardiac Ca(2+) sparks: an investigative mathematical model of calcium-induced calcium release

A Ca(2+) spark arises when a cluster of sarcoplasmic reticulum (SR) channels (ryanodine receptors or RyRs) opens to release calcium in a locally regenerative manner. Normally triggered by Ca(2+) influx across the sarcolemmal or transverse tubule membrane neighboring the cluster, the Ca(2+) spark has been shown to be the elementary Ca(2+) signaling event of excitation-contraction coupling in heart muscle. However, the question of how the Ca(2+) spark terminates remains a central, unresolved issue. Here we present a new model, "sticky cluster," of SR Ca(2+) release that simulates Ca(2+) spark behavior and enables robust Ca(2+) spark termination. Two newly documented features of RyR behavior have been incorporated in this otherwise simple model: "coupled gating" and an opening rate that depends on SR lumenal [Ca(2+)]. Using a Monte Carlo method, local Ca(2+)-induced Ca(2+) release from clusters containing between 10 and 100 RyRs is modeled. After release is triggered, Ca(2+) flux from RyRs diffuses into the cytosol and binds to intracellular buffers and the fluorescent Ca(2+) indicator fluo-3 to produce the model Ca(2+) spark. Ca(2+) sparks generated by the sticky cluster model resemble those observed experimentally, and Ca(2+) spark duration and amplitude are largely insensitive to the number of RyRs in a cluster. As expected from heart cell investigation, the spontaneous Ca(2+) spark rate in the model increases with elevated cytosolic or SR lumenal [Ca(2+)]. Furthermore, reduction of RyR coupling leads to prolonged model Ca(2+) sparks just as treatment with FK506 lengthens Ca(2+) sparks in heart cells. This new model of Ca(2+) spark behavior provides a "proof of principle" test of a new hypothesis for Ca(2+) spark termination and reproduces critical features of Ca(2+) sparks observed experimentally.