Coherent beat to beat variability of self-similar Ca2+ and surface membrane's signaling mechanisms determines the spontaneous action potential firing rate and rhythm of cardiac pacemaker cells

自相似 Ca2 和表面膜信号传导机制的连贯逐搏变异性决定了心脏起搏细胞的自发动作电位放电率和节律

• 批准号: 10007322
• 负责人: Edward Lakatta
• 金额: $ 23.22万
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10007322
• 关键词: Action Potentials; Acute; Adrenergic Receptor; Adult; Affect; Behavior; Blood flow; Ca(2+)-Transporting ATPase; Calmodulin; Cell Culture Techniques; Cell physiology; Cell surface; Cells; Characteristics; Charge; Clock protein; Coupled; Couples; Coupling; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Data Set; Disease; Environment; Heart; Heart Rate; In Vitro; Individual; Ion Channel; Ion Pumps; Ions; Kinetics; Laws; Length; Link; Measures; Membrane; Membrane Potentials; Modeling; Nodal; Oryctolagus cuniculus; Pacemakers; Phosphorylation; Principal Component Analysis; Proteins; Pump; Regression Analysis; Ryanodine Receptors; Sarcoplasmic Reticulum; Signal Transduction; Sinoatrial Node; Stimulus; Surface; System; Testing; Time; Tissues; Variant; calmodulin-dependent protein kinase II; feeding; flexibility; heart rhythm; insight; nodal myocyte; receptor; response; symposium; voltage

The heart rate and rhythm are regulated by rate and rhythm of spontaneous action potential (AP) firing of pacemaker cells that reside within sinoatrial node tissue. Early reductionist studies of mechanisms that underlie pacemaker automaticity had focused upon behaviors of individual surface membrane ion channels. We put forth the idea that spontaneous action potentials generated by single, isolated sinoatrial nodal cells (SANC) are generated by a coupled-clock system: An ensemble of surface membrane electrogenic molecules that directly controls the membrane potential and trans-membrane ion flux, and indirectly regulates intracellular Ca2+ cycling; and a Ca2+ clock, the sarcoplasmic reticulum (SR) and its decorator proteins, that directly control intracellular Ca2+ cycling and indirectly regulate transmembrane ion flux. The two clocks operate as a coupled system in which the coupling fidelity is controlled by voltage, time, Ca2+, intrinsic cAMP signaling, and cAMP-PKA and Ca2+-calmodulin-associated, PKA and CaMKII-dependent clock protein phosphorylation. Surface membrane ion channels, ion electrogenic exchange proteins, e.g., Na-Ca exchanger and ion pumps, e.g., Na-KATPase, comprise the ensemble of M clock molecules: Ion channels are both voltage and time dependent, and are also regulated by phosphorylation and trans membrane ion concentration gradients; the Na-Ca exchanger is voltage-dependent, but unlike M clock ion channels, is not time-dependent. The SR Ca2+ clock operates as a Ca2+ capacitor: its Ca2+ charge is regulated by an energy dependent Ca2+ ATPase, (Serca2), that pumps Ca2+ into the SR lumen, and by ryanodine receptors (RyRs), that dissipate the Ca2+ charge via releasing Ca2+ beneath the cell surface membrane. Ion pumps within each clock are energy dependent. Because both clocks either directly or indirectly regulate both the surface membrane voltage and intracellular Ca2+, neither M nor Ca2+ clock functions can operate independently of each other. Rather, changes in activation states kinetic functions of molecules within either clock in response to extrinsic stimuli affects functions of molecules of the other clock. We have referred to such clock interactions as clock-coupling. The fidelity of clock coupling in a given steady state regulates the mean AP firing rate (cycle length characteristic of that steady state). Clock coupling fidelity, by necessity, is not fixed, however, but, by necessity, must be variable, in order to rapidly confer heart rate flexibility required to match variations in blood flow from the heart to acute variation in the bodys blood flow demands. Clock coupling fidelity is regulated by numerous external signals that impact on intrinsic coupled clock functions of SANC, including: autonomic receptor stimulation and downstream signaling by cAMP and PKA and CAMKII dependent phosphorylation of clock proteins; the concentration of the oscillatory substrate, Ca2+ itself (regulated in part by the SANC transmembrane Na+ gradient); and importantly, the AP firing rate or cycle length that emerges as a given steady state is achieved, ie, feed forward signaling, emanating from the firing rate, per se, regulates the fidelity of clock-coupling. Thus, AP cycle length is not only regulated by but also regulates the fidelity of M and Ca2+ clock coupling rate. Although by convention we refer to an average AP cycle length that characterizes a given steady state, AP cycle lengths vary from cycle to cycle indicating that a true steady state AP cycle length is never achieved. Prior to the elucidation of the coupled-clock system it had been discovered that AP cycle to cycle length variability could be linked to beat to beat homogeneity of activation states of SANC M clock ion channel molecules leading to beat to beat variability in channel availability. More recently, cycle to cycle variability in the rhythm of of local Ca2+ releases of the Ca2+ clock of SANC has also been demonstrated to be linked to action potential cycle length variability and to cycle to cycle variability of clock coupling. We hypothesized that concordant beat to beat variability of order (or disorder) among intrinsic mechanisms that regulate SANC M and Ca clock functions and their coupling determines the average AP firing rate and rhythm (cycle to cycle variability) that emerge in a given apparent steady state. We employed two external perturbations of the clock functions known to markedly effect steady state AP firing rate: (1) adrenergic receptor stimulation (bARs) and (2) an in vitro cell culture environment, in which mean APCL of cultured SANC cSANC) becomes about twice that of freshly isolated SANC (fSANC) and remains stable for several days ( ). bARs acutely restores APCL cSANC to that of fSANC. In response to bARs in single SANC, (f-SANC). In addition to recording average AP cycle lengths and AP cycle to cycle variability, we measured prior to and during bARs mean of M clock kinetic functional parameters (time to 90% AP repolarization (AP90) and time from maximum diastolic potential (MDP) to onset of non-linear diastolic depolarization (DD), and their cycle to cycle variability: and Ca2+ clock kinetic parameter the time to 90% decay of the AP-induced global cytosolic Ca2+ transient (CaT90) and diastolic LCR periods, measured as the time elapse between the prior preceding of AP induced Ca2+ transient to an LCR onset). We assessed cycle to cycle parameter variability under each condition in c and f SANC and their cycle to cycle variabilities as coefficient of variation (CV) about the mean, ie standard deviation divided by the mean. We employed linear correlation analyses, followed by principal component analyses (to determine the relationship of cycle to cycle variability of each function to its mean. To assess the degree of concordance among the means and CVs of measured M and Ca2+ clock parameters in c- and f-SANC and the concordance of these parameters to mean APCL and its CV. We used multiple regression analyses to determine whether the concordance among mean functions and concordance variability of each function) could predict the mean APCL and its cycle to cycle variability of the entire data set, ie, in C and f-SANC in control in response bARs. Finally, we employed power-law analyses to determine whether concordant degrees of order (variability) of M and Ca2+ clock kinetic functions prior to and during bARs in both c- and f-SANC are self-similar. In addition to measuring and analyzing mean and variability of AP characteristics, we explored the variability of the simulated ion currents (predicted by numerical modelling) that underlie APs in order to derive mechanistic insights into cycle variability of in currents that generates cycle variability of AP waveforms and the APFIV. And we found that the cycle to cycle variabilities of ion currents differ from each other and also differ to the experimentally measured APFIV both in control and in response to autonomic receptor stimulation.

心率和节奏受自发作用电位的速率和节奏（AP）的调节，该动作电位（AP）放电位于窦淋巴结组织内的起搏器细胞。 早期还原论对起搏器自动化基础的机制的研究集中在单个表面膜离子通道的行为上。 We put forth the idea that spontaneous action potentials generated by single, isolated sinoatrial nodal cells (SANC) are generated by a coupled-clock system: An ensemble of surface membrane electrogenic molecules that directly controls the membrane potential and trans-membrane ion flux, and indirectly regulates intracellular Ca2+ cycling;和Ca2+时钟，肌浆网（SR）及其装饰蛋白，该蛋白直接控制细胞内Ca2+循环并间接调节跨膜离子通量。 这两个时钟是一个耦合系统，其中耦合保真度由电压，时间，Ca2+，固有的cAMP信号以及CAMP-PKA以及CA2+-Calmodulin相关，PKA和CAMKII依赖性时钟蛋白磷酸化。 表面膜离子通道，离子电源交换蛋白，例如Na-Ca交换器和离子泵，例如Na-katpase，构成了M时钟分子的合奏：离子通道是电压和时间依赖的，并且也受磷酸化和跨膜膜纤维化和跨膜晶体元素离子浓缩量剂量调节； Na-Ca交换器与电压有关，但与M时钟离子通道不同，不是时间依赖性。 SR Ca2+时钟作为CA2+电容器的作用：其Ca2+电荷受能量依赖性Ca2+ ATPase（SERCA2）（SERCA2）的调节，该电容器将Ca2+泵入SR Lumen，并由Ryanodine受体（RYRS）调节，并通过在细胞表面循环CAC2+ BEAN CAS2+电荷来调节Ca2+电荷。每个时钟内的离子泵取决于能量。 由于两个时钟都直接或间接调节表面膜电压和细胞内Ca2+，因此M和Ca2+时钟功能都无法彼此独立地运行。相反，两个时钟内分子的激活状态的动力功能会影响外在刺激会影响另一个时钟分子的功能。 我们已经提到了时钟交互，例如时钟耦合。 时钟耦合在给定稳态中的保真度调节平均AP触发速率（该稳态的循环长度特征）。 需要时钟耦合忠诚度并不是固定的，但是，必须有必要是可变的，以便迅速赋予心率灵活性，以匹配从心脏的血液流量变化到身体血液流量需求的急性变化。 时钟耦合的忠诚度受到影响固有耦合时钟功能的许多外部信号的调节，包括：CAMP，PKA和PKA和CAMKII依赖性时钟蛋白的自主受体刺激和下游信号传导；振荡底物的浓度Ca2+本身（部分由跨膜Na+梯度调节）；重要的是，达到给定稳态的AP点火速率或周期长度，即从发射速率发出的馈电信号传导，本身可以调节时钟耦合的忠诚度。 因此，AP循环长度不仅受其调节，还调节M和Ca2+时钟耦合速率的保真度。 尽管按照惯例，我们指的是特征给定稳态的平均AP周期长度，但AP周期长度因周期而异，表明从未实现真正的稳态AP循环长度。 在阐明耦合 - 锁系统之前，已经发现，可以将AP周期链接到循环长度可变性与BEAT链接以击败Sanc M Clock M Clock ION通道分子激活状态的均匀性，从而使Beat在通道可用性中击败可变性。最近，也已证明，SANC Ca2+时钟局部Ca2+释放节奏的循环变异性也已被证明与动作电位循环长度的可变性以及循环与时钟耦合的循环变异性有关。 我们假设在调节sanc M和Ca时钟功能的内在机制中，一致的节拍击败了秩序（或无序）的变异性及其耦合决定了在给定的明显稳态下出现的平均AP触发率和节奏（循环到周期变异）。我们采用了两种外向函数的外部扰动，已知可以显着影响稳态AP发射速率：（1）肾上腺素能受体刺激（bars）和（2）一个体外细胞培养环境，其中培养的sanc csanc的平均APCL的平均APCL变为两倍的两倍，即新鲜分离的sanc（FSANC（FSANC）），并且保持了稳定的稳定。 酒吧急性恢复APCL csanc。为了响应单个sanc中的条（F-sanc）。除了记录平均AP周期长度和AP循环到周期变异性外，我们还测量了M时钟动力学功能参数（时间为90％AP复极化（AP90）（AP90）的时间（AP90），以及从最大舒张力潜力（MDP）到非线性舒张期舒张极变（DD）的时间（DD）的时间（dd）的时间：和CAC2+ KIN的时间：全局的胞质Ca2+瞬态（CAT90）和舒张期LCR周期，被测量为AP先前诱导的Ca2+瞬时到LCR发作之间的时间流逝）。 我们评估了C和F SANC中每个条件下的周期参数变异性及其循环变异性的循环变异性，以循环变异性作为变异系数（CV）围绕平均值，即标准偏差除以平均值。 We employed linear correlation analyses, followed by principal component analyses (to determine the relationship of cycle to cycle variability of each function to its mean. To assess the degree of concordance among the means and CVs of measured M and Ca2+ clock parameters in c- and f-SANC and the concordance of these parameters to mean APCL and its CV. We used multiple regression analyses to determine whether the concordance among mean functions and concordance variability of每个函数）可以预测平均APCL及其周期，以在响应栏中在控制中的C和F-SANC中整个数据集的循环变异性。最后，我们采用了幂律分析来确定在c - 和f-sanc的条形之前和bar中，M和Ca2+时钟动力函数的一致程度（可变性）是否是自相似的。除了测量和分析AP特性的平均值和变异性外，我们还探索了基于AP的模拟离子电流（通过数值建模预测）的变异性，以便将机械性见解到产生AP -波形和APFIV的循环变化的电流的机械循环变异性。我们发现，离子电流的循环变化循环彼此不同，并且在对照方面和对自主受体刺激的反应中的实验测量的APFIV也有所不同。