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.

心率和节律由窦房结组织内起搏细胞自发动作电位（AP）放电的速率和节律调节。 早期对起搏器自动性机制的还原论研究主要集中在单个表面膜离子通道的行为上。 我们提出这样的想法：单个孤立的窦房结细胞（SANC）产生的自发动作电位是由耦合时钟系统产生的：直接控制膜电位和跨膜离子通量的表面膜生电分子的集合，以及间接调节细胞内 Ca2+ 循环； Ca2+ 时钟、肌浆网 (SR) 及其装饰蛋白，直接控制细胞内 Ca2+ 循环并间接调节跨膜离子通量。 这两个时钟作为耦合系统运行，其中耦合保真度由电压、时间、Ca2+、内在 cAMP 信号传导以及 cAMP-PKA 和 Ca2+-钙调蛋白相关、PKA 和 CaMKII 依赖性时钟蛋白磷酸化控制。 表面膜离子通道、离子生电交换蛋白（例如 Na-Ca 交换器）和离子泵（例如 Na-KATPase）组成了 M 时钟分子的集合：离子通道既依赖于电压又依赖于时间，并且还受到磷酸化和磷酸化的调节。跨膜离子浓度梯度； Na-Ca 交换器是电压依赖性的，但与 M 时钟离子通道不同，它不是时间依赖性的。 SR Ca2+ 时钟作为 Ca2+ 电容器运行：其 Ca2+ 电荷由能量依赖性 Ca2+ ATP 酶 (Serca2) 和兰尼碱受体 (RyRs) 调节，前者将 Ca2+ 泵入 SR 腔，后者通过释放 Ca2+ 耗散 Ca2+ 电荷位于细胞表面膜下方。每个时钟内的离子泵都依赖于能量。 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 时钟功能和 Ca2+ 时钟功能都不能彼此独立运行。相反，响应于外部刺激的任一时钟内的分子的激活状态动力学功能的变化会影响另一个时钟的分子的功能。 我们将这种时钟交互称为时钟耦合。 给定稳态下时钟耦合的保真度调节平均 AP 发射率（该稳态的周期长度特征）。 然而，时钟耦合保真度必然不是固定的，而是必须是可变的，以便快速赋予匹配来自心脏的血流变化与身体血流需求的急剧变化所需的心率灵活性。 时钟耦合保真度由影响 SANC 内在耦合时钟功能的众多外部信号调节，包括：自主受体刺激以及 cAMP 和 PKA 以及时钟蛋白依赖的 CAMKII 磷酸化的下游信号传导；振荡底物 Ca2+ 本身的浓度（部分由 SANC 跨膜 Na+ 梯度调节）；重要的是，当达到给定稳态时出现的AP发射率或周期长度，即，从发射率发出的前馈信令本身调节时钟耦合的保真度。 因此，AP周期长度不仅受M和Ca2+时钟耦合率的调节，而且还调节M和Ca2+时钟耦合率的保真度。 尽管按照惯例，我们指的是表征给定稳态的平均 AP 周期长度，但 AP 周期长度因周期而异，这表明永远无法实现真正​​的稳态 AP 周期长度。 在阐明耦合时钟系统之前，已经发现AP周期与周期长度的变异性可能与SANC M时钟离子通道分子激活状态的逐搏同质性相关，导致通道可用性的逐搏变异性。最近，SANC 的 Ca2+ 时钟局部 Ca2+ 释放节律的周期变异性也被证明与动作电位周期长度变异性和时钟耦合的周期变异性相关。 我们假设调节 SANC M 和 Ca 时钟功能的内在机制中顺序（或无序）的一致心跳变异性及其耦合决定了在给定的表观稳定状态下出现的平均 AP 放电率和节律（周期间变异性） 。我们采用了已知显着影响稳态 AP 放电率的两种时钟功能的外部扰动：(1) 肾上腺素能受体刺激 (bAR) 和 (2) 体外细胞培养环境，其中培养的 SANC 的平均 APCL (cSANC) 变为约是新分离的 SANC (fSANC) 的两倍，并且可以保持稳定数天 ( )。 bAR 能够迅速将 APCL cSANC 恢复到 fSANC。响应单个 SANC 中的 bAR（f-SANC）。除了记录平均 AP 周期长度和 AP 周期间变异性外，我们还在 bAR 之前和期间测量了 M 时钟动力学功能参数的平均值（90% AP 复极时间 (AP90) 和从最大舒张电位 (MDP) 到开始的时间）非线性舒张去极化 (DD) 及其周期变异性：和 Ca2+ 时钟动力学参数 AP 诱导的整体胞质 Ca2+ 瞬变衰减 90% 的时间(CaT90) 和舒张期 LCR 期，测量为前一次 AP 诱导的 Ca2+ 瞬态到 LCR 开始之间经过的时间。 我们评估了 c 和 f SANC 中每种条件下的周期间参数变异性及其周期间变异性，作为平均值的变异系数 (CV)，即标准差除以平均值。 我们采用线性相关分析，然后进行主成分分析（以确定每个函数的周期变异性与其平均值的关系。评估 c- 和 Ca2+ 时钟参数的平均值和 CV 之间的一致性程度） f-SANC 以及这些参数与平均 APCL 及其 CV 的一致性，我们使用多重回归分析来确定平均函数之间的一致性以及每个函数的一致性变异性是否可以预测平均 APCL 及其周期。整个数据集的循环变异性，即在响应 bAR 中的 C 和 f-SANC 中的控制。最后，我们采用幂律分析来确定 c- 和 f-SANC 中 bAR 之前和期间 M 和 Ca2+ 时钟动力学函数的有序度（变异性）是否一致。除了测量和分析 AP 特性的平均值和变异性之外，我们还探索了 AP 背后的模拟离子电流的变异性（通过数值建模预测），以便获得对产生 AP 波形循环变异性的电流循环变异性的机制见解和 APFIV。我们发现离子电流的周期变化彼此不同，并且在控制和对自主受体刺激的响应方面也与实验测量的 APFIV 不同。