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: 10688760
• 负责人: Edward Lakatta
• 金额: $ 9.66万
• 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/10688760
• 关键词: Action Potentials; Acute; Adrenergic Receptor; Adult; Behavior; Biophysical Process; Calmodulin; Cell Culture Techniques; Cell membrane; Cell physiology; Cells; Characteristics; Clock protein; Coupled; Couples; Coupling; Cues; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Data; Data Set; Disease; Environment; Heart; Heart Rate; Ice; In Vitro; Individual; Ion Channel; Ions; Kinetics; Laws; Length; Link; Measures; Membrane; Membrane Potentials; Modeling; Nodal; Oryctolagus cuniculus; Pacemakers; Phosphorylation; Principal Component Analysis; Proteins; Regression Analysis; Sarcoplasmic Reticulum; Signal Transduction; Sinoatrial Node; Surface; System; Testing; Time; Tissues; Variant; calmodulin-dependent protein kinase II; experimental study; heart rhythm; insight; models and simulation; molecular clock; molecular scale; nodal myocyte; receptor; response; simulation; 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. 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. Variability of Vm and Ca2+ parameters measured experimentally in cells within and across autonomic states is linked to the respective variabilities of clock molecular availability to respond to Vm and Ca2+ cues that cannot be directly measured experimentally during AP firing. To gain further insight into the variability of these biophysical mechanisms, we performed numerical simulations using a modified Maltsev-Lakatta model that features the coupled-clock mechanism (Maltsev & Lakatta, Am J Physiol. Heart Circ Physiol. 2009). We investigated the variability of peak amplitudes and amplitudes at -40 mV during DD of 6 major currents (If, INCX, IKr, ICaL, ICaT and IKACh) and Ca under cell membrane in each of the three autonomic states: basal, ISO 100nM, and CCh 100nM. Observation of model simulations applied to experimental data indicated that (as was the case for experiment data) the simulated variables are self-similar to each other across broad range of APFI within the three autonomic states. Our numerical model simulations further extended our perspectives to the molecular scale and demonstrated that many ion currents also behave self-similar across autonomic states.

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。 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。 在自主状态内和跨自主状态的细胞中通过实验测量的 Vm 和 Ca2+ 参数的变异性与响应 Vm 和 Ca2+ 线索的时钟分子可用性的各自变异性有关，而这些线索在 AP 放电期间无法直接通过实验测量。为了进一步了解这些生物物理机制的可变性，我们使用修改后的 Maltsev-Lakatta 模型进行了数值模拟，该模型具有耦合时钟机制（Maltsev & Lakatta, Am J Physiol. Heart Circ Physiol. 2009）。我们研究了 DD 期间 6 个主要电流（If、INCX、IKr、ICaL、ICaT 和 IKACh）和细胞膜下 Ca 在三种自主状态（基础、ISO 100nM、和CCh 100nM。对应用于实验数据的模型模拟的观察表明（与实验数据的情况一样）模拟变量在三种自主状态内的广泛 APFI 范围内彼此自相似。我们的数值模型模拟进一步将我们的视角扩展到分子尺度，并证明许多离子电流在自主状态下也表现出自相似性。