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

Lakatta, Edward

Abstract

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.