A coupled SYSTEM of intracellular Ca²⁺ clocks and surface membrane voltage clocks controls the timekeeping mechanism of the heart’s pacemaker

Abstract

Ion channels on the surface membrane of sinoatrial nodal pacemaker cells (SANC) are the proximal cause of an action potential. Each individual channel type has been thoroughly characterized under voltage clamp, and the ensemble of the ion channel currents reconstructed in silico generates rhythmic action potentials. Thus, this ensemble can be envisioned as a surface “membrane clock” (M clock). Localized subsarcolemmal Ca²⁺ releases are generated by the sarcoplasmic reticulum via ryanodine receptors during late diastolic depolarization and are referred to as an intracellular “Ca²⁺ clock”, because their spontaneous occurrence is periodic during voltage clamp or in detergent-permeabilized SANC, and in silico as well. In spontaneously firing SANC, the M and Ca²⁺ clocks do not operate in isolation, but work together via numerous interactions modulated by membrane voltage, subsarcolemmal Ca²⁺, and PKA and CaMKII-dependent protein phosphorylation. Through these interactions the two subsystem clocks become mutually entrained to form a robust, stable, coupled-clock system that drives normal cardiac pacemaker cell automaticity. G-protein coupled-receptors signaling creates pacemaker flexibility, i.e. effects changes in the rhythmic action potential firing rate, by impacting on these very same factors that regulate robust basal coupled-clock system function. This review examines evidence that forms the basis of this coupled-clock system concept in cardiac SANC.

Introduction

The major electrogenic surface membrane molecules (and their ion currents) in sinoatrial nodal pacemaker cells (SANC) include ion channels and transporters: L-type Ca²⁺ channels (I_CaL), T-type Ca²⁺ channels (I_CaT), delayed rectifier K⁺ channels (I_K), hyperpolarization-actiavted, funny channels (I_f), Na⁺/Ca²⁺exchanger (NCX, I_NCX), and Na⁺/K⁺ATPase (I_NaK). The structure and function of these, and of other electrogenic molecules, have been extensively studied and comprehensively described in numerous reviews (e.g.^1-3). Figure 1A presents a graphic scheme of the coupled-clock system concept: temporal profiles of sarcolemmal ion currents, membrane potential (V_m), and intracellular Ca²⁺ cycling during SANC duty cycles. When voltage and time-dependent properties of each individual ion channel derived from voltage clamp studies are entered into numerical models, the ensemble of ion channels in silico can generate rhythmic spontaneous AP’s⁴, and thus the ensemble can be envisioned as a surface “membrane clock” (M clock). But direct experimental characterization of the ensemble function of the M clock cannot be made during the normal SANC duty cycle, i.e. in the course of normal interactions with intracellular processes.

Fig.1. The coupled-clock pacemaker system.

A. Schematic illustration of key phases of the functional interactions between M clock and Ca²⁺ clocks. Modified from¹⁰³. B. Schematic illustration of interactions of molecules comprising the full coupled-pacemaker clock. Note that the same regulatory factors (red lettering) of the SR Ca²⁺ clock (gray intracellular area, black lettering) couple the Ca²⁺ clock to the M clock (blue membrane area, blue lettering). G protein-coupled receptors (green lettering) regulate both the Ca²⁺ clock and membrane clock via those same factors (red lettering) and other coupling factors (green shapes). See text for numerous additional details. Modified from⁶⁷.

Numerous studies over the last two decades have investigated the role of intracellular Ca²⁺ cycling in cardiac pacemaker function^5-12. Specific, detailed mechanisms of Ca²⁺ cycling contributions have become available in more recent studies (reviews^13-15 and numerical modeling¹⁶). The sarcoplasmic reticulum (SR) is wired to oscillate Ca²⁺ via its Ca²⁺ pumps (SERCA-2) and Ca²⁺ release channels, ryanodine receptors (RyR)(Fig.2). Because Ca²⁺ oscillations generated by the SR during experiments in the absence of sarcolemmal function^17,18, and in silico¹⁶ are rhythmic, the SR has been referred to as an intracellular “Ca²⁺ clock” (Fig.1B). But in nature, neither clock functions in the absence of the other. Abundant evidence indicates that functional interactions that are critical for normal automaticity occur between the two clocks (reviews^13,14). Specific surface membrane proteins not only effect changes in membrane potential, but also directly or indirectly regulate intracellular Ca²⁺ cycling; and conversely, intracellular Ca²⁺ cycling proteins also regulate V_m via Ca²⁺-modulation of surface membrane electrogenic molecules. Furthermore, other coupling factors, in addition to Ca²⁺, i.e. protein phosphorylation by protein kinase A (PKA) or Ca²⁺-calmodulin-dependent protein kinase II (CaMKII), that affect function of proteins of both clocks (Fig.1B), are critical for regulation of normal automaticity by the coupled-clock system.

Fig.2. CaMKII, SERCA, RyR and NCX immunolabeling in rabbit SANC.

A. The intracellular distribution of total and active CaMKII in SANC. A uniform distribution of the total CaMKII immunolabeling (top); the localization of active CaMKII beneath the sarcolemmal membrane (middle); the negative control (bottom), i.e., in the absence of the primary antibodies (from⁴⁹). B SERCA labeling in the single rabbit SANC. C, Confocal image of SANC double immunolabeled for NCX and RyR. D, pixel-by-pixel fluorescence intensities of labeling along an arbitrary (white) line in panel C. The horizontal dashed lines show the average pixel intensity. E, Topographical profiles of the pixel intensity levels of each antibody labeling and overlay in the small SANC in panel C. The maximum height represents the brightest possible pixel in the source image. B-E from²⁰.

Specific voltage- time- Ca²⁺- dependent coupling within and between surface membrane and intracellular Ca²⁺ cycling proteins during the SANC AP duty cycle

M and Ca²⁺ clock events and coupling during late diastolic depolarization

Spontaneous diastolic depolarization (DD) is the essence of cardiac pacemaker cell automaticity. The DD occurs in two phases: the early and late DD (Fig.1A). Confocal imaging of Ca²⁺ in mammalian SANC and atrial subsidiary pacemaker cells combined with non-invasive perforated patch-clamp electrophysiology^8,19 and imaging of toad sinus venosus cells⁷, has documented the occurrence of subsarcolemmal Local Ca²⁺ Releases (LCR’s) during the late DD (Fig.1A and Fig.3). SANC exhibit robust SERCA2 and RyR immunolabeling^11,20 (but see also²¹). SERCA2 immunolabeling in SANC is located diffusely throughout the cytoplasm and per nuclear area, whilst RyR immunolabeling is most intense in the subsarcolemmal space (Fig.2)^11,20,21. In spontaneously firing rabbit SANC, LCRs emanate from SR via RyRs and in confocal line-scan images appear as 4-10 μm Ca²⁺ wavelets; they emerge following the dissipation of the global systolic transient effected by the prior AP, and crescendo during the DD, peaking during the late DD, as they merge into the global cytosolic Ca²⁺ transient triggered by the next AP (Fig.3)^17,19. LCRs (Fig.3A-D), or the integral of LCR’s (Fig.3E), i.e., late diastolic Ca²⁺ elevations (LDCAE) (Fig.3F), have now been documented in numerous species^7,8,19,22-24.

Fig. 3. LCR appear during late diastolic depolarization in SANC of different species.

A. Line-scan image of LCRs with superimposed spontaneous APs in rabbit SANC. White arrowheads show LCRs. The LCR period is defined as indicated. Modified from²⁷. B. local Ca²⁺ releases in cat latent pacemaker cells (from⁸, with permission). C, A line-scan image of LCRs with superimposed spontaneous APs in a mouse SANC (from²², with permission). D. Ca²⁺ sparks in toad pacemaker cells: (left) a line-scan image showing regions of localised, transient increase in fluorescence along the upper border of the toad pacemaker cell; (right) intensity map of fluorescence of a composite spark obtained by superimposing 11 sparks (from⁷, with permission). E. Confocally measured subsarcolemmal LCRs (upper panel) in rabbit SANC. The temporal average of the individual LCRs within each cycle (lower panel) generates Late Diastolic Ca²⁺ Elevation (LDCAE) that precedes Ca²⁺ transient induced by action potential. Modified from¹⁰⁴. F. Superimposed APs (red trace) and associated Ca²⁺ transients (blue trace) recorded in rabbit SANC (LDCAE are indicated by arrows) (from²⁴, with permission).

While there is some evidence to indicate that I_CaT may activate LCR’s in cat latent pacemaker cells⁸, voltage clamp studies in rabbit SANC indicate that LCR’s are not appreciably affected by I_CaT inhibition²⁵, do not require membrane depolarization (Fig.4A), but occur spontaneously^17,18. In Fig.4A, note that following acute voltage clamp at the MDP of spontaneously firing SANC, several oscillatory cycles of Ca²⁺ release occur prior to their damping out, due to SR and cytosolic Ca²⁺ depletion¹⁷. Rhythmic or periodic spontaneous Ca²⁺ oscillations persist during acute voltage clamp at a potential that prevents Ca²⁺ loss from the cell and are accompanied by membrane current fluctuations of the same periodicity (Fig.4B). Similar perspectives regarding the characteristics of spontaneous LCRs are gleaned from studies in saponin-skined rabbit SANC, in which intracellular [Ca²⁺] is buffered at a constant physiological level^17,18. LCR-initiated, late DD inward current in rabbit SANC varies from 0.3 pA/pF²⁶ to 1.6 pA/pF¹⁹, yielding an I_NCX range from 9 to 48 pA for a 30 pF SANC. This range of inward I_NCX is sufficient to broadly modulate the DD^16,26,27, because the SANC membrane resistance is high, and extremely small net ion current change (a few pA in rabbit SANC²⁸) has marked effects on V_m. At the resolution of the confocal microscope²⁰ NCX and RyRs molecules colocalize across the ~12 nm subsarcolemmal gap between RyR’s on the SR and NCX molecules on the sarcolemma (Fig.2). While I_NCX is voltage dependent, it does not exhibit its own time-dependent gating mechanisms, as do most surface membrane ion channels; rather time-dependent changes in [Ca²⁺] in the subsarcolemmal space regulate the timing of I_NCX activation. Subsarcolemmal Ca²⁺ release induces an inward I_NCX, as each Ca²⁺ that is transported out of the cell is exchanged for 3Na⁺. The rate of Na⁺/Ca²⁺ exchange by NCX, and hence the instant value of I_NCX, is determined by V_m and the instant Na⁺/Ca²⁺ gradient across the sarcolemma (Fig.1A).

Fig. 4. LCR occur in SANC the absence of changes in membrane potential.

A. (top) Simultaneous recordings of APs, linescan image and normalized subsarcolemmal fluorescence averaged spatially over the band indicated by doubleheaded arrow in a representative SANC prior to, during, and following acute voltage clamp at the maximum diastolic potential (from¹⁷); (bottom) Average total LCR signal mass in 9 cells in control during spontaneous beating, and during each “would-be” cycle of voltage clamp. B, Simultaneous recordings of membrane potentials or current (top), confocal line-scan image (middle), and normalized fluo-3 fluorescence (bottom) averaged over the line-scan image, in a representative spontaneously beating SANC before and during voltage clamp to −10 mV. Fast Fourier transform (FFT) of Ca²⁺ and membrane current fluctuations during voltage clamp to −10 mV (from¹⁸).

The membrane current fluctuations generated by LCRs during late DD (Fig.4B) drive miniature V_m oscillations (Fig.5A). When these are suppressed by disabling RyRs with ryanodine (Fig.5A inset), the exponential character of the late DD is abolished^13,27 (Fig.5B). (Of note, ryanodine blocks neither I_f¹², nor I_CaL²⁵). The membrane current and voltage responses to the LCR’s, and, most importantly, AP generation by SANC, require extracellular Na⁺, because when Na⁺ is acutely removed from the bathing milieu of rabbit¹⁹ (Fig.5C) or guinea pig²⁹ SANC (Fig.5D) following a prior AP, generation of the subsequent AP acutely fails (Fig.5C,D), while LCR’s persist (Fig.5C). This indicates that forward mode NCX, by generating inward I_NCX, couples LCRs to the late DD acceleration¹⁹. Activation of I_CaL during the late DD (activation threshold is about −50 mV to −40 mV) results in the generation of the AP rapid upstroke, which triggers a global SR Ca²⁺ release (see next section for detailed interactions during the AP).

Figure 5. Spontaneous miniature DD voltage fluctuations and beating of SANC are inhibited when either LCRs are suppressed by ryanodine or I_NCX is acutely inhibited by a Na⁺ poor solution.

A. Time course of DD variance (dots) and of the relative LCRs occurrence (bars) observed at different times during the measurement period (24 SANC). Inset shows that the average amplitude of DD fluctuations, calculated for the 50-ms segment preceding AP upstroke, is suppressed by ryanodine (from²⁷). B. Superimposed APs of representative rabbit SANC before and after treatment with 3 μmol/L ryanodine (from¹³). C. Linescan image of Ca²⁺ release with superimposed AP records during rapid sprits with Na⁺ free solution. Red curve superimposed on the last AP preceding spritz of Na⁺ free solution is a copy of the residual membrane potential oscillation observed during Na⁺-free solution spritz. Note that this maneuver suppressed late DD (see inset) and blocked the subsequent AP firing. (from¹⁹). D. A rapid switch to low-Na⁺ solution (during the time indicated by the bar) caused immediate cessation of spontaneous action potentials in guinea-pig SANC. Action potentials speedily reappeared following rapid switch back to normal Na⁺ solution (with permission from²⁹).

L-type Ca channels are a multi-subunit complex, and α1C represents the major isoform of the channel central pore subunit in the cardiovascular system. However, recently α1D has been discovered in the mouse SA node^30,31. In comparison to α1C subunit as a central pore, L-type Ca²⁺ current with α1D subunit is activated at lower voltages, suggesting that this channel might be involved in the generation of DD. A substantial contribution of Cav1.3 channel in total I_CaL in mouse SANC was confirmed in Cav1.3 knockout mice in which the density of I_CaL compared with wild-type mice was reduced by 79%³⁰. Low-voltage-activated I_CaL has also been shown in rabbit SANC, but the contribution of this current to the total I_CaL in rabbit SANC is still unknown³².

The combined perspectives about interactions between the Ca²⁺ and M clocks gleaned thus far are that: 1) there is a rhythmic spontaneous Ca²⁺ oscillator or clock that generates LCRs within SANC; 2) during spontaneous AP firing, LCRs, generated by the Ca²⁺ clock emerge during late DD; 3) the intracellular Ca²⁺ clock requires recurring APs to provide Ca²⁺ to sustain its rhythmic oscillator function; 4) LCRs generated by the Ca²⁺ clock interact with the surface membrane and, via activation of I_NCX, generate miniature current and voltage oscillations that confer the exponential increase to the late phase of DD; 5) This late DD acceleration by I_NCX is required for sufficient I_CaL activation to generate the subsequent timely rapid AP upstroke. In the absence of this LCR-activated I_NCX “prompt” to the membrane, rhythmic AP’s do not occur.

M - Ca²⁺ clock events and coupling during the AP

Ca²⁺ influx via L-type channels during the AP induces Ca²⁺ release from SR via RyRs (i.e. Ca²⁺ -induced Ca²⁺ release, CICR)^33,34. SANC do not have T-tubules, so that AP-triggered Ca²⁺ release, resulting in a global subsarcolemmal and cytosolic Ca²⁺ transient, occurs via subsarcolemmal RyR’s^19,34. Ca²⁺ that accumulates within the subsarcolemmal space binds to calmodulin to inactivate I_CaL (Fig.1B) and modulates the NCX throughout the AP, contributing to the Ca²⁺ transient decay by causing Ca²⁺ efflux from the cell. SR pumps Ca²⁺ continuously throughout the AP cycle, at a rate modulated by the amount of Ca²⁺ to be pumped. A substantial component of Ca²⁺ influx via the L-type Ca²⁺ channels is pumped directly into the SR³⁵, and replenishes cell Ca²⁺ load to balance Ca²⁺ efflux via NCX exchange. Ca²⁺ influx may also occur via Ca²⁺ store-operated via TRPC channels that have been identified in mouse SANC³⁶.

The occurrence of AP-triggered Ca²⁺ release from the SR (via CICR) synchronizes the global SR in a relatively Ca²⁺ depleted state that temporarily suspends generation of spontaneous LCRs. In Figure 4A, note that in the absence of the timely occurrence of APs during voltage clamp, the integrated LCR signal mass (i.e. LCR number, amplitude and width) generated by the Ca²⁺ clock continues to grow in magnitude, and passes through a maximum prior to its damping out (Fig.4A, lower panel). This transient exaggeration of the LCR signal mass can be construed as a fail-safe “prompt” for the membrane in the event that restitution processes of M clock (e.g. I_CaL) becomes sluggish and cannot respond to the initial LCR signal.

Membrane depolarization during the AP activates K⁺ channels which effect AP repolarization. Intracellular Ca²⁺ also modulates K⁺ channel gating^37,38 (Fig.1B). As NCX flux is both voltage- and Ca²⁺-dependent, K⁺ channels, via AP repolarization, modulate the activation of forward mode NCX exchange. Since NCX assists in reducing the cytosolic and subsarcolemmal Ca²⁺, K⁺ channels, by activating NCX, indirectly modulate cell Ca²⁺ balance. The membrane repolarization effected by K⁺ channel activation also inactivates L-type Ca²⁺ channels, limiting Ca²⁺ influx and thus modulates Ca²⁺ balance via this effect.

M and Ca²⁺clock events and coupling following the AP in early and mid DD

The [Ca²⁺] within the cytosolic and subsarcolemmal compartments decays (due to Na⁺-Ca²⁺ exchange and SR Ca²⁺ pumping) to a nadir near the end of early DD (Fig.1A). Inward I_NCX concomitantly decays and also reaches its nadir near the end of early DD (just before LCRs emerge) (Fig.1A).

Following achievement of the maximum diastolic potential (MDP), I_K conductance (g_K) decreases (Fig.1A) and this is one mechanism of the early DD in pacemaker cells. This “g_K decay” is thought to unmask an inward current, initially described as a background current (I_bNa) (presumably of Na⁺ selectivity)³⁹. A large amplitude background current is a critical component of all numerical SANC models: I_bNa is not only a partner in g_K mechanism, but also balances the outward I_NaK. It was speculated that window currents of I_CaL and I_CaT and I_Na (when present in SANC)⁴⁰, a chloride current⁴¹, or a Ca²⁺-activated nonselective (TRPM4) cation channel⁴² contribute to the background currents in SANC. It has also been speculated that a Na⁺-H⁺ mechanism contributes to background current because it is reduced by amiloride⁴³. An interesting idea is that background current is generated by a non-transported Na⁺-Ca²⁺ leak of NCX⁴⁴. Additionally, since I_bNa is a Na⁺ current that is present during early and mid DD, it could be postulated that a substantial inward NCX Na⁺ current driven by the decaying Ca²⁺ transient (as predicted by many numerical SANC models⁴), also contributes to background current during this period. I_bNa may also embrace the Na⁺ current of I_st (see below). A radical idea is that a background current within the DD range does not exist, but is an artifact of the seal leak that always accompanies the whole-cell patch clamp recording²⁸. In short, despite extensive electrophysiological and pharmacological studies^28,40,43, the molecular identity of the underlying “background channel” remains unknown.

A non-selective, sustained or steady current (I_st)⁴⁵ has also been suggested as a DD mechanism. I_st has some characteristics of I_CaL, e.g. it is sensitive to dihydropyridines and has a reversal potential close to +37 mV. However, I_st is activated at more negative potentials than I_CaL and achieves peak amplitude at about −50 mV⁴⁶. Since I_st is mainly carried by Na⁺, increased by lowering extracellular [Ca²⁺], and reduced by lowering extracellular [Na]⁴⁵, it behaves as I_NCX and, hence, might, in part at least, reflect I_NCX activation by LCRs during mid DD (because LCRs just begin to occur also at about −50 mV). The reported I_st sensitivity to dihydropyridines is not surprising, then, because a reduction of I_CaL reduces Ca²⁺ influx, the cell Ca²⁺ and SR Ca²⁺ loads, and hence reduces LCRs. Thus, it is possible that I_st reflects combined (direct and indirect) effects of I_NCX and I_CaL.

The later repolarization phase of the AP also activates I_f (Fig.1A). I_f is a non-selective current (carried by both Na⁺ and K⁺). Since it has a reversal potential of about −25 mV, it generates an inward current during DD, and is yet another early-mid DD mechanism (Fig.1A)^28,47. Cytosolic Ca²⁺ does not directly regulate I_f⁴⁸.

The mid DD eventually gives way to late DD, a new cycle begins, and the events during late DD, as described above, recur. The numerous interactions between the M and Ca²⁺ clock subsystems, as illustrated in Fig.1, and discussed above, are summarized in Online Table I. These numerous interactions result in a mutual entrainment of the molecular function of both clocks that confers robustness to coupled-clock pacemaker system (a numerical study¹⁶ and review¹⁴). Indeed, by releasing Ca²⁺ into the subsarcolemmal space with precise timing during late DD, the spontaneous Ca²⁺ clock interacts with membrane proteins to cause an inward I_NCX current that facilitates the “on-time” occurrence of the next AP. By “igniting” the membrane depolarization that facilitates the production of a timely AP, the Ca²⁺ clock guarantees its own existence during future cycles: the AP (that the Ca²⁺ clock facilitates) resets the Ca²⁺ clock via CICR-mediated SR Ca²⁺ depletion, and refuels the Ca²⁺ clock with Ca²⁺, via Ca²⁺ influx through L-type Ca²⁺ channels. The M clock, in turn, not only generates the AP, the sine-qua-non of SANC function, but, by ensuring the continual function of the Ca²⁺ clock, the M clock also insures its future normal function to generate on-time rhythmic APs via surface membrane ignitions by its partner, the Ca²⁺ clock.

The crucial role of phosphorylation of Ca²⁺ cycling and membrane proteins in subsystem clock functions and coupling

The voltage- time- Ca²⁺ - interactions in Fig 1A are modulated by phosphorylation of surface membrane and SR proteins by both PKA and CaMKII (Fig.1B). This protein phosphorylation is required for normal coupled-clock basal function, because spontaneous AP firing ceases when either kinase is inhibited^18,49 (see below for further details).

Phosphorylation-dependent regulation of intracellular Ca²⁺ cycling and surface membrane proteins of the coupled-clock system is complex, with numerous functional redundancies (Fig.1B). Ca²⁺ ion begets protein kinase activation, and the latter leads to both an increase in Ca²⁺ influx, via modulation of surface membrane molecular functions, and acceleration of SR Ca²⁺ cycling, via modulation of SR Ca²⁺ cycling proteins. SANC have a high constitutive activation of adenylyl cyclase (AC) that results in a high level of basal cAMP¹⁸. Although SANC, like ventricular myocytes, express high levels of Ca²⁺-inhibited AC types 5 and 6⁵⁰, the discoveries of Ca²⁺-activated AC types, i.e. AC1 and AC8, in rabbit and guinea-pig SANC^48,51,52, and localization of the basal Ca²⁺-activated AC activity within lipid raft microdomains⁵², link Ca²⁺ to localized cAMP production (Fig.1B). Ca²⁺ binds to calmodulin to activate AC, leading to a high basal level of cAMP-mediated, PKA-dependent phosphorylation of surface membrane and intracellular proteins involved in cell Ca²⁺ balance and SR Ca²⁺ cycling^18,52 (Fig.1B).

Basal PKA-dependent phospholamban phosphorylation modulates kinetics of SR Ca²⁺ pumping, and phosphorylation of RyRs alters threshold of spontaneous activation of RyRs by Ca²⁺ within the SR. PKA-dependent mechanisms (Fig.1B, red; Online Table I) also regulate function of surface electrogenic proteins (Fig.1B, blue; Online Table I) and couple M and Ca²⁺ clocks. PKA is targeted to the L-type Ca²⁺ channel through the membrane associated anchoring protein, AKAP15/18^53,54. In rabbit SANC, there appears to be a high basal PKA-dependent phosphorylaton of L-type Ca²⁺ channels, as a specific PKA inhibitor peptide, PKI, suppresses I_CaL by ~80%⁵⁵. I_Ks is also modulated by cAMP-mediated, PKA-dependent phosphorylation⁵⁶ (Fig.1B). Augmented Ca²⁺ influx and accelerated Ca²⁺ cycling via PKA-dependent protein phosphorylation enhances basal CaMKII activity⁵⁷ which further promotes Ca²⁺ influx and intracellular Ca²⁺ cycling. Key roles of Ca²⁺-CaMKII-dependent phosphorylation on surface membrane proteins in SANC are the facilitation of I_CaL amplitude⁵⁸ and the acceleration of the removal of I_CaL inactivation⁴⁹. Ca²⁺-CaMKII dependent phosphorylation of SERCA stimulates Ca²⁺ pumping⁵⁹, and phosphorylation of RyR modulates Ca²⁺ release characteristics. In isolated rabbit SANC activated CaMKII is localized beneath the cell membrane, whereas the total CaMKII is uniformly present throughout the cell⁴⁹(Fig.2A). This restricted localization of active CaMKII to the subsarcolemmal space of SANC is consistent with the idea that CaMKII targets sarcolemmal and subsarcolemmal compartments, and that CaMKII activity is likely regulated by local Ca²⁺ gradients in submembrane microdomains^49,60. Note that unlike PKA or CaMKII-dependent protein phosphorylation, cAMP, per se, does not directly regulate SR Ca²⁺ cycling, but it indirectly does so via regulation of PKA-dependent phosphorylation of SR and surface membrane proteins.

This “feed-forward” Ca²⁺ signaling (Fig.1B), i.e., Ca²⁺ release begets Ca²⁺ release via both Ca²⁺-activated AC-cAMP-dependent PKA activity and CaMKII activity of both surface membrane and SR Ca²⁺ cycling proteins, is kept in check by factors that regulate Ca²⁺ and kinase activates, so that, basal state Ca²⁺ influx and Ca²⁺ cycling kinetics remain stable. High constitutive basal phosphodiesterase (PDE) activity within SANC²⁶, which degrades cAMP, is one such control check point (Fig.1B). Indeed, inhibition of basal PDE within SANC markedly elevates cAMP-mediated, PKA-dependent phosphorylation of SR proteins (c.f. Fig.7A) and surface membrane proteins (that generate I_CaL and I_K) (Fig.1B), resulting in an acceleration the spontaneous AP firing rate of rabbit SANC²⁶. Phosphoprotein phosphatase activity is likely another control point (Fig.1B)⁶¹.

Figure 7. Experimental perspectives on how the AP cycle length relates to the LCR period via phosphorylation-dependent mechanisms.

A. The relative effects of a PKA inhibitor PKI, carbachol (CCh), PDE inhibition (IBMX, milrinone) and isoproterenol (ISO) to alter the LCR period are linked to their effects to alter phospholamban (PLB) phosphorylation. The dashed line is the best fit least squares logarithmic function through the points: Y= −52.01 ln(X) + 107.11. B. The relative effects of PKI, CCh, PDE inhibition and ISO to alter the spontaneous cycle length over a wide range are linked to their effects on the LCR period. The best fit least squares linear functions (dashed line) through the points is: cycle length = 0.89 LCR period + 11.43 msec.

How the coupled pacemaker clock keeps time

The delay between the onset of the global SR Ca²⁺ depletion triggered by an AP (via CICR) and the spontaneous emergence of an LCR during the subsequent DD is the LCR period (Fig.3A). The LCR period is the master integrated function of coupled-clock system, the LCR period determines the “ticking speed” of the coupled-clock SYSTEM. The LCR period does not report Ca²⁺ clock function, per se, but reports the function of the coupled-clock system, because of intimate interactions of the electrogenic sarcolemmal molecules with the intracellular Ca²⁺ cycling apparatus described above and depicted in Fig.1B. For example, the Ca²⁺ available for SR pumping, which is regulated by PKA and CaMKII dependent phosphorylation of SR Ca²⁺ cycling proteins (Fig.1), is critically dependent on beat to beat Ca²⁺ influx via L-type Ca²⁺ channels. While an occurrence of an AP-induced global SR Ca²⁺ depletion causes the spontaneous LCRs to stop, the Ca²⁺ clock does not stop: after being synchronized in the Ca²⁺ depleted state, it continues to measure time from the onset of its Ca²⁺ depletion and when threshold conditions for spontaneous Ca²⁺ release are achieved begins to generate LCRs again. Thus, the LCR period is determined by the duration of this restitution process.

The schematic in Figure 6A depicts the concept that the restitution process that determines the LCR period is regulated (i) by the kinetics of SR Ca²⁺ cycling, i.e., by the rate of Ca²⁺ pumping into the SR, and (ii) the threshold of SR Ca²⁺ load required for spontaneous RyR activation. Ca²⁺ and PKA- and CaMKII-dependent phosphorylation by controlling cell Ca²⁺ balance and SR Ca²⁺ cycling (Fig.1B) regulates the restitution process. This concept is supported by numerical model simulations of the SANC coupled-clock system¹⁶ (Fig.6B). Ca²⁺, cAMP-mediated, PKA-dependent and CaMKII-dependent phosphorylation (Fig.1B, red) are crucial nodes within the coupled-clock system because their joint action on Ca²⁺ and M clock proteins determines the LCR period in a given steady-state.

Figure 6. Conceptual and numerical modeling perspectives on how SR Ca²⁺ pumping and the cycle length relate to the LCR period.

A. Schematic illustration of the concept that the rate of SR Ca²⁺ refilling and Ca²⁺ release threshold determine the LCR period, and the timing of the LCR induced diastolic depolarization (Modified from¹⁰⁵). B. A novel numerical SANC model¹⁶ of a dynamically integrated system of Ca²⁺ and membrane clocks predicts the wide range of pacemaker rate modulation via variations in SR Ca²⁺ pumping rate (color coded, 1 to 30 mmol/s), mimicking various degrees of PKA-dependent phospholamban phosphorylation. Shown are simulations of SR [Ca²⁺], SR Ca²⁺ release flux, I_NCX, and V_m.

Manipulation of the coupled-clock systems critical nodes alters its ticking speed and spontaneous AP firing rate

Disabling the major coupled-system nodes, i.e. the regulatory factors of the subsystem clocks and their coupling to each other (Fig.1B red) has marked effects on the basal LCR period and AP cycle length. As noted above, I_NCX-Ca²⁺ coupling (Fig.1) is clearly required for spontaneous AP firing (Fig.5C,D)^19,29. Since acute augmentation of I_NCX during DD requires an acute increase in subsarcolemmal [Ca²⁺], it would be expected that intracellular Ca²⁺ buffering, by disrupting numerous Ca²⁺ dependent functions in Fig.1B that regulates the LCR period, would lead to severe disregulation of the coupled-clock system. Indeed, buffering intracellular Ca²⁺ in rabbit SANC by BAPTA (but not the slower Ca²⁺ chelater, EGTA) severely impairs or blocks spontaneous AP firing, i.e. normal automaticity^20,49. Since subsarcolemmal Ca²⁺ regulation during DD depends upon SR Ca²⁺ pumping, it would be also expected that impairment of the SR Ca²⁺ pumping or release mechanisms would impair the LCR period and impair normal automaticity^11,29. As expected, SERCA2 inhibition, by cyclopiazonic acid, prolongs the LCR period and reduces the SANC beating rate^11,29.

The plant alkaloid ryanodine, which locks RyR’s in an open sub-conductance state, effecting depletion of SR Ca²⁺ content, impairs Ca²⁺ release, also prolongs the LCR period (and can abolish LCRs in concentration-dependent manner), flatters the late DD (Fig.5B), and results in impairment of normal automaticity (see Online Table II for data and references).

Although ryanodine significantly reduced the spontaneous beating rate in all 20 studies in isolated SA node tissue or isolated SANC or the intact heart reviewed in Online Table II, the extent of ryanodine-induced suppression varies from 12% to 100%, average ~40 % refer to Online Table II. The response to ryanodine varies with the [ryanodine], the time of ryanodine exposure, and species. A small suppression of the SAN beating rate by ryanodine in some studies may be due to an insufficient ryanodine concentration, or insufficient time of exposure. The kinetics of the ryanodine effect are especially important because a rapid application of ryanodine to isolated SANC or SAN tissue initially increases RyR Ca²⁺ release, and the spontaneous beating rate concomitantly increases; then as the SR becomes depleted with time, the spontaneous beating slows^5,62. This poses problems if sampling of the ryanodine effect on beating rate among different cells within or among studies is made at different relative times of the evolution of the ryanodine response. For example, a study that failed to detect any average change in the spontaneous beating of small SANC, thought to be isolated from the central area of the SA node⁶³, reported a large variation around the null average change in the beating rate of small SANC, indicating that in half of the cells ryanodine increased beating rate, whilst in the other half it decreased the beating rate⁶³. A subsequent study reported the ryanodine effect to suppress spontaneous AP firing was independent of cell size²⁰. Finally, a long exposure to a high ryanodine concentration that causes a severe SR Ca²⁺ depletion, can lead to a compensatory increase in Ca²⁺ influx⁶⁴.

Manipulation of PKA or CaMKII-dependent phosphorylation of coupled-clock proteins via basal PDE or PKA inhibition, have marked impacts on the LCR period and AP cycle length. But direct experimental elucidation of the specific role of any single coupling factor is not possible, because of the complex interactions within the coupled system (Fig.1B). Figure 7A illustrates the dependence of the LCR period in spontaneously beating SANC upon PKA-dependent phosphorylation of phospholamban across a broad range of phospholamban phosphorylation at serine 16. Shifts in the phosphorylation status of proteins other than phospholamban, e.g. RyR or of surface membrane molecules, e.g. I_CaL, I_K (Fig.1B) are also involved in regulation of the LCR period, as noted above. But phosphorylation of these proteins has not yet been measured directly. Thus, phospholamban phosphorylation presently serves as a general index of phosphorylation of proteins of the coupled pacemaker clock systems. Changes in phosphorylation status, reported by phospholamban phosphorylation, and changes in LCR period are closely correlated with changes in the spontaneous SANC cycle length (Fig 7B).

Simulations of a novel numerical model that predicts the LCR period of the coupled-clock system (LDCAE in Fig.6B) can isolate and explore coupling factor contributions^16,65. Model simulations support the concept that the LCR period, in its role as the master integrated function of numerous tightly coupled processes in SANC (Fig.1), regulates the pacemaker system “ticking speed” (Note the tight correlation between the timing of LDCAE emergence, the timing of DD acceleration and the cycle length in Fig.6B)^16,65.

Pacemaker rate modulation via G protein-coupled receptor (GPCR) signaling

In addition to robustness or “fail-safe”, stable basal operation, the heart’s pacemaker clock must tick over a wide range of frequencies that encompass the physiologic range of heart rates. This flexibility of the pacemaker clock’s “throttle” is achieved via modulation of the LCR period by GPCR signaling (reviews^13-15). G protein-coupled receptors modulate the coupled system of M and Ca²⁺ clocks (Fig.1, green) via same factors (Ca²⁺ and protein phosphorylation) that regulate the basal state clock function (Fig.1 red). Specifically, β adrenergic receptor (β-AR) stimulation further increases, and Cholinergic receptor (ChR) stimulation reduces basal levels of phosphorylation of coupled-clock proteins (Fig.7A), leading to an increase and a reduction, respectively, in LCR period. Note in Fig.7B, modulation of LCR period and cycle length by experimental maneuvers that interrupt basal PKA-dependent signaling and those effected by GPCR stimulation form a continuum. Novel numerical modeling simulations of coupled-clock function predict this continuum on the basis of phosphorylation-dependent gradations in the SR Ca²⁺ cycling rate (Fig.6B)¹⁶.

β-adrenergic receptor stimulation effects on the LCR period and AP cycle length

β-AR stimulation (or PDE inhibition) reduces the LCR period (Fig. 7A), shifting LCR occurrence to an earlier time during DD, i.e., a time occupied solely by early DD mechanisms during basal state beating¹⁸. In addition to the phase shift, the integrated LCR Ca²⁺ release signal mass also increases as RyR activation becomes more synchronized via local recruitment^25,26. This earlier and stronger Ca²⁺ release results in earlier and stronger I_NCX and DD acceleration, resulting in a reduction in cycle length. Thus pacemaker rate acceleration by β-AR stimulation is linked to both the early and late DD phases^27,66,67. The resultant cycle length modulation, effected by β-AR signaling is due to phosphorylation- dependent effects on both ion channels and SR Ca²⁺ cycling, as discussed above.

Numerous recent studies also have shown that in order to increase the beating rate, β-AR stimulation (or PDE inhibition) requires a link between β-AR-induced increases in PKA or CaMKII modulation of surface membrane molecules and SR Ca²⁺ cycling molecules within the coupled-clock system (Fig.8)^9,18,25. In rabbit SANC, when the function of intracellular RyRs is disabled by ryanodine, neither β-AR stimulation (nor PDE inhibition) augments I_NCX or accelerates the DD^25,27, or produces the expected increase in the spontaneous SANC firing rate (Fig.8A). In the presence of ryanodine there is an approximate three-fold reduction in the ability of stimulation of β-ARs with isoproterenol to accelerate the firing rate, and about a six-fold increase in the isoproterenol EC₅₀²⁵, even though the effect of these maneuvers to increase I_CaL amplitude remains intact^25,26 (Fig.8B). Thus, β-AR stimulation (or PDE inhibition) effects to augment the M clock function alone, via I_CaL augmentation (or I_K activation^26,56), are not sufficient to effect the normal increase in AP firing rate. In other words, SERCA2 Ca²⁺ pumping and Ca²⁺ release within the coupled-clock system is a required “switchboard” that links Ca²⁺ influx via M clock proteins in response to β-AR stimulation (or PDE inhibition) to an increase in the SANC firing rate. The specific link function of the switchboard is to generate an increased LCR signal mass, that occurs at an earlier time during DD. The requirement of intact RyR function within the coupled-clock system for β-AR stimulation-induced positive chronotropic effect has also been confirmed in both single SANC and intact SA node of numerous species (Fig. 8) (the results of 8 studies are summarized in Online Table VA and VB, respectively).

Figure 8. β-AR stimulation or PDE-inhibition induced increase in spontaneous beating rate of isolated rabbit SANC in vitro or of isolated or intact canine SA node requires intact RyRs function.

A. Changes of rabbit SANC firing rate by β-AR stimulation or PDE inhibition in the presence and absence of ryanodine. B. The increase in L-type Ca²⁺ current by β-AR stimulation or PDE inhibition is not affected when RyR Ca²⁺ release in rabbit SANC is inhibited by ryanodine. C. Changes in the canine heart rate (percent from baseline) induced by different pharmacological interventions. The gray bars show the changes during 3 μmol/L ZD 7288 (I_f channel inhibitor, ZD), 3 μmol/L ryanodine (Ryd), and 10 μmol/L ryanodine plus 200 nmol/L thapsigargin (Ryd+Thap) infusion without isoproterenol (ISO), and black bars during 1 μmol/L ISO infusion (with permission from²³). D. The increase of the heart rate of intact canines in response to sequential ISO infusions (two control infusions followed by a third graded-dose infusion in the presence of ryanodine) delivered by microdialysis into the SA nodal artery: ISO produces a brisk, reproducible dose-dependent tachycardia, and sequential dose responses in control are superimposable. Disabling of RyRs with ryanodine (5 nmol/min) following the second control ISO infusion dramatically reduces the effects of the next ISO infusion to increase the in vivo heart rate. Local nodal dialysis with ryanodine prior to the third ISO infusion reduced resting heart rate by 12% (from 108 ± 5 to 96 ± 6 bpm; P < 0.05), and suppressed the subsequent response to ISO by 75% (from¹⁸).

In the presence of a sub-maximal [ryanodine] the ability of β-AR stimulation to increase the spontaneous SANC beating rate in most species is strikingly suppressed (up to 100%), Online Table VA. In contrast, in the presence of a very high concentration of ryanodine (30 μmol/L), i.e. a concentration that exceeds that used in the most studies in conjunction with β-AR stimulation (Online Table V), a high SANC firing rate is observed during β-AR stimulation^68,69. This may be attributable to the idea that high [ryanodine] locks RyRs in a closed state, preventing SR Ca²⁺ depletion⁶⁸. An additional issue that complicates the interpretation of a failure of ryanodine to suppress the isoproterenol induced increase in AP firing rate in⁶⁹ is that the AP cycle length prior to β-AR stimulation was not reported.

Importantly, the chronotropic response to β-AR stimulation in vivo, requires intact SR Ca²⁺ cycling within the coupled-clock system: in open chest dogs, a robust, dose-dependent increase in the spontaneous beating rate effected by microdialysis of the SA node artery with isoproterenol is virtually abolished when RyR is inhibited by ryanodine (Fig.8D, Online Table VB).

In spite of the robust evidence detailed above, an alternative view of the mechanism of β-AR-modulated increase in AP firing rate denies the requirement for a change in PKA-dependent Ca²⁺ regulation of the complex coupled-clock for a normal chronotropic response to β-AR stimulation. This alternative view point attributes β-AR-induced chronotropy to a direct cAMP-dependent facilitation of I_f channel opening, via a cAMP-dependent shift of the voltage dependence of its activation curve^70,71. Numerous studies, however, employing pharmacologic (Online Table III), or genetic (Online Figure I) manipulations have demonstrated that when I_f current is suppressed or deleted, the resting heart rate or cell beating rate is modestly decreased (~16% in SA node or isolated heart and ~18% in SANC, Online Table III), and a full or major part of the positive chronotropic response to β-AR stimulation still remains intact (Online Table IV and Online Figure I, c.f.^15,67 for review). While the I_f current does not appear to have a major effect to accelerate the beating rate of SANC during β-AR stimulation^15,67, it may have a key role in stabilizing the pacemaker rhythm, particularly during transition states^16,67,72-75.

Cholinergic receptor stimulation effects on the LCR period and AP cycle length

Two recent studies in rabbit SANC^24,61 have demonstrated that ChR stimulation suppresses LCR’s (or LDCAE), and that LCR or LDCAE suppression contributes substantially (35%-75%) to the slowing of the beating rate. In isolated rabbit SANC, the ChR stimulation-induced beating rate reduction is entirely dependent on G_i activation⁶¹ (Fig.1 green; Online Table I). At low [carbachol], I_KACh activation is not evident, and the prolongation of the LCR period and cycle are attributable to a suppression of cAMP-mediated, PKA-dependent Ca²⁺ signaling: a 50% reduction in phospholamban phosphorylation is accompanied by ~60% prolongation of LCR period (Fig.7A) that is linked to prolongation in the cycle length (Fig.7B). Phosphatase inhibition reverses the effect of carbachol on SANC LCR period and cycle length⁶¹. At higher [carbachol] I_KACh activation underlies a more pronounced reduction in the beating rate⁶¹.

It had previously been thought that ChR stimulation induced chronotropic effect (similar to that of β-AR stimulation) is due to an impact on sarcolemmal membrane ion channels, specifically I_KACh, I_f, and I_CaL^70,76-79. In rabbit SANC under voltage clamp, a ChR stimulation-induced suppression of I_CaL in the absence of prior β-AR stimulation^55,61,80 has been ascribed to a reduction of basal PKA-dependent L-type Ca²⁺ channel phosphorylation⁵⁵. (Note that such an effect would be expected to have a marked effect on intacellular Ca²⁺ cycling, vide supra.) I_f suppression has specifically been considered to be an important mechanism in the ChR stimulation-induced decrease of the spontaneous SA node beating rate, particularly at low ChR agonist concentrations⁷⁶ (but see61,79,81). Studies performed either in the isolated Langendorff hearts or in isolated SANC using tertiapin Q to block I_KACh^24,61,82,83 provide different perspectives on the contribution of I_KACh and I_f to the bradycardic response to carbachol. In one study in isolated rabbit SANC, I_f blockade did not affect the beating rate reduction at any level of stimulation with carbachol⁶¹; in another study the combined contribution of I_f and of I_K,ACh currents has been estimated to be about 25% of ACh-induced decrease in SANC beating rate²⁴.

To summarize this section, graded changes in the steady-state phosphorylation status of proteins that regulate Ca²⁺ within the coupled-clock system lead to gradations in the LCR period by GPCR signaling (Fig.7A), cause graded changes in the timing of the onset and amplitude of the late DD exponential increase, and thus cause concomitant gradations of the steady-state AP cycle length^18,26,27,61 (Fig.7B). It is necessary to emphasize, however, that modulation of the LCR period and SANC beating rate by coupled-clock functions in response to GPCR stimulation depends not only on the direct modulation of protein phosphorylation, per se, that is produced by GPCR stimulation (Fig.1), but also depends upon the secondary modulation via rate and rhythm of AP occurrence that results from GPCR stimulation: changes in the AP firing rate or AP shape, per se, due to any cause, indirectly effect changes in intracellular Ca²⁺cycling characteristics via an effect on steady state cell Ca²⁺ levels, i.e., via the Bowditch treppe effect⁸⁴.

A coupled-clock system also regulates the automaticity of embryonic heart

Ca²⁺ cycling proteins (RyR2, SERCA2a, NCX) are abundantly expressed very early in development of cardiomyocytes and embryonic stem cell-derived cardiocytes (ESC’s). SR Ca²⁺ cycling is crucially important in the developmental regulation of Ca²⁺ transients and contraction in these cells⁸⁵, which are presently considered to be a prime substrate for engineering biological pacemakers⁸⁶. Numerous studies indicate that spontaneous intracellular Ca²⁺ releases via RyRs drive automaticity (likely via NCX) and contraction of embryonic cardiomyocytes^87,88 and ESC’s^85,89-94, In addition to RyR, Ca²⁺ release channels activated by IP3, are also involved in spontaneous Ca²⁺ release in embryonic cardiomyocytes and ESCs^87,91,92. Genetic manipulation of Ca²⁺ cycling proteins (see below), or pharmacologic inhibition of Ca²⁺ cycling (ryanodine, thapsigargin, cyclopiazonic acid, BAPTA-AM), or of NCX function, substantially slows or halts automaticity of embryonic cardiomyocytes^87,88 and ESCs⁹⁴.

Genetic manipulation of RyR, NCX, CaMKII or ankyrin B

Genetic manipulation of RyR or NCX, like pharmacologic maneuvers discussed above, also markedly impairs normal automaticity and markedly impairs the chronotropic response to β-AR stimulation. RyR2 knockout in mice results in embryonic lethality, which was initially interpreted to result from generally perturbed Ca²⁺ homeostasis, due to a Ca²⁺ overloaded SR, that resulted in contractile impairment and cell death⁹⁵. A more specific explanation of this lethality was later gleaned from studies of mouse ESCs⁹⁰, in which RyR2 knock-out not only causes a failure of spontaneous LCR’s, markedly suppresses basal spontaneous cell beating and the response to β-AR stimulation, but also leads to a marked depression of the obligatory developmental increases in heart rate and cardiac output required to support continued cardiac embryonic differentiation⁹⁰. Recent data also demonstrate that RyR mutations can initiate severe abnormalities in human cardiac pacemaker function: genomic deletion of RyR2 exon-3 leads to sinoatrial and atrioventricular node dysfunctions, atrial fibrillation and atrial standstill⁹⁶.

The importance of NCX in automaticity of embryonic cardiomyocytes is supported by observations that NCX knockout mice are embryonic lethals, without evidence that the heart ever having a single beat^97,98. Interestingly, mice with NCX knockout only in ventricular myocytes (with NCX in SANC remaining intact) live to adulthood with only modestly reduced cardiac function⁹⁹

CaMKII has been genetically manipulated, via conditional expression of CaMKII peptide inhibitor, AC3-I. These mice have the same basal heart beating rate as wild type mice²³, but during stress exhibit significantly less heart rate acceleration than their wild type counterparts. Ryanodine significantly and equivalently reduces the beating rate in SANC isolated from either wild type or AC3-I mice. In the AC3-I mouse SANC, the positive chronotropic effect of β-AR stimulation are severely reduced. Thus, the β-AR chronortopic response in mouse SANC requires CaMKII-dependent activation and its concomitant effects to increase SR Ca²⁺ load and to increase diastolic RyR Ca²⁺ release in the context of the coupled-clock system²². (Of note, the results of this study also strongly suggest that the reduced response to β-AR stimulation in AC3-I mice is independent of I_f, since this current, as well as its response to isoproterenol, is fully preserved²².)

Reduced expression of AnkB in mice (heterozygous AnkB mice) results in SA node dysfunction, severe bradycardia and heart rate variability, associated with a substantial decrease in I_NCX and I_CaL and abnormal Ca²⁺ cycling in SANC, while I_CaT and I_f remain unchanged¹⁰⁰.

In contrast to knockout of RyR2, NCX or CaMKII function, or Ankyrin-B, or mutations in human RyRs, genetic manipulation in mice including HCN2 or HCN4 (general or cardiac-specific SA node and AV node) knock outs, or inhibition of cAMP binding to HCN4 subunits, have minimal or no effects on resting heart rate, or on increases in heart rate during exercise, or chronotropic effect of β-AR stimulation (Online Figure I). Conditional, global deletion of HCN4 isoform in adult mice (Online Figure IA) results in a significantly augmented response to ChR stimulation by carbachol⁷³. But when such conditional HCN4 deletion only occurred within the SA and atrio-ventricular nodes (Online Figure IB), carbachol markedly and equally decreased the heart rate in both genotypes. This strongly suggests that a full response to ChR stimulation does not require I_f⁷⁴.

Summary

Over 30 years ago, Rapp and Berridge¹⁰¹, in a theoretical treatise on biological rhythms, in the absence of experimental data, made an intriguing prediction about the heart’s rhythm:

“The heart is a complicated organ containing different regions of specialized tissue. Unfortunately, the region of greatest interest form the point of view of oscillations, the sino-atrial node, is least accessible experimentally. Accordingly, anything said about the heart beat must be stated circumspectly. However, the case for a central role for calcium and cyclic AMP is now very strong, indeed. We wish to carry the argument one stage further and suggest that oscillation in cyclic AMP and calcium constitutes the basic driving signal for the initiation of heart beat. Should this theory be correct, it would resolve what must be one of the oldest and most intriguing scientific questions.”

The material reviewed herein indicates that the Rapp-Berridge prediction is accurate, in large measure. Prolific experimental evidence, supported by novel numerical modeling, permits the conclusion that SANC normal automaticity is regulated by constitutive Ca²⁺ activation of AC, and that the resultant increase in cAMP activates PKA- , and that PKA/CaMKII-dependent protein phosphorylation govern a complex clock system comprised of intracellular SR and surface membrane molecules that regulate intracellular Ca²⁺ cycling (Fig.1B). This coupled pacemaker clock system is robust, because the same factors that regulate SR Ca²⁺ cycling and sarcolemmal molecular function, also couple SR Ca²⁺ cycling to sarcolemmal protein function to effect normal basal automaticity. G protein-coupled receptor signaling insures pacemaker flexibility, effecting rate regulation by impacting on these very same factors that regulate coupled-clock Ca²⁺ cycling to guarantee basal state pacemaker stability and robustness. Thus, intimately intertwined properties of robustness and flexibility of the heart’s coupled pacemaker system insure stable heart rates of a wide range that is required for peaceful rest or flight or fight.

Where do we go from here?

It has become clear (to us) that normal SANC function results from a complex integration of its component subsystem clocks. In this context, issues, such as, “what is the most important mechanism that underlies pacemaker cell function”, or references to a specific ion channel as “THE PACEMAKER CHANNEL”^2,102 cease to have substance. Still, numerous experimental gaps exist with respect to a complete understanding of the molecular functions of the individual components of this complex clock system, and in the complete conceptualization of how these components interact with each other. In our opinion, for this field to move forward, novel data derived from reductionist approaches must be integrated, via implementation of multifaceted protocols, and via numerical modeling of a complex pacemaker clock systems such as that as illustrated in Fig.1B and summarized in Online Table I. A few specific items for a future research agenda that relates to specific subsystem components and their interactions are listed below.

• What are the molecular identities of I_bNa and I_st? Is it NCX?

• Do TRPC channels exist in SANC of other species besides mouse³⁶; and, if so, what is their function?

• Are low-voltage activation L-type Ca²⁺ channels functional in SANC of larger mammals?

• What is the molecular mechanism of spontaneous RyR Ca²⁺ release during DD? How does local control of RyR’s work in SANC? How do stochastic RyR openings become partially synchronized to generate periodic LCRs?

• Are there “orphan” RyR’s in SANC, as in ventricular cells?

• Are there functional RyR3′s in SANC?

• What is the precise spatial arrangement of L-type Ca²⁺ channels, RyRs, and NCX molecules across the ~12 nm subsarcolemmal gap? What is the precise interplay of the different system components, i.e. Ca²⁺ release via RyRs, Ca²⁺ influx via Ca²⁺ channels and Ca²⁺ removal via NCX to regulate [Ca²⁺] in the subsarcolemmal space during very late DD and the onset of the rapid AP upstroke,

• Do periodic cAMP oscillations occur within SANC, and is their steady-sate period the same as the LCR period.

• Can specific actions of phospholamban phosphorylation to modulate SR Ca²⁺ pumping and SR Ca²⁺ load be distinguished from phosphorylation dependence of RyR-mediated Ca²⁺ release?

• How do Ca²⁺-calmodulin-activated and Ca²⁺-inhibited AC types interact in cAMP generation, e.g. with respect to GPCR receptor stimulation? Specifically, how does PKA-signaling interact with Ca²⁺-CaMKII signaling (in time and space)?

• In addition to activating PKA in SANC, does cAMP activate Exchange Proteins directly Activated by Cyclic AMP (EPAC), as in some cell types?

• To what extent does basal phosphatase activity regulate basal PKA and CaMKII-dependent protein phosphorylation in SANC?

• What is the geometrical arrangement of micro-scaffold complexes within SANC that link function of specific subtypes of AC’s, PDE’s, PKA, CaMKII, and phosphatases? How are these micro-scaffolds compartmentalized within SANC.

• Experiments that define transient state kinetics of cAMP-, PKA-, CamKII- signaling in spontaneously firing SANC, are required for more definitive systems modeling of biochemistry kinetics to match V_m and Ca²⁺ transient state kinetics of the AP firing rate.

• In addition to GPCR, how do naturally occurring biochemical/biophysical stimuli that are external to the cell, i.e. mechanical deformation (stretch) and temperature, regulate SANC pacemaker function?

• How is the Na⁺ equilibrium potential (E_Na) regulated in SANC? What are the roles of Na⁺/K⁺ ATPase and Na⁺/H⁺ exchanger? What is the phosphorylation status of NCX, or of Na⁺/K⁺ ATPase, and its accessory proteins?

• Mitochondria cycle both Ca²⁺ and Na⁺: Is there a mitochondrial Ca²⁺ clock? How does it interact with the SR Ca²⁺ clock?

• Ditto for myofilament Ca²⁺ clock.

• Since SANC have a prominent mitochondrial density, but a sparse myofilament density, why are ATP production and consumption so high? Is this required for continual formation and degradation of cAMP by constitutively active AC’s and PDE’s?

• Will cultured rabbit SANC models enable genetic manipulation of coupled-clock system proteins to progress beyond the mouse?

• Are there resident precursor cells within the SA node cell system to replenish damaged cells?

• Will those who aspire to design biologic pacemakers begin to address the complex pacemaker clock system (Fig.1 and Online Table.I)?

• Given the wide range of literature values for parameters of a given ion current and Ca²⁺ cycling, don’t numerical models need to explore the entire range of all parameters, including those of local late diastolic subsarcolemmal Ca²⁺ releases, by performing extensive parametric sensitivity analyses?

• Can stochastic, locally propagating LCRs be numerically modeled?

• Can theoretical modeling of the intact SA node function incorporate the concept of the interacting Ca²⁺ and M clocks within individual SANC comprising the node?

List of non-standard abbreviations and acronyms

SANC

sinoatrial nodal cell

LCR

subsarcolemmal Local Ca²⁺ Releases

SR

sarcoplasmic reticulum

‘Ca²⁺ clock’

SR generating rhythmic, spontaneous Ca²⁺ releases

Surface “membrane clock” or ‘M clock’

the ensemble of sarcolemmal ion currents generating spontaneous action potentials

DD

diastolic depolarization.