Cardiac Impulse Is Initiated by a Coupled System of Membrane Ion Channels and Ca2+ Cycling Proteins

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Cardiac Impulse Is Initiated by a Coupled System of Membrane Ion Channels and Ca²⁺ Cycling Proteins

Edward G. Lakatta, Yael Yaniv and Victor A. Maltsev

Chapter Outline

Different types of heart cells (i.e., pacemaker cells within the sinoatrial node [SAN] and contractile myocytes within myocardium) determine how fast and strong, the heart beats. The heart’s pacemaker cells normally generate spontaneous rhythmic changes in their membrane potential, thereby producing almost perfectly periodic spontaneous action potential (APs; i.e., normal automaticity). Pacemaker cells within the heart having “clocks” with the briefest rhythmic periods “capture” or trigger other excitable cells. SAN cells (SANC) are the dominant cardiac pacemaker cells, because they exhibit shorter periods between spontaneous APs than do atrioventricular nodal cells or His-Purkinje cells. Thus, SANC normally initiate the cardiac impulse by generating spontaneous APs that are conducted to the ventricle and entrain the duty cycle of ventricular myocytes.

The essence of cardiac pacemaker function is diastolic depolarization (DD) that is absent in ventricular myocytes. DD represents a slow spontaneous decrease in membrane polarization until it reaches the membrane excitation threshold, culminating in generation of an AP. SANC robustness (i.e., “fail safe” properties conserved during evolution of the animal kingdom) and flexibility (the ability to react to demands for faster or slower AP firing rate) result from the timely integration of signaling events at multiple levels within the SAN, including subcellular, cellular surface membrane, and tissue architecture. The interactive network of mechanisms intrinsic to SANC must also interpret and react to signals arising extrinsic to the cell (e.g., stretch, electrotonic impulses, neurotransmitter or hormonal stimulation of surface membrane receptors).

The present perspective views SANC, per se, as a system comprising several levels of complexity and integrated components. Sarcolemmal ion currents (Figure 25-1 and Table 25-1) have been extensively studied in the past (see review¹), and the ensemble of these currents re-created in silico from experimental voltage-clamp data can generate spontaneous APs.² The subsystem of sarcolemmal molecules forming a voltage membrane oscillator was conceptualized as a membrane clock³ (M-clock, for short) and has been the predominant feature in at least 12 SANC numerical models.² During rhythmic spontaneous AP firing by SANC, the M-clock is dynamically coupled to Ca²⁺ cycling (see Figure 25-1, gray area) via multiple voltage-, Ca²⁺-, cyclic adenosine monophosphate (cAMP)-, and phosphorylation-dependent mechanisms (coupling factors).⁴ SANC sarcoplasmic reticulum (SR), however, in isolation from the M-clock can generate its own spontaneous local (submembrane) rhythmic Ca²⁺ signals. Thus, similar to the M-clock, the SR is intrinsically also an almost perfectly periodic oscillator that can be envisioned as a Ca²⁺-clock.³ During spontaneous AP firing by SANC, the M- and Ca²⁺-clocks do not exist as separate entities: They mutually entrain each other via aforementioned coupling factors. These coupled oscillators that differ in nature thus represent heterogeneous redundancy within SANC that confers a physiological robust, yet extremely flexible, united pacemaker cell system.^4,5

Table 25-1

Major Ion Currents Reported in SA Node and SANC of Various Species

Figure 25-1 Schematic illustration of interactions of identified molecules comprising the fully coupled-pacemaker cell clock. Note that common regulatory factors (purple large lettering) govern the function of both the SR Ca²⁺-clock (gray intracellular area) and the M-clock (light-blue cell membrane area with blue labels depicting electrogenic proteins). These common factors act as nodes within the system to couple the function of the activities of both clocks. The system is balanced as illustrated by traffic light–like colors: Green arrows designate signaling driving AP firing, but red arrows show suppression, maintaining a given steady state level of cAMP and protein phosphorylation. G protein–coupled receptors (green and red shapes within the membrane) modulate both Ca²⁺-clock and M-clock function via those same crucial signaling nodes of the system. (Modified from Lakatta EG, Maltsev VA, Vinogradova TM: A coupled SYSTEM of intracellular Ca²⁺ clocks and surface membrane voltage clocks controls the timekeeping mechanism of the heart’s pacemaker. Circ Res 106:659–673, 2010.)

Realization of Importance of Ca²⁺ Signaling for Cardiac Pacemaker Function

In the late 1970s to the early 1980s, as the importance of an intracellular Ca²⁺ transient (as the trigger for ventricular myocyte contraction) came into focus, the idea that an intracellular Ca²⁺ oscillator could also drive membrane excitations in Purkinje fibers was suggested.⁶ But because oscillatory current in Purkinje fibers became manifest only in a Ca²⁺ overload state, such current fluctuations were interpreted in the context of “abnormal automaticity” (i.e., they were attributed to a disturbance in normal cardiac function⁷).

In the late 1980s, afterdepolarizations and aftercontractions were recorded in Purkinje fibers under normal Ca²⁺ loading conditions,⁸ and these fibers demonstrated localized, spontaneous myofilament motion caused by spontaneous local Ca²⁺ oscillations⁹ in the absence of Ca²⁺ overload. A cytosolic Ca²⁺ transient is evoked by each spontaneous SANC AP (see Figure 25-2, D), and that Ca²⁺ influx via L-type Ca²⁺ channels (LCCh) (see Figure 25-1) affects SR Ca²⁺ loading. Chelation of intracellular Ca²⁺ in SANC^10–12 markedly slowed or abolished spontaneous AP firing. The importance of SR Ca²⁺ release and Na⁺/Ca²⁺ exchanger (NCX) current (I_NCX) for pacemaker cell pacemaker function was also demonstrated directly by effects of ryanodine on Ca²⁺ cycling.^13,14 Ryanodine (by disabling the ryanodine receptor [RyR] and the SR release channel [Figure 25-2], and by depleting SR Ca²⁺ content] had a profound negative chronotropic effect on the automaticity of subsidiary atrial pacemakers¹⁵ and in SANC.^13,14 Additional voltage-clamp experiments in perforated patch configuration in isolated cat atrial latent pacemaker cells demonstrated the occurrence of an inward current during late DD that was sensitive to ryanodine, and presumably was linked to I_NCX activated by SR Ca²⁺ release.¹⁶

Figure 25-2 A, Line-scan image of LCRs (white arrowheads) with superimposed spontaneous APs recorded in rabbit SANC. B, Upper panel, LCRs imaged by confocal microscopy; lower panel, Temporal average of LCRs creates ensemble LCR signal or late diastolic Ca²⁺ elevation (LDCaE) that precedes AP-induced Ca²⁺ transient.²⁹ C, LDCaE in single SANC of rabbit⁵⁷ and guinea pig.²⁰ D, Novel numerical model predicts “yin-yang” types of interactions within the coupled-clock system of SANC (see text for details). D, Diastolic depolarization; SR, sarcoplasmic reticulum. (Modified from Maltsev VA, Lakatta EG: Synergism of coupled subsarcolemmal Ca²⁺ clocks and sarcolemmal voltage clocks confers robust and flexible pacemaker function in a novel pacemaker cell model. Am J Physiol Heart Circ Physiol 296:H594–H615, 2009.)

Ca²⁺-Clocks in Pacemaker Cells

Confocal imaging of Ca²⁺ in mammalian SANC and atrial subsidiary pacemaker cells combined with noninvasive perforated patch-clamp electrophysiology 17,18 and imaging of toad sinus venosus cells¹⁹ has documented the occurrence of spontaneous, localized, diastolic Ca²⁺ release in pacemaker cells in the absence of Ca²⁺ overload. These local Ca²⁺ releases (LCRs) during DD are initiated beneath the cell surface membrane via spontaneous activation of RyR (Figure 25-2A,B). SANC exhibit robust SERCA2 and RyR immunolabeling,^20,21 and although SERCA2 immunolabeling in SANC occurs diffusely throughout the cytoplasm and in perinuclear area, RyR immunolabeling is most intense in the subsarcolemmal space.^20,21 LCRs appear as 4- to 10-µm Ca²⁺ wavelets in confocal line-scan images of spontaneously firing rabbit SANC and emerge following dissipation of the global systolic transient effected by the previous AP; they crescendo during DD, peaking during late DD, as they merge into the global cytosolic Ca²⁺ transient triggered by the next AP (see Figure 25-2A).^18,22 LCRs and the ensemble LCR signal (i.e., integral of all LCRs), that is, late diastolic Ca²⁺ elevations (LDCaE), have now been documented in numerous species (see review⁴) (see Figure 25-2, B-D).

LCR occurrence does not require triggering by depolarization of the surface membrane: Persistent rhythmic oscillatory membrane currents can be activated by rhythmic LCRs during voltage-clamp (at potentials that prevent cell Ca²⁺ depletion, e.g., −10 mV). Both persistent LCRs and the currents activate simultaneous fluctuations of the same frequency,22,23 and both are abolished by ryanodine. Sustained LCR activity is also observed in chemically “skinned” SANC (i.e., having a detergent-permeabilized cell surface membrane) bathed in a physiological [Ca²⁺] of 100 nM.^22,23 Because LCRs are generated as rhythmic events at rates of 1 to 5 Hz (i.e., encompassing those of spontaneous AP firing in SANC), SR was conceptualized as an intracellular “Ca²⁺-clock.”³ The ability of SANC SR to generate sustained intracellular Ca²⁺ oscillations under normal physiological conditions within this broad frequency range has also been demonstrated in numerical model simulations that have embraced the occurrence of experimentally determined LCRs.⁵

RyR2 knockout not only causes failure of spontaneous Ca²⁺ releases in embryonic cardiac cells and suppresses spontaneous cell beating, it also leads to a marked depression of the obligatory developmental increase in heart rate and cardiac output that, in the absence of knockout, support continued cardiac embryonic differentiation.²⁴ As in adult SANC, a crucial regulatory role of RyR2-mediated Ca²⁺ releases in pacemaker function is demonstrated in RyR2 knockout cells, as β-adrenergic receptor (β-AR) stimulation is ineffective in these cells.²⁵ An essential role of RyR2 in pacemaker function has been recently demonstrated in tissue-specific RyR2 knockout mice with acute ≈50% loss of RyR2 protein in the heart, but not in other tissues.²⁶ The RYR2 loss-of-function causes bradycardia and arrhythmia. Furthermore, cardiac RyR2 knockout mice exhibit some functional and structural hallmarks of heart failure, including sudden cardiac death.

Crosstalk of Ryanodine Receptor and Na⁺/ Ca²⁺ Exchanger to Transfer Intracellular Ca²⁺ Signals to M-Clock

NCX is not an ion channel, and its amplitude almost instantly follows changes in membrane potential or intracellular [Ca²⁺]. NCX function is both voltage- and Ca²⁺-dependent. When intracellular [Ca²⁺] is high as the result of AP-induced Ca²⁺ transient, AP repolarization activates NCX forward mode, generating inward current I_NCX by exchanging one Ca²⁺ (going out) to 3 Na⁺ (going into the cell). In contrast to hyperpolarization-activated “funny” current, I_f (see Table 25-1), I_NCX is activated earlier, as it almost instantly follows membrane hyperpolarization (see Figure 25-2, D). Furthermore while the non-selective I_f is decreasing and reversing during later DD, I_NCX is substantially increasing further by waxing diastolic Ca²⁺ release from the SR (see Figure 25-1 and Figure 25-2, D, described later in detail). Thus, I_NCX has been realized as a major transduction mechanism of intracellular Ca²⁺ signaling that crucially contributes to DD and normal automaticity of SANC. The NCX is one of the earliest functional genes that exist during early embryonic heart development,²⁷ and NCX1 is required for normal pacemaker activity. Atrial-specific NCX knockout mice that lack NCX1 but have intact I_f, and express NCX1 in ventricular myocytes,²⁸ live to adulthood, and exhibit a markedly reduced heart rate. However APs can be evoked under current-clamp conditions in SANC isolated from these mice, indicating that knockout cells retain electrical excitability.

At the resolution of the confocal microscope ²¹ NCX and RyRs, molecules appear co-localized across the ≈12-nm subsarcolemmal gap between RyRs on the SR and NCX molecules on the sarcolemma. The crosstalk of LCRs and NCX activates a forward mode NCX operation that generates local inward I_NCX currents, which produce miniature membrane voltage fluctuations.^18,29,30 The net late DD inward current initiated by the LCR ensemble (measured as ryanodine-sensitive current) in rabbit SANC varies from 0.3 pA/pF³¹ to 1.6 pA/pF,¹⁸ yielding a whole-cell I_NCX range from 9.6 to 51.2 pA for a 32-pF SANC (see Figure 25-2, D). Because an extremely small net ion current change (a few pA in rabbit SANC) has marked effects on membrane potential, an inward current of this magnitude is sufficient to broadly modulate the DD.^5,30,31 For comparison, the peak of I_f achieved during DD in SANC ranges from 0.02 to 0.23 pA/pF as predicted by 12 different numerical models.² LCR-activated inward I_NCX, together with other DD mechanisms (such as L-type Ca²⁺ current, I_CaL), imparts the exponentially rising phase to the DD^18,29,30 that initiates the subsequent AP (see Figure 25-2, A-C). Although voltage-activated L-type Ca²⁺ currents surely contribute to late DD, and L-type Ca²⁺ currents are required for AP initiation, failure to generate diastolic I_NCX signals results in pacemaker failure when I_CaL density is normal (e.g., when RyRs are disabled ryanodine or during short-term NCX blockade¹⁸). Thus available experimental and numerical modeling data clearly indicate an important role of Ca²⁺ cycling molecules and their “crosstalk” in normal physiological function.

The Essence of Pacemaker Function Is a Coupled Function of an Intracellular Ca²⁺-Clock and Membrane Ion Channel Clock

The LCR Period Reports the Functional State of the Coupled System (Not That of the Ca²⁺-Clock Per Se)

The LCR period is the delay between the AP-triggered global cytosolic Ca²⁺ transient and LCR emergence during DD (Figure 25-2, A). It reports the time at which AP ignition is prompted via I_NCX activation, and it closely predicts the AP cycle length in numerous experimental perturbations of SANC function (Figure 25-3, B). Over the wide range of conditions and widely varying AP firing rates from about 1 to 4 Hz, the relationship of LCR periods to the spontaneous cycle lengths of SANC is nearly identical, with a slope being close to 1 and an offset “ignition” time of 10 to 100 ms (review⁴). The LCR period and the cycle length remain strongly coupled, not only in the steady state beating, but also during stringent transitions, such as the transient state after removal of voltage-clamp at the maximum diastolic potential (MDP),²² and during intrinsic beat-to-beat variations in AP cycle length.³²

Figure 25-3 A, PKA-dependent changes in phosphorylation of phospholamban predict changes in the SANC LCR period. B, Changes in the steady state LCR period in response to numerous perturbations of the coupled-clock system predict concomitant changes in the steady state spontaneous AP cycle length. (Modified from Lakatta EG, Maltsev VA, Vinogradova TM: A coupled SYSTEM of intracellular Ca²⁺ clocks and surface membrane voltage clocks controls the timekeeping mechanism of the heart’s pacemaker. Circ Res 106:659-673, 2010. Updated with data from Vinogradova TM, Brochet DX, Sirenko S, et al: Sarcoplasmic reticulum Ca²⁺ pumping kinetics regulates timing of local Ca²⁺ releases and spontaneous beating rate of rabbit sinoatrial node pacemaker cells. Circ Res 107:767–775, 2010.)

Although LCRs are indeed generated by SR, the LCR period is not an exclusive property of the SR function per se, but rather is the result of the mutual entrainment of both Ca²⁺– and M- clocks regulating Ca²⁺ balance of the system (including Ca²⁺ available for pumping by the SR) via multiple Ca²⁺-, voltage-, and enzyme-dependent processes (described in detail later). Thus, the LCR period characterizes the integration and kinetics of the clock-system, rather than just the function of a Ca²⁺-clock alone. These ideas have recently evolved into a concept of a coupled-clock pacemaker system 4,5 featuring a “yin-yang”–type coexistence via interdependence³³ (see Figure 25-2, D).

Symmetrical Importance of Ca²⁺– and M-Clocks: Clocks Coupling Guarantees Each Other’s Timely Existence and Confers Robustness to SANC Normal Automaticity

Although spontaneous Ca²⁺ release is intrinsically stochastic, its potentially deleterious, unlimited regenerative nature is quenched by induction of an AP, thus shifting Ca²⁺ release properties from stochastic to periodic. The SR Ca²⁺ release periodicity is entrained by rhythmically occurring APs via rhythmically triggered depletion of the SR. While the M-clock–generated AP initially depresses SR Ca²⁺ release by effecting global Ca²⁺ SR depletion, this Ca²⁺ depletion simultaneously initiates the coupled-clock restitution process: Ca²⁺ influx via LCCh restores Ca²⁺ lost from the cell via NCX earlier in the prior cycle. At the same time, SR begins to refill Ca²⁺ (via sarco/endoplamic reticulum Ca²⁺-ATPase [SERCA] Ca²⁺pumping and, likely, inter-SR Ca²⁺ diffusion toward junctional SR) and then generates LCRs again when restitution is completed with the achievement of a threshold intra-SR [Ca²⁺] and removal of RyR inactivation, both of which are required for subsequent spontaneous RyR activation. The growth of submembrane LCRs activates I_NCX (i.e., the electrogenic component of the coupled system), which drives DD to reach a threshold of LCCh activation that continues to further accelerate DD, ultimately leading to the generation of rapid upstroke of the next AP. Of note, LCCh activation accelerates the DD of a given cycle not only directly via its inward current, but also indirectly via its Ca²⁺ influx (during DD, before the AP upstroke), resulting in Ca²⁺-induced Ca²⁺ release and, hence, in additional LCRs and greater NCX activation, creating (for a short time) a regenerative, positive-feedback loop. These chain reaction types of interactions (revealed by numerical modeling ⁵; see Figure 25-2D) that become apparent during late DD may be construed as being akin to a “rocket launch” (i.e., likely important for robust late DD acceleration and spontaneous AP generation).

Hence, during spontaneous AP firing, the SR does not oscillate Ca²⁺ independently, as it can in the absence of M-clock, but rather operates as a “stop-watch” to generate a timely crucial LCR/I_NCX signal during DD that is likely further amplified by the Ca²⁺-induced Ca²⁺ release. After each successful “rocket launch” (i.e., AP ignition), SR function is reset by the AP. Thus, in a given steady state, the electrochemical interactions of the ensemble proteins of the system recur during each cycle (i.e., M-clock [or Ca²⁺-clock]), initiating Ca²⁺-clock (or M-clock); this guarantees its own future periodic existence and thereby robust spontaneous AP firing. Thus, the coupled-clock concept and the symmetrical importance of both clocks have actually “married” the efforts of two earlier reductionist approaches to explain cardiac pacemaker function based predominantly on either Hodgkin-Huxley–type ion current kinetics or on the periodic Ca²⁺ pumping-release paradigm of the SR function, igniting APs simply via an I_NCX electrogenic link.

Beat-to-Beat Ca²⁺-Dependent Regulation of SAN Pacemaker Cell Rate and Rhythm

If the Ca²⁺– and M-clocks were indeed coupled, as has been described, then Ca²⁺ changes must regulate the AP firing rate on a beat-to-beat basis. Evidence for the role of Ca²⁺ in beat-to-beat regulation stems from experiments in rabbit SANC in which spontaneous AP firing is interrupted by a voltage-clamp to −65 mV.²² As voltage-clamp is applied, the ensemble LCR signal or LCR signal mass (a product of LCR amplitude, size, and duration) during the time interval, corresponding to the inter-AP interval (i.e., “would-be” cycle length), remains as during spontaneous AP firing but then wanes after a few “would-be” cycles as SR Ca²⁺ becomes depleted in the absence of regularly occurring APs. Upon removal of the voltage-clamp, spontaneous AP firing is restored, and this produces Ca²⁺ influx for pumping into the Ca²⁺-depleted SR. Beat-to-beat Ca²⁺-dependent regulation is revealed during the transition process, as SR Ca²⁺ load increases: The beat-to-beat increase in spontaneous AP firing rate closely follows the concomitant beat-to-beat increase in diastolic LCR activity (assessed as the LCR signal mass²²).

A more direct type of evidence for beat-to-beat Ca²⁺ dependent regulation of SANC has been recently provided by acute photolysis of the intracellular caged Ca²⁺ compound.³⁴ In response to loading of the caged Ca²⁺ buffer, the kinetics of Ca²⁺ removal from the cytosol is markedly slowed, the integral of Ca²⁺ release in the form of LCRs becomes substantially reduced, and LCRs become uncoupled from AP generation, which becomes markedly slowed and then dysrhythmic. When Ca²⁺ is acutely released from the caged compound by flash photolysis, intracellular Ca²⁺ dynamics are acutely restored, and rhythmic APs resume immediately at a normal rate. Thus, when Ca²⁺ is photo-released, a small increase in Ca²⁺ acutely affects SANC function: The initial effect of the flashes markedly accelerates DD and acutely reduces spontaneous cycle length and synchronous LCRs. After a few rhythmic cycles, however, these effects of the flash wane as interference with Ca²⁺ dynamics by the caged buffer is reestablished.

Another important piece of evidence for the beat-to-beat Ca²⁺-dependent regulation of SANC function is provided by two-dimensional (2D)-camera measurements of the whole-cell ensemble of LCRs simultaneously with perforated-patch AP recordings.³² These experiments demonstrate that the beat-to-beat variations in average LCR period (measured each cycle) of the whole-cell LCR ensemble predict variations in AP cycle length.

Basal Levels of cAMP and Protein Phosphorylation by PKA and CaMKII Drive the Coupled-Clock System

SANC have a high constitutive level of activation of adenylyl cyclases (ACs) that results in a high level of basal cAMP ²³ and PKA-dependent protein phosphorylation. Although SANC, like ventricular myocytes, express high levels of Ca²⁺-inhibited AC types 5 and 6, the discoveries of Ca²⁺-activated AC types (i.e., AC1 and AC8), in rabbit and guinea pig SANC^35,36 and localization of the basal Ca²⁺-activated AC activity within lipid raft microdomains³⁶ link Ca²⁺ to localized cAMP production (see Figure 25-1). 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^23,36 (see Figure 25-1).

Confocal imaging of immunolabeled proteins demonstrates that active CaMKII is highly localized beneath the surface membrane.¹¹ Thus, a geographical association has been noted between local CaMKII activity and AC activities and LCRs. CaMKII inhibition reduces I_CaL in isolated SANC, thereby reducing the rate of AP firing.¹¹ Increased CaMKII activity is also essential for the increase in SR Ca²⁺ release in SANC in response to β-AR stimulation.³⁷ BayK 8644 (a LCCh agonist) requires CaMKII activation to increase SANC AP firing.³⁸ CaMKII is activated in rabbit SANC during AP firing in the basal state (likely as the result of active basal intracellular Ca²⁺ cycling).

The likelihood for spontaneous SR Ca²⁺ release to occur increases as a function of the SR Ca²⁺ load, determined, in a physiological context, by the Ca²⁺ that is available for pumping into the SR and the phosphorylation status of the Ca²⁺ cycling proteins, including phospholamban (which regulates the activity of SR Ca²⁺ pumping protein SERCA), RyR (the SR release channel), and LCCh (see review ³⁹). SR Ca²⁺ refilling kinetics regulates the LCR period and the spontaneous beating rate of rabbit SANC.⁴⁰ PKA- and CaMKII-dependent phosphorylation of SR cycling proteins and LCCh leads to synchronization of spontaneous RyR activation. The schematic in Figure 25-4A depicts the concept that the restitution process that determines the LCR period is regulated (1) by the kinetics of SR Ca²⁺ cycling (i.e., by the rate of Ca²⁺ pumping into the SR); and (2) by the threshold of SR Ca²⁺ load required for spontaneous RyR activation. This concept is also supported by numerical model simulations of the SANC coupled-clock system (Figure 25-4B).

Figure 25-4 A, Schematic illustration of the concept of how the rate of SR Ca²⁺ refilling and the Ca²⁺ release threshold determine the LCR period, as well as the timing of the LCR-induced DD.⁴ B, A new numerical of the SANC model⁵ predicts the wide range of pacemaker rate modulation via variations in SR Ca²⁺ pumping rate (1 to 30 mM/s), mimicking various degrees of PKA-dependent phospholamban phosphorylation.

Experimental data clearly support the critical importance of Ca²⁺-dependent, basal protein phosphorylation for normal SANC automaticity. Reducing the phosphorylation state of these proteins in rabbit or guinea pig SANC by buffering intracellular [Ca²⁺] or directly inhibiting AC, or basal PKA or CaMKII activities (including by cholinergic receptor [ChR] stimulation, see below), has marked effects in slowing or even abolishing rhythmic APs (see review ⁴). And vice versa—increasing PKA signaling via stimulation of β-ARs, or exposure to a membrane-permeable analog of cAMP, or inhibition of phosphodiesterase or phosphatase activity (see later) accelerates the AP firing rate. It is important to note that independent of the type of perturbation of protein phosphorylation, the LCR period always closely predicts the cycle length (see Figure 25-3B). Maneuvers that increase protein phosphorylation require an intact Ca²⁺-clock to effect an increase in the spontaneous AP firing rate.^23,41 Normal automaticity of SANC, in fact, can be envisioned as a phenomenon that emerges from the phosphorylation state of the coupled-clock systems proteins; this synchronizes their functions to result in rhythmic spontaneous AP firing.

Mechanisms Intrinsic to the Coupled-Clock System That Restrain Its Basal AP Firing Rate

The coupled-pacemaker system dynamics are driven by a positive-feedback Ca²⁺ signaling, that is, Ca²⁺release begets more Ca²⁺ release via PKA and CaMKII pathways (see Figure 25-1, green arrows):

This positive-feedback signaling scheme obviously would drive the AP firing rate to its maximum. Thus, mechanisms to damp this signaling (see Figure 25-1, red arrows) must function continuously and concurrently to keep in check cell cAMP level and the phosphorylation of molecules that drive AP firing. Basal phosphodiesterase activity is one such restraining mechanism to reduce the high constitutive AC activity in SANC.³¹ Such a system of a high rate of cAMP production by Ca²⁺-activated ACs is balanced to a high rate of cAMP degradation by phosphodiesterase to still maintain high basal levels of cAMP and, hence, basal phosphorylation of the signaling proteins of both clocks. Basal activation of phosphoprotein phosphatases in SANC⁴² is another mechanism that limits PKA- and CaMKII-dependent phosphorylation. The net result of basal activation of phosphodiesterase and phosphoprotein phosphatases is that the basal LCR period and the AP cycle length are maintained near the midpoint of their functional ranges (see Figure 25-3A,B).

Other restraining mechanisms of the system limit Ca²⁺ influx and hence cell Ca²⁺ load, for example, calmodulin-mediated LCCh inactivation, which limits Ca²⁺ influx via LCCh during each AP. It is interesting to note that membrane ion channels and transporters regulate Ca²⁺ balance of the coupled system not only directly, by impacting on Ca²⁺ influx/efflux of LCCh/NCX, but also indirectly, for example, by I_K activation, which repolarizes AP, thereby limiting Ca²⁺ influx of LCCh and simultaneously increasing Ca²⁺ efflux via voltage-dependent activation of the NCX forward mode. Even I_f activation regulates cell Ca²⁺ balance, by limiting the MDP, thereby limiting voltage-dependent Ca²⁺ efflux via NCX.

Thus, the robustness of the coupled-clock system is imparted by its numerous functional redundancies and by dual regulation of SR Ca²⁺cycling and membrane ion current generators by a common set of factors (coupling factors or nodes [see Figure 25-1, purple labels]) kept in check by phosphodiesterase and phosphatase activities, voltage-dependent negative feedback on ion fluxes, and calmodulin-dependent modulation of LCCh. These complex interactions (depicted in Figure 25-1) are the essence of the system’s ability to function as a clock (i.e., despite substantial transient cell Ca²⁺ changes during each AP cycle, the steady state average cell Ca²⁺ balance and levels of phosphorylation of M- and Ca²⁺-clock proteins remain stable), so that all events recur during each cycle in the same sequence and magnitude, ensuring a stable AP cycle length.

Autonomic Receptor Modulation of the Coupled-Clock System

Nature imparts flexibility to the coupled clock of the cardiac pacemaker to vary its AP firing rate over a wide physiological range (i.e., from 60 to 240 bpm in humans) via variation in GPCR signaling (see Figure 25-1, green and red shapes) that links both adrenergic and cholinergic receptors to the very same nodes of the coupled-clock system (see Figure 25-1, purple labels) that regulate the basal state LCR period⁴: Stimulation of sympathetic β-ARs in SANC, via G_s activation, increases the spontaneous AP firing rate via effects on the proteins of both clocks. It is well established that β-AR stimulation can modulate ion channels of the M-clock,¹ specifically, I_K and I_CaL (via PKA-dependent phosphorylation) and I_f (via direct effects of cAMP; see Figure 25-1). However, β-AR stimulation, via changes in cAMP/PKA-dependent phosphorylation due to AC activation and phosphatase inhibition, can also alter SR Ca²⁺ efflux via RyR, as well as Ca²⁺ sequestration via SERCA.^14,20,41 Thus, β-AR stimulation modulates cAMP and protein phosphorylation levels of both clocks, simultaneously increases Ca²⁺ within the system via more frequent and larger I_CaL activation, and accelerates refilling of SR with Ca²⁺ to generate stronger LCRs with shorter LCR periods^23,40,41 that herald stronger I_NCX, thus accelerating DD.⁴³ CaMKII-dependent phosphorylation of phospholamban and RyR also increases in response to β-AR stimulation (likely as the result of changes in cell Ca²⁺ dynamics and concomitant calmodulin activation, effected primarily by PKA activation). Thus, CaMKII-dependent phosphorylation likely has an important role as a Ca²⁺-calmodulin–dependent amplifier of the initial effect of β-AR stimulation.

ChR act as a physiological “break” on the heart rate. ChR signaling activates G_i protein that couples to several downstream targets: (1) G_i-βγ activation of I_KACh (see Table 25-1; see review¹ for details), and (2) a G_i-α_s–coupled reduction in AC activity that limits cAMP/protein kinase A (PKA)-dependent phosphorylation. The lack of effect of pertussis toxin (PTX), which prevents G_i activation, on basal AP firing rate indicates an absence of any significant basal G_i activity that relates to AP firing. In isolated, single rabbit SANC, the threshold for a cholinergic agonist, carbachol, to reduce the AP firing rate is ≈10 nM, and half maximal inhibition is achieved at 100 nM. I_KACh activation becomes apparent at higher concentrations of carbachol (>30 nM).⁴⁴ A reduction in AP firing rate in response to carbachol concentrations between 10 and 30 nM theoretically can be explained by a reduction in I_f via reduced cAMP, but stimulation remains intact in I_f-deficient mice. Alternative powerful, cAMP-dependent mechanisms include a concurrent reduction in PKA-dependent phosphorylation of Ca²⁺ cycling proteins (e.g., phospholamban, RyR)^44,45 and likely the M-clock (e.g., LCCh and I_K channels). Thus, in contrast to β-AR stimulation, ChR stimulation decreases the Ca²⁺ balance of the system, which leads to a reduction in AP firing rate. It is important to note that the transition to a new steady state of the reduced AP firing rate is a feed-forward process. Specifically, during this transition, each spontaneous cycle is affected by prior cycles featuring less frequent and smaller I_CaL activation that further decelerates refilling of SR with Ca²⁺ to generate weaker LCRs with longer LCR periods,^44,45 decreasing and delaying I_NCX activation and decelerating DD. Thus, the feed-forward mechanism of the AP firing rate reduction reinforces the initial effect of ChR stimulation on cell Ca²⁺ balance, which likely extends the regulation range and confers robustness to the ChR-mediated bradycardic effect.

The ideas of the Ca²⁺-clock and the coupled-clock system have served as the basis for a novel type of robust and flexible biological pacemakers.46–48 One way to actually mimic the clock coupling that naturally occurs in SANC is to express Ca²⁺-activated AC type 1 or 8^35,36 to activate proteins of both clocks via cAMP and cAMP-activated PKA-dependent phosphorylation.^47,49 Another interesting approach is to reactivate within ventricular myocytes the genetic program of the SAN.⁴⁸

Mitochondrial Function in SANC

Rabbit SANC specifically had been referred to as “empty” cells with sparse and scattered myofilaments and mitochondrial densities compared with atrial and ventricular cells. These conclusions, however, were based on rough-cut sections of the SAN visualized by electron microscopy requiring a thin tissue slice, in which mitochondria can be damaged, and on the fact that artifacts can be created, in part as the result of different cut angles, which can change the tissue morphology, giving rise to the impression of “empty” space. Mitochondrial density visualized in isolated intact rabbit SANC by specific mitochondrial labeling revealed the presence of abundant mitochondria and mitochondrial density in SANC that is similar to that in atrial and ventricular myocytes ⁵⁰; hence, mitochondria are an important part of the coupled-clock system (Figure 25-5).

Figure 25-5 Illustration of the interplay of AC- and cAMP/PKA-dependent signaling to the myofilaments, SR Ca²⁺-cycling proteins, ion channels, and basal state ATP demand and supply in SANC.

The respiration or oxygen consumption rate in a physiologically coupled mitochondrial system reflects the rate of mitochondrial adenosine triphosphate (ATP) production. The respiration rate of SANC is comparable with that of unloaded, ventricular myocytes that are electrically stimulated at 3Hz (i.e., similar to spontaneous SANC AP firing rate).⁵⁰ Therefore, the ability of the mitochondria to produce ATP in SANC is comparable with that of ventricular myocytes, but “the energy budget” is spent differently in the two cell types. The AP-initiated cytosolic Ca²⁺ transient delivers Ca²⁺ to the contractile proteins to induce myofilament force production and displacement, and myofilaments are the major ATP consumers in ventricular myocytes. But SANC myofilament density is relatively low compared with that of ventricular myocytes.⁵¹ In SANC, ATP consumption by spontaneous myofilament contraction is lower than in ventricular myocytes, and most ATP in SANC is consumed in the synthesis of cAMP and Ca²⁺-cycling maintenance of cell ionic homeostasis (see Figure 25-5). Short-term metabolic inhibition by cyanide, which inhibits cytochrome oxidase in the mitochondria, reduces the intracellular Ca²⁺ transient amplitude and the AP firing rate in primary pacemaker cells.^50,52

Cytosolic Ca²⁺ enters mitochondria via the mitochondrial uniporter and is extruded by the mitochondrial NCX (see Figure 25-5). In ventricular myocytes, changes in mitochondrial Ca²⁺ alter the activity of several mitochondrial enzymes that take part in ATP production (see review cf⁵³). In SANC, however, reducing Ca²⁺ influx into the mitochondria does not significantly alter the ATP level. These results strongly suggest that mitochondrial Ca²⁺ does not directly regulate ATP supply to demand. In contrast to ventricular myocytes, Ca²⁺ activation of cAMP/PKA signaling in SANC has a major role in linking basal ATP utilization and mitochondrial ATP production. A gradual reduction in cAMP/PKA signaling is accompanied by a gradual reduction in ATP and a reduction in AP firing rate, suggesting that the Ca²⁺-activated cAMP/PKA signaling cascade that drives spontaneous AP firing of SANC, as has been described, is a unique core feed-forward system that not only drives basal ATP consumption but also regulates ATP production. cAMP/PKA signaling can effect ATP production by phosphorylation of several mitochondrial proteins (e.g., voltage-dependent anion carrier [VDAC]) and complexes I through V (see review⁵⁴). An increase in phosphorylation of these complexes in the electron transport chain by cAMP/PKA enhances the flux of protons and drives them to complex V to increase the rate of ATP production. Phosphorylation of VDAC can also increase the delivery rate of ATP from the mitochondria to the cytosol. Therefore, a decrease in basal cAMP/PKA signaling in SANC appears to signal to mitochondria that ATP production should be reduced.

ATP can be produced in the cytosol by glycolysis. In SANC, inhibition of glycolysis does not affect the basal spontaneous AP firing rate.50,55 Moreover, inhibition of glycolysis in SANC does not affect either ATP level or O₂ consumption; therefore under basal conditions, glycolysis is not the major mechanism to produce ATP in SANC. However, during high demand, or hypoxic conditions (i.e., when the amount of oxygen to the cell is limited), glycolysis may have an important role in ATP production in SANC.

In ventricular myocytes, mitochondrial Ca²⁺ flux plays a fundamental role in buffering cytosolic Ca²⁺ and modulates the SR Ca²⁺ load in both normal and pathological conditions. Similar to ventricular myocytes, mitochondrial-SR Ca²⁺ crosstalk is present within SANC, and mitochondrial Ca²⁺ flux in SANC affects the SR Ca²⁺ load, thus indirectly affecting SR Ca²⁺ release.⁵⁶An increase in mitochondrial Ca²⁺ attained by inhibition of mitochondrial NCX decreases the SR Ca²⁺ load and reduces the ensemble LCR Ca²⁺ signal. In contrast, a reduction in mitochondrial Ca²⁺ by inhibition of mitochondrial uniporter increases the SR Ca²⁺ load and increases the ensemble LCR Ca²⁺ signal. Basal coupled-clock system automaticity is modulated by changes in Ca²⁺ cycling into and out of mitochondria in SANC, impacting on cytosolic Ca²⁺, SR Ca²⁺ loading, and, indirectly, SR Ca²⁺ release characteristics. Changes in the spontaneous AP cycle length in response to changes in mitochondrial Ca²⁺ are predicted by concurrent changes in the periodicity of the spontaneous rhythmic LCR Ca²⁺ signal. Because basal Ca²⁺ is also linked to cAMP-PKA signaling within SANC that regulates ATP supply/demand matching, mitochondrial Ca²⁺-cytosolic Ca²⁺ crosstalk indirectly links to ATP supply and demand.