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

Chapter Outline

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

Ca²⁺-Clocks in Pacemaker Cells

Confocal imaging of Ca²⁺ in mammalian SANC and atrial subsidiary pacemaker cells combined with noninvasive perforated patch-clamp electrophysiology17,18 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 microscope²¹ 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⁺

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