The mechanism of spontaneous diastolic depolarization has traditionally been attributed to a "voltage clock" mechanism, mediated by voltage-sensitive membrane currents, such as the hyperpolarization-activated pacemaker current (I_f) regulated by cyclic adenosine monophosphate (cAMP).2)3) This current is also referred to as a "funny" current because, unlike the majority of voltage-sensitive currents, it is activated by hyperpolarization rather than depolarization. The funny channel becomes activated at the end of the action potential (at voltages from -40/-50 mV to -100/-110 mV), which corresponds to the range in which diastolic depolarization occurs. The funny channel then depolarizes the membrane to a level at which Na⁺ channels open to initiate the action potential.4)5) I_f is a mixed Na⁺-K⁺ inward current activated by hyperpolarization and modulated by the autonomic nervous system. Because the membrane ionic channels open and close according to the membrane potential, this process is referred to as a membrane voltage clock. The f-channels are encoded by the hyperpolarization-activated, cyclic nucleotide-gated (HCN) channel gene family. Of the four known HCN subunits, HCN4 is the most highly expressed in the mammalian SAN. The major role of I_f has been reinforced by the fact that mutations in the I_f channel are associated with a reduced baseline heart rate,6) and drugs which block I_f (such as ivabradine) do the same.7)8) However, while point mutations of HCN4 are associated with baseline sinus bradycardia, the maximum heart rate achieved during exercise is normal.9) The latter finding implies that I_f is not the only mechanism of SAN automaticity, especially during sympathetic activation.

The traditional mechanism for heart rhythm generation has been restricted to sarcolemmal ion channels. Recently, spontaneous Ca²⁺ release from the sarcoplasmic reticulum (SR) has been suggested to be the mechanism for sinus rhythm generation. When the SR is full, the probability of spontaneous Ca²⁺ release increases. On the other hand, when the SR is empty, the chances for spontaneous Ca²⁺ release decreases. The rhythmic alterations of SR Ca²⁺ release is referred to as the Ca²⁺ clock. Because the SR Ca²⁺ content is controlled in part by the membrane voltage, it is important to recognize that the activation of the Ca²⁺ clock and the membrane ionic clock are interdependent. The involvement of Ca_i in heart rhythm generation was first suggested by the observation that application of ryanodine slowed subsidiary pacemakers in cat right atria.10) Subsequent work showed the involvement of the electrogenic Na-Ca exchange current (I_NCX) in pacemaker activity and also raised the possibility that increased Ca²⁺ release, followed by activation of I_NCX, could play a role in the positive chronotropic effect of β-adrenergic stimulation in latent pacemaker cells.11-16) Lakatta et al. suggested that spontaneous rhythmic Ca²⁺ release from the SR in SAN cells, manifested as Ca2+ sparks, work as the "Ca²⁺ clock."17-21) The elevated Ca_i causes diastolic depolarization via I_NCX activation, which coordinately regulates the sinus rate along with the voltage clock.17-21)

The idea of a Ca²⁺ clock suggests that the mechanism of automaticity is the same as that of delayed afterdepolarization (DAD), which occurs when there is a SR Ca²⁺ overload. This suggests that the SAN must exist normally in a state of calcium overload. Vinogradova et al.21) also demonstrated that a high basal level of protein kinase A (PKA) activity in the SAN might contribute to a state of Ca²⁺ overload. A disease associated with Ca²⁺ clock malfunction has also been reported in patients with a genomic deletion of the RyR2 exon-3. Patients with that mutation develop catecholaminergic polymorphic ventricular tachycardia, along with SAN and atrioventricular node dysfunction, atrial fibrillation, and atrial standstill.22) It is possible that Ca²⁺ clock malfunction might contribute to the bradycardia and atrial arrhythmias in the latter patients.

Cardiac automaticity at the organ level is a very complex phenomenon and, in addition to cellular mechanisms, integrative factors are involved in cardiac pacemaking. The intact SAN is a heterogeneous structure that includes multiple different cell types interacting with each other.23-26) The relative importance of the voltage and Ca²⁺ clocks for pacemaking in different regions of the SAN, and in response to neurohumeral stimuli, such as β-agonists, may be different. Indeed, activation maps in intact canine right atria (RA) have shown that the SAN impulse origin is multicentric,27) and sympathetic stimulation predictably results in a cranial (superior) shift of the pacemaking site in humans and dogs.28)29) Based on evidence from isolated SAN myocytes, late diastolic Ca_i elevation (LDCAE) relative to the action potential upstroke is a key signature of pacemaking by the Ca²⁺ clock.10-21) It is possible that the same phenomenon could provide insight into the relative importance of the Ca²⁺ and voltage clock mechanisms in pacemaking in intact SAN tissue. We therefore designed a study to determine the role of Ca²⁺ and voltage clocks on heart rhythm generation and on the mechanisms of pacemaker hierarchy in the intact SAN.30)

At baseline, the spontaneous diastolic SR Ca²⁺ release, which is manifested by the LDCAE, was observed in only a small percentage of the preparations. However, the LDCAE occurred in all preparations during isoproterenol infusion, and was associated with a superior shift of the leading pacemaker site coincident with the appearance of robust LDCAEs (Fig. 1) in this region. Most importantly, the site of maximum LDCAE slope always co-localized with the leading pacemaking site, suggesting a shift in which the voltage clock now lagged the Ca²⁺ clock (Fig. 2). This observation indicates a strong association between LDCAE and pacemaking during β-adrenergic stimulation, and provides new insight into pacemaker hierarchy in the canine RA.27-29)

The Ca_i dynamics of SAN were characterized not only by the earliest onset of LDCAE, but also by the fastest Ca_i reuptake as compared with other RA sites. The baseline 90% Ca_i relaxation time was shorter at the superior SAN than at other RA sites. This resulted in the formation of the Ca_i sinkhole, which was facilitated by a rapid decline (short relaxation time) of the Ca_i fluorescence at the superior SAN during isoproterenol infusion and suggests that Ca_i reuptake by the SR is fastest in the superior SAN (Fig. 1D). The key protein regulator of SR Ca²⁺ uptake is phospholamban, which inhibits SERCA2a in the dephosphorylated state. There was a significantly lower SERCA2a/phospholamban ratio at SAN sites than at RA sites, suggesting more phospholamban molecules are available to regulate SERCA2a molecules in SAN than in RA. Isoproterenol infusion phosphorylates phospholamban and relieves phospholamban inhibition of SERCA2a, which may account for more robust Ca²⁺ uptake in SAN than in RA during isoproterenol infusion.30)

The LDCAE was closely related to SR Ca²⁺ release. Caffeine sensitizes the ryanodine receptor 2 to activation, resulting in increased SR Ca²⁺ release.18) The superior shift of the LDCAE and the pacemaking site was also consistently observed with caffeine infusion. Ryanodine, which blocks ryanodine receptors, caused a dose-dependent suppression of sinus node activity, and impaired isoproterenol-induced LDCAE.

The combination of ryanodine and thapsigargin also suppressed sinus node activity, and impaired isoproterenol-induced LDCAE. In contrast, the I_f blocker, ZD 7288 (3 µmol/L), did not prevent the LDCAE in the superior SAN.

Multiple time-and voltage-dependent ionic currents have been identified in cardiac pacemaker cells, which contribute to diastolic depolarization, including I_Ca-L, I_Ca-T, I_ST, and various types of delayed rectifier K currents.31) Many of these membrane currents are known to respond to β-adrenergic stimulation. Some of these currents, such as I_Ca-L, also promote the LDCAE and the acceleration of the sinus rate by the Ca²⁺ clock, as well as the voltage clock. In an intact SAN, SR inhibitors and I_f blockade slowed the sinus rate under basal conditions and blunted the isoproterenol-induced increase in the sinus rate. Therefore, the interdependence and synergy between the two clocks was evident.

It is known that heart failure is frequently associated with SAN remodeling, resulting in decreased SAN reserve.32) We performed nerve recording in a canine model of pacing-induced heart failure and found intermittent tachybradycardia episodes.33) Interestingly, the prolonged (>3 s) sinus pauses were triggered not by vagal activation, but by short bursts of sympathetic activity (Fig. 3). Typically, a burst of sympathetic activity is associated with tachycardia. When there is sympathetic withdrawal, the tachycardia terminates, followed by prolonged pauses during which no activation was observed. Preliminary studies34) showed that cryoablation of the stellate ganglia markedly reduces the prolonged sinus pause episodes in the same model. These findings suggest that the left stellate ganglion nerve activity is causally related to the tachybrady syndrome. The molecular mechanism of this association, however, remains unclear.

Our understanding of cardiac automaticity has progressed considerably during the last 10 years. Several questions regarding how the heart rhythm is generated and controlled in physiologic and pathologic conditions remain unanswered. A recent study by Yeh et al.35) showed funny current downregulation is assocciated with atrial tachyarrhythmias. The authors suggested that the down-regulation of funny current may underlie the mechanisms tachybrady syndrome. Whether or not Ca²⁺ clock malfunction also contributes to the mechanisms of tachybrady syndrome remains unclear. Our laboratory is currently evaluating whether the Ca²⁺ clock is impaired in a canine atrial fibrillation model with sinus dysfunction. Secondarily, we are trying to reproduce the human sick sinus syndrome model by manipulating the Ca²⁺ and voltage clocks in a canine isolated RA preparation. We anticipate that these studies will help us better understand the mechanisms of SAN dysfunction and facilitate design mechanism-based therapy of this disease.