Novel Perspectives on the Beating Rate of the Heart

To the Editor:

Recent studies^1–3 have demonstrated that local Ca²⁺ release during the later part of diastolic depolarization (DD) via ryanodine receptors (RyRs) in the sinoatrial nodal cells (SANCs) activates the electrogenic Na⁺-Ca²⁺ exchanger (NCX). The resultant inward current enhances the rate of DD, leading to an earlier occurrence of the subsequent action potential, ie, to an increase in the beating rate.³ Thus, a beating rate is a result of synchronization of two “ionic generators”: (1) a set of sarcolemmal ionic channels and (2) an array of RyRs, which, when activated, can generate rhythmic oscillations of intracellular [Ca²⁺].

Other recent studies indicate that the effect β-adrenergic receptor (β-AR) stimulation to enhance the rate of DD and to increase the beating rate requires intact RyR function.^4,5 Specifically, β-AR stimulation by isoproterenol recruits additional RyRs to release Ca²⁺ during DD; this activates NCX current and increases the rate of DD.⁵ Partial inhibition of normal RyR function by ryanodine, which results in sarcoplasmic reticulum Ca²⁺ depletion, blunts the dose response of isoproterenol to increase the beating rate.⁵

DiFrancesco and Robinson⁶ point out that prior studies have indicated that β-AR receptor stimulation augments the hyperpolarization-activated current, I_f, in SANCs. While this indeed may be the case, the real issue at hand is to what extent a change in I_f relates to a change in the beating rate. In our experiments, the contribution of I_f to the beating rate either in control conditions or during β-AR stimulation was minimal compared with the effect of suppression of intact RyR function.⁵ Although in our study⁵ we used 2 mmol/L Cs⁺ (a concentration that nearly completely blocks I_f),⁷ other I_f current blockers, UL-FS49 and ZD7288, which exhibit opposite voltage dependence of I_f block than Cs⁺, suppress the spontaneous beating rate of intact rabbit sinoatrial node equally as Cs⁺, but the reduction varies only from 12% to 15%.⁸ Given that rabbit primary SANCs are relatively insensitive to block of I_f,⁸ one of the reasons for such a small effect of I_f suppression in our experiments could be that our cells are mainly primary pacemaker cells.

A second issue raised by DiFrancesco and Robinson⁶ is that ryanodine, which specifically binds to RyRs, and, at the concentration used in our study, functionally locks RyRs in a subconducting open state, somehow “reflects an uncoupling for the β -adrenergic cascade from their normal target channels.” Although there is no experimental evidence to substantiate this intriguing possibility, the fact that such an effect would be perpetrated by interfering with normal sarcoplasmic reticulum Ca²⁺ cycling and RyR function by ryanodine would still necessitate the conclusion that normal RyR function is required to regulate cell Ca²⁺ to permit various aspects of β-AR signaling required to increase the beating rate. This is the de facto interpretation we assigned to our result. We are thus puzzled by the fact that DiFrancesco and Robinson take issue with it, because, via their argument, they would, by necessity, reach the same conclusion.

In addition, DiFrancesco and Robinson⁶ inquire about Ca²⁺-dependent modulation of the L-type Ca²⁺ channel. We interpret our recent observation that removal of L-type Ca²⁺ channel inactivation by CaMKII⁹ during DD enables these channels to be more responsive to a given depolarization. This mechanism, however, appears to be insufficient to effect an increase in DD rate or beating rate in response to β-AR stimulation in the absence of RyR Ca²⁺ release to activate NCX.⁵ Furthermore, the source of Ca²⁺ that activates NCX and modulates L-type Ca²⁺ channel may differ: Ca²⁺ release from RyRs to activate NCX current is crucial for the β-AR effect to enhance the beating rate; Ca²⁺-dependent activation of CaMKII appears to be closely linked to Ca²⁺ influx via L-type Ca²⁺ channel (CaMKII even associates with the C-terminal tail of L-type Ca²⁺ channel¹⁰) than to RyR Ca²⁺ release.⁹

In summary, although the role of specific ion channels in the spontaneous membrane potential depolarization of SANCs has been firmly established in earlier studies with respect to modulation of the DD and the beating rate, the advent of Ca²⁺-sensitive indicators, coupled to confocal imaging and to simultaneous measurement of membrane potential or current, has elucidated a major modulatory role of the local [Ca²⁺] beneath the cell membrane on pacemaker rate.