A Novel Mechanism of Pacemaker Control That Depends on High Levels of cAMP and PKA-Dependent Phosphorylation
A Precisely Controlled Biological Clock
- local calcium release
- sarcoplasmic reticulum
- Na–Ca exchanger
- ryanodine receptor
- diastolic depolarization
See related article, pages 505–514
The mammalian heart has remarkable intrinsic rhythmic properties. It is widely agreed that spontaneous diastolic depolarizations (DDs) in sino-atrial node cells (SANCs) periodically initiate action potentials (AP), which set the rhythm of the heart.1 Efforts to understand the origin of the pacemaker activity have a lengthy history and the subject has, for various technical reasons, proved somewhat intractable.
Any explanation of pacemaker activity must address three central issues. First, how do DDs arise? Second, what determines their periodicity? And third, how is the rate modulated? In this issue of Circulation Research, Vinogradova and her colleagues2 offer some novel observations that go far toward explaining these issues. The article, which is the most recent of an exhaustive series of experiments from Dr Lakatta’s group, offers an explanation of the control of pacemaker activity based on both biophysical and biochemical observations, integrated with appropriate mathematical modeling (see supplement). This work depends on the central idea that pacemaking involves complex interactions within a multi-molecular complex that resides in both sarcolemmal and SR membranes. An attractive feature of this work is that it suggests a number of interesting structural and functional avenues of investigation that are amenable to contemporary biophysical methods, particularly confocal microscopy.
No single current by itself is responsible for DD. It is the sum of at least 6 ionic currents: Ikr, If, Ist, ICa (with two components: ICa-T and ICa-L), and INCX.1,3 In a previous study, Bogdanov et al4 show that sodium–calcium exchanger (NCX) is of crucial importance to maintaining pacemaker activity. A more complete discussion of the temporal relationships between these various currents is reviewed elsewhere.5
Dr Lakatta’s group have emphasized the importance of the involvement of intracellular Ca2+ (particularly SR Ca2+) in the regulation of pacemaker activity. Although their work is extremely provocative, it is worth pointing out that this view of pacemaker activity is not unanimous. It is in principle possible to obtain pacemaker activity with only three time- and voltage-dependent currents.12 The implication of this is that Ca2+ homeostasis need not be involved in pacemaker activity. Moreover, recently Lancaster et al13 have pointed out that smaller centrally located SANCs continue to pace in the presence of ryanodine. Clearly the involvement of Ca homeostasis in pacemaker activity is controversial.
Dr Lakatta’s group has developed the idea that in SANCs there are periodic spontaneous local Ca2+ release events (LCRs), through RyR2 which cause a rise in Ca2+ in a domain that is closely opposed to NCX. These exchangers preferentially extrude Ca2+ which produces an inward current (INCX) which contributes to DD. INCX, although not the only cause of DDs, clearly augments the later part of DD. In an earlier publication Vinogradova et al7 showed that the Fast Fourier Transform of membrane current fluctuations displayed a similar periodicity to LCRs, with a dominant power at 2.9 Hz. The key finding of the current article is that this mechanism depends crucially on a high level of basal cAMP and its attendant PKA phosphorylation. These high levels of cAMP seem, at least within the heart, to be characteristic of SANCs but not of ventricular cells.
How Does NCX Produce Periodic Activity: The LCR Clock
Vinogradova et al propose in this study that the LCR period functions as a clock during spontaneous beating. The period of this clock is the time from the onset of triggered SR Ca2+ release during the prior AP to LCR onset during subsequent DDs.2 We begin our consideration of the LCR clock with the LCR events depicted in the Figure. This release from the SR is imaged with line scans along the longitudinal axis and beneath the sarcolemma of the cell (see Figure 3c of reference 2).
These local Ca2+ release events appear to be larger than sparks in rabbit cardiac myocytes.6 They display a temporal separation from the putative global release event which appears to be evoked during the AP. The LCRs are abolished by ryanodine but remain under voltage-clamp and in skinned cells. Bogdanov et al4 showed that LCRs appeared as sub-microscopic wavelike patterns in SANCs. These LCRs may resemble the small Ca2+ wavelets that Stuyvers et al9 detected in canine Purkinje cells. The LCRs studied by Vinogradova et al2 do not seem to be necessarily responsible for triggering the putative global release events for reasons that are not clear but may be related to geometric and structural constraints. LCR events clearly warrant further investigation.
In the cascade that comprises the clock the next event of importance is the activation of NCX. Maltsev et al8 have calculated that these LCRs activate sufficient DD to ensure that an AP is evoked. The rate of this depolarization will depend on the magnitude of LCRs and as such is dependent on SR Ca2+ content.
Relationship between the LCR and SR Ca2+ content
The current article provides evidence that phosphorylation of numerous control points modulates the clock periodicity. We will now consider the involvement of the SR as one of the control points. Ca2+ is principally extruded from the cell by NCX. SR Ca2+ content will then depend mainly on the balance between the extrusion of Ca2+ and Ca2+ influx through L-type Ca2+ channels during each duty cycle. In particular, the integrated flux through local L-type Ca2+ channels depends on the extent of activation and inactivation of ICa. If in successive beats net influx increases, SR content will also increase, with reasonably predictable effects on the clock. The studies by Vinogradova et al2 seem to imply that SR content and hence the magnitude and properties of LCRs are controlled exquisitely. How can SR content control LCRs? It seems that the finding by these authors that high basal cAMP and PKA-dependent phosphorylation in SANCs may suggest that SR Ca2+ content is somewhat high in these cells during normal activity. Most recently Gyorke et al10 have suggested that the concentration of luminal calcium influences the open probability (Po) of the RyR by an allosteric mechanism involving a complex of SR proteins. This increase in Po will on average increase the chances that a RyR opens with a shorter latency. Changes in RyR latency provide another point where the timing of the clock can be modified. Moreover, with an increased Po and possibly elevated luminal Ca2+, the increase in SR permeability and the increased driving force for SR Ca2+ release could enlarge LCRs.
The studies depicted in this article invite quantitative consideration of the relationship between SR Ca2+ content and the size of the Ca2+ transient, which presumably includes LCR events. Trafford et al11 have suggested that the relationship between Ca2+ release and SR Ca2+ content is extremely steep such that the Ca2+ transient is proportional to the 6th power of SR Ca2+ content. This means that if, as a result of the extent of phosphorylation suggested by this study, the SR Ca2+ content is high in SANCs, small changes in SR Ca2+ content would have rather large effects on the magnitude of LCRs. For this reason, the modification of the clock periodicity will be dependent on SR Ca2+ content and the magnitude and timing of the LCRs (supplemental Figure, available online at http://circres.ahajournals.org). There are a number of implications to these assumptions. First, small changes in transmembrane fluxes can have significant effects on the clock without a high metabolic cost in Ca2+ pumping. Secondly, the dynamic range of the clock can, in principle, be significantly influenced by small changes in the extent of phosphorylation at the various control points of the clock. Third, because clock function may depend on SR Ca2+ content, dramatic reductions in SR Ca2+ content would seriously disrupt the function of the clock. These reductions are unlikely because they would require large imbalances in sarcolemmal Ca2+ fluxes.
Vinogradova et al2 also indicate that both phospholamban phosphorylation at serine 16 and phosphorylation on the RyRs is increased. The former will increase SR Ca2+ uptake. The latter will increase the Ca2+ leak through RyRs. Thus it is possible that SR Ca2+ content does not increase. How will this modify LCR generation? If Ca2+ accumulates in an unstirred layer adjacent to the RyRs, it might reach a threshold value where regenerative responses of RyR aggregates could produce LCRs. However, this increased cycling of Ca2+ through the SR would be at an increased energetic cost to pacemaker activity. It is clear that the entire issue of SR content in SANCs may be of considerable functional significance. As such, it may become central to future studies.
The authors are grateful for the support of the National Institutes of Health Research grants No. HL62690 and HL70828. We also appreciate the continuing support of the Nora Eccles Treadwell Foundation.
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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