High Basal Protein Kinase A–Dependent Phosphorylation Drives Rhythmic Internal Ca2+ Store Oscillations and Spontaneous Beating of Cardiac Pacemaker Cells
Local, rhythmic, subsarcolemmal Ca2+ releases (LCRs) from the sarcoplasmic reticulum (SR) during diastolic depolarization in sinoatrial nodal cells (SANC) occur even in the basal state and activate an inward Na+-Ca2+ exchanger current that affects spontaneous beating. Why SANC can generate spontaneous LCRs under basal conditions, whereas ventricular cells cannot, has not previously been explained. Here we show that a high basal cAMP level of isolated rabbit SANC and its attendant increase in protein kinase A (PKA)-dependent phosphorylation are obligatory for the occurrence of spontaneous, basal LCRs and for spontaneous beating. Gradations in basal PKA activity, indexed by gradations in phospholamban phosphorylation effected by a specific PKA inhibitory peptide were highly correlated with concomitant gradations in LCR spatiotemporal synchronization and phase, as well as beating rate. Higher levels of basal PKA inhibition abolish LCRs and spontaneous beating ceases. Stimulation of β-adrenergic receptors extends the range of PKA-dependent control of LCRs and beating rate beyond that in the basal state. The link between SR Ca2+ cycling and beating rate is also present in vivo, as the regulation of beating rate by local β-adrenergic receptor stimulation of the sinoatrial node in intact dogs is markedly blunted when SR Ca2+ cycling is disrupted by ryanodine. Thus, PKA-dependent phosphorylation of proteins that regulate cell Ca2+ balance and spontaneous SR Ca2+ cycling, ie, phospholamban and L-type Ca2+ channels (and likely others not measured in this study), controls the phase and size of LCRs and the resultant Na+-Ca2+ exchanger current and is crucial for both basal and reserve cardiac pacemaker function.
Recent studies have demonstrated that in sinoatrial (SA) nodal cells (SANC) generate local, rhythmic, subsarcolemmal Ca2+ releases (LCRs) under basal conditions, ie, even in the absence of experimental Ca2+ loading or stimulation of β-adrenergic receptors (β-ARs).1–3 In rabbit SANC, spontaneous, rhythmic LCRs occur during the late diastolic depolarization and activate Na+-Ca2+ exchanger (NCX) to generate an inward current that accelerates the depolarization rate, and, thus, LCRs are involved in control of spontaneous beating rate of SANC.1 The mechanisms that permit SANC, but not ventricular myocytes, to generate rhythmic LCRs under basal conditions, however, have not been delineated.
Spontaneous SR Ca2+ release is facilitated by factors that increase the rate at which the SR can pump Ca2+, foremost among which are elevated cell Ca2+ or elevated cAMP and its attendant protein kinase A (PKA)-dependent protein phosphorylation that results from intense β-AR stimulation. Whereas the cytosolic Ca2+ concentration does not appreciably differ in rabbit ventricular cells and SANC,2,4 the cAMP level of the intact SA node is high,5 and it has been suspected that the basal cAMP level within SANC is elevated.6,7 The SA node, however, is highly innervated, and neither the basal cAMP level nor PKA-dependent protein phosphorylation has been directly measured in isolated SANC.
The present study demonstrates that cAMP and basal cAMP-mediated, PKA-dependent phosphorylation of phospholamban (PLB) are markedly elevated in SANC compared with other cardiac cell types. Basal PKA-dependent phosphorylation is obligatory for both, the occurrence of spontaneous basal LCR and spontaneous basal beating. The variation of basal LCR characteristics and spontaneous beating rate in response to graded reduction of basal PKA activity and that following β-AR stimulation form a continuum. Finally, SR Ca2+ cycling is relevant to PKA-dependent control of heart rate in vivo, as the increase in the heart rate in response to microdialysis of the SA node of intact dogs with a β-AR agonist is markedly suppressed when normal SR Ca2+ cycling is interrupted.
Materials and Methods
An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org.
SA Node Cell Preparations and Electrophysiological Recordings
Single spindle-shaped spontaneously beating SANC or atrial or ventricular myocytes were isolated from the rabbit hearts as previously described.8 All drugs used in this study are listed in the online data supplement.
A subset of SANC was permeabilized with 0.01% saponin as previously described.2
Confocal Imaging of LCRs
SANC were loaded with fluo-3–acetoxymethyl ester (Molecular Probes, Eugene, Ore). All images were recorded in the line-scan mode, using confocal microscopy as previously described.1,2
The detection of site-specific PLB phosphorylation was performed in isolated SANC or ventricular myocytes using a phosphorylation site-specific P-Ser-16PLB antibody (Badrilla), as previously described.9
SA nodal, atrial, or ventricular cells were homogenized, and cAMP was estimated using a cAMP (3H) assay system (Amersham Biotech).
A primary SA node pacemaker cell model featuring diastolic Ca2+ release10 was modified to simulate the effects of experimentally observed PKA modulation of LCR on spontaneous beating rate.
Nodal Microdialysis In Vivo
Microdialysis was performed as previously described.11
Data are presented as mean±SEM. The statistical significance of effects was evaluated by Student t test or ANOVA where appropriate. A value of P<0.05 was considered statistically significant.
Basal cAMP Concentration, PKA-Dependent Phosphorylation, and Spontaneous Beating of SANC
The basal cAMP concentration is substantially higher in isolated SANC than in atrial or ventricular myocytes (Figure 1a) and is markedly suppressed by an adenylyl cyclase (AC) inhibitor, MDL-12,330A, and increased by isoprotezenol (ISO) (Figure 1a).
The phosphorylation status of PLB was used as a marker for PKA-dependent protein phosphorylation. Basal PLB phosphorylation at the PKA-dependent serine 16 site is markedly increased in SANC but very low in ventricular myocytes (Figure 1b). Either AC or PKA inhibition reduces basal PLB phosphorylation (Figure 1c through 1e). The high basal PKA-dependent PLB phosphorylation is further augmented by β-AR stimulation (Figure 1d and 1e).
Maneuvers known to regulate the basal cAMP-mediated, PKA-dependent signaling affect the basal SANC spontaneous beating rate. Figure 2a illustrates, in a representative cell, that a membrane-permeable, specific peptide inhibitor of the PKA catalytic subunit, 14 to 22 amide, PKI,12 slows then, abolishes spontaneous beating. Following PKI-induced cessation of spontaneous beating SANC responded to external stimulation at rate equivalent to the pre drug spontaneous beating rate (results are not shown). The PKI effects are reversible on washout. Figure 2b illustrates that H-89, another PKA inhibitor, also abolishes spontaneous beating. Gradations of basal PLB phosphorylation, effected by graded concentrations of PKI, are highly correlated with concurrent gradations in the spontaneous beating rate (Figure 2c). The functionally relevant basal PKA activity is driven by a high basal activity of AC, because a potent AC inhibitor, MDL-12,330A, like PKI, also abolishes spontaneous SANC beating (Figure 2b). Constitutive activation of β-AR receptors, ie, in the absence of receptor ligands, is not a mechanism driving this high basal cAMP-PKA activity that is crucial for spontaneous beating of nodal cells, because neither the β1-AR antagonist, CGP-20712A, nor the β2-AR subtype inverse agonist, ICI 118,551, affect the spontaneous beating rate (Figure 2b).
PKA Signaling Controls Spontaneous LCRs
To determine the role of the high basal cAMP and PKA-dependent phosphorylation of PLB on the basal LCR characteristics of SANC in the absence of spontaneous action potentials (APs), avoiding concurrent effects of sarcolemmal ion channel gating, we permeabilized SANC with saponin or prevented AP occurrence by voltage clamping the cell membrane potential. Figure 3a shows, in a representative saponin permeabilized cell, that spontaneous LCRs are present in a physiologic (100 nmol/L) cytosolic free [Ca2+]; exposure to PKI reduces the LCR frequency and size. The average effects of PKI on LCR frequency and size are depicted in Figure 3b.
Figure 3c and 3d show a representative nodal cell before and following voltage clamping. Note, in the line-scan images and Ca2+ waveforms below each image, that during spontaneous beating before voltage clamp, LCRs occur slightly in advance of an AP. Note also (Figure 3c versus 3d) that during spontaneous beating, LCRs (arrowheads) are less numerous in the presence of the specific PKA inhibitor peptide, PKI. During voltage clamp at −10 mV, a potential that prevents rapid cell Ca2+ loss and SR Ca2+ depletion in SANC in the absence of APs,2 spontaneous, roughly periodic LCRs persist and their dominant frequency is reduced by PKI (Figure 3e). The local increase in submembrane [Ca2+] resulting from LCRs during voltage clamp generates an inward current via NCX.1,13–15 The green lines in Figure 3c and 3d depict inward current fluctuations generated by LCR occurrence. Note, that the periodicity of these current fluctuations, is the same as that of LCRs and is similarly shifted by PKI (Figure 3f).
During spontaneous beating both spontaneous LCRs and AP triggered SR Ca2+ releases mutually impact on each other because they occur from the same Ca2+ pool via the same release channel. The LCR period or “clock” during spontaneous beating can be characterized as the time from the onset SR Ca2+ release triggered by the prior AP to LCR onset during the subsequent diastolic depolarization2 (see Figure 4a). Figure 4b and 4c depict histograms of LCR periods and sizes during spontaneous beating, before, and during inhibition of PKA activity by PKI, which shifts the LCR distribution to longer periods and smaller sizes, prolonged exposure to PKI abolishes LCRs.
The effects of PKA inhibition on the LCR period are, likely, mediated by a reduction in phosphorylation of multiple proteins that regulate cell Ca2+ balance, including L-type Ca2+ channels and SR Ca2+ cycling proteins. Changes in SR Ca2+ pumping in other cardiac cells15 are linked to changes in the level of PKA-dependent phosphorylation of PLB (Figure 8c). The decay rate of the transient increase in cytosolic Ca2+ triggered by an AP reflects, in part, the rate at which the SR pumps Ca2+.15 Changes in this decay rate following PKI, therefore, in part at least, reflect changes in PLB phosphorylation status induced by this maneuver and, thus, indirectly reflect the effect on the rate at which the SR reloads with Ca2+. PKI significantly increased the average 90% decay time of AP-induced Ca2+ transient (T90) from 308±34 to 425±36 ms (P<0.05, n=5), and there was a strong correlation between the PKI-induced increase in T90 and increase in LCR period (r2=0.81). This effect of PKI on the decay of the AP triggered cytosolic Ca2+ transient and LCR period indirectly confirms that PKA-dependent modulation of the SR Ca2+ pumping rate is one critical determinant of the LCR “clock.”
The PKA-dependent effects on basal LCR and the concurrent PKA-dependent changes in beating rate are tightly correlated. Figure 4d (blue symbols) shows that the PKI-induced increase in LCR period directly correlates with the PKI induced increase in the spontaneous cycle length, and beating stops when PKA-dependent phosphorylation reaches sufficiently low level. Figure 5 shows that inhibition of basal PKA signaling by another PKA inhibitor, H-89, proportionally prolongs both LCR period and spontaneous cycle length in the same cell and that changes in LCR period that occur over time during exposure to the PKA inhibitor are highly correlated with time-dependent changes in the spontaneous cycle length. The effects of H-89 on both LCR period and cycle length are reversible on drug washout.
Stimulation of β-AR Extends the Range of PKA-Dependent Control of LCR Characteristics and Beating Rate
Figure 6a and 6b show that the addition of cAMP to “skinned” SANC increases the frequency (reduces the period) of LCRs. Figure 6c and 6d illustrate typical responses to ISO during spontaneous beating and during voltage clamp in intact SANC. β-AR stimulation by ISO shifts the LCR periodicity and that of associated current fluctuations measured during voltage clamp to a higher frequency (Figure 6e and 6f). Figure 7a and 7b show that during spontaneous beating β-AR stimulation reduces the LCR period and increases LCR size. The shift in LCR period is highly correlated with the β-AR induced change in beating rate (Figure 7c). Further, the PKA dependence of the basal LCR period and spontaneous beating rate effected by basal PKA inhibition and that following activation of PKA following β-AR stimulation form a single continuous function conforming to the line of identity (r2=0.97) (Figure 7d).
Numerical Modeling of PKA-Dependent Effects on LCRs and Spontaneous Beating of SANC
Prior experimental and numerical modeling results in SA nodal pacemaker cells have demonstrated that the potent control of the spontaneous beating rate by LCRs is attributable to LCR activation of NCX, generating an inward current that accelerates the later diastolic depolarization.1,10,13,14,16 Model simulations provide a unique opportunity to disable selected PKA targets during simulation. To estimate relative contributions of each to the spontaneous beating rate following a change in PKA signaling, we modified our prototype numerical model10 to incorporate experimentally measured PKA-dependent changes in individual LCRs phase and size as well as known changes of ion channel activity produced by PKI or β-AR stimulation described in the literature (see the online data supplement). The model simulation allowing PKA-dependent effects on both LCRs and ion channels predicts the experimentally obtained continuum of basal and reserve beating rate regulation by PKA (Figure 7d). Model simulations allowing only PKA-dependent changes in ICa,L or changes only in ion channels (ICa,L, If, and IKr), in the absence of change in LCRs, exhibit a markedly damped PKA-dependent chronotropic response (Figure 2, online data supplement). In contrast, simulations that allow PKA-dependent effects on ICa,L and LCRs, exhibit robust regulation on the cycle length, over a range nearly identical to experimentally measured indicating that changing both ICa,L and LCR are crucial for the PKA-dependent chronotropic effect.
cAMP/PKA Control of Beating, Both In Vitro and In Vivo, Requires Intact SR Ca2+ Cycling
If PKA-dependent effects on SR Ca2+ cycling were required for the PKA-dependent control of nodal cell beating, as suggested by the strong correlation between LCRs and the spontaneous beating rate (Figures 4, 5, and 7⇑⇑) and by model simulations (Figure 7d and online data supplement), then disabling normal SR function while leaving the cAMP/PKA signaling cascade intact ought to interfere with the ability of cAMP/PKA signaling to increase the beating rate. We observed that the ability of a membrane permeable cAMP analogue (CPT-cAMP) to increase the SANC beating rate is indeed markedly reduced by ryanodine (Figure 3, online data supplement).
To determine whether the ryanodine suppression of the effects of PKA-dependent signaling is relevant to heart rate regulation in vivo we microdialyzed the interstitium of the heart SA node in intact, open chest dogs with the β-AR agonist ISO. Figure 8a shows that a robust, dose-dependent increase in beating rate when ISO was administered locally into the node. The response was easily repeated in time controls (Figure 8a). However, the subsequent instillation of ryanodine, which reduced the basal heart rate by 12%, nearly eliminated the heart rate response to β-AR stimulation (Figure 8b). This result confirms results of studies in isolated SANC,13 in which the effect of β-AR stimulation with ISO to increase the SANC beating rate is markedly reduced in the presence of ryanodine, which does not affect the ability of ISO to increase ICaL.
Basal cAMP PKA-Dependent Phosphorylation and Spontaneous Beating
The first novel finding of the present study is that the basal level of cAMP is markedly higher in SANC than in other cardiac cell types. Increased basal cAMP is critical to SANC basal pacemaker function, because when basal AC is inhibited, cAMP is reduced, and basal SANC beating is abolished. It had previously been hypothesized that catecholamines liberated from within SA nodal cells act as neurotransmitters to affect an autocrine activation of β-ARs and AC that underlies the pacemaker function of these cells.6 The present results, demonstrating that blockade of β-ARs does not affect basal spontaneous beating, however, negates this intriguing hypothesis, and, in addition, the failure of the inverse agonist to reduce the spontaneous beating rate discounts the idea that constitutive β-AR activation, ie, that in the absence of β-AR ligands, drives cAMP production and PKA activation.
The second novel finding of the present study is that the high basal cAMP, per se, is not directly linked to the basal pacemaker activity in SANC: basal PKA-dependent protein phosphorylation is high in SANC, and inhibition of PKA activity, ie, signaling distal to cAMP (Figure 8c), abolishes beating. Therefore, the obligatory role of high basal cAMP with respect to basal pacemaker function is not direct but lies in the high level of PKA-dependent protein phosphorylation that it supports. In this regard, whereas cyclic nucleotide gated channels (HCN) in SANC may be affected by the high basal level of cAMP, suppression of beating by PKA inhibition apparently rules out the idea that cAMP gating of HCN channels, per se, is sufficient for the basal spontaneous beating in rabbit SANC.
Basal PKA-Dependent Phosphorylation and Spontaneous LCRs
A third novel finding of the present study is that high basal PKA-dependent protein phosphorylation in SANC is obligatory for the occurrence of basal spontaneous, rhythmic submembrane Ca2+ releases, as inhibition of PKA prolongs the LCR period, reduces LCR size, and ultimately abolishes LCRs. In the present study, PKA-dependent PLB phosphorylation at serine 16 was used as a convenient index of phosphorylation of a protein involved in SR Ca2+ cycling. Our preliminary data indicate that basal phosphorylation of ryanodine receptor (RyR) in rabbit SANC, assessed by an anti–phosphorylated RyR2 antibody is also substantially higher in rabbit SANC than in rabbit ventricular cells (D.Y., unpublished results, 2005). The cAMP PKA dependence of spontaneous LCR characteristics in saponin-permeabilized cells (Figures 3, a and b, and 6, a and b) demonstrates that phosphorylation of proteins that directly determine SR Ca2+ pumping and release is involved in regulation of the basal LCR period (Figure 8c). This regulation is independent of sarcolemmal ion channels. However, in the spontaneously beating cells with intact surface membrane function, the basal phosphorylation of additional proteins, not measured in the present study, that affect the cell Ca2+ load and Ca2+ cycling is also a crucial determinant of LCR occurrence and characteristics. Specifically, L-type Ca2+ channels are major targets of PKA-dependent phosphorylation, and, although not directly attached to the SR Ca2+ cycling machinery, their activation during spontaneous beating is necessary for LCR occurrence and characteristics. The absence of Ca2+ influx via L-type Ca2+ channel during voltage clamp at −60 mV of SANC depletes the SR Ca2+ load, damping and then abolishing LCRs within a few seconds.2 Additionally, ICa,L activation during an AP initiates global SR Ca2+ release, depleting the SR Ca2+ load and resetting the LCR clock. A prior study in SANC has observed a marked reduction in basal L-type Ca2+ current in response to PKI, suggesting a high basal PKA phosphorylation of L-type Ca2+ channels17 in these cells. High basal phosphorylation of L-type Ca2+ channel augments basal Ca2+ influx during ICa,L activation, leading to an increase in SR Ca2+ load,13 and markedly affects basal LCR characteristics on this basis. Thus, during spontaneous beating, PKI effects on LCRs are mediated, in part at least, by an effect to reduce L-type Ca2+ current.
Further, elevations of PKA activity, or the change in Ca2+ itself that results from PKA-dependent protein phosphorylation, also impact on the function of other proteins that control membrane potential and affect AP repolarization characteristics or the maximum diastolic potential. Moreover, the delayed rectifier K+ channel is phosphorylated by PKA. Several time- and voltage-dependent ion currents that become activated during diastolic depolarization, ie, T-type and L-type Ca2+ currents, hyperpolarization activated inward current (If), chloride current, the delayed rectifier K+ current, a time-independent background current carried by Na+ are modulated by Ca2+.15,18 However, because cAMP/PKA protein phosphorylation or the associated Ca2+ modulation of ion channels and membrane potential also indirectly affect the net cell Ca2+ balance and SR Ca2+ load, these channels may also be indirectly linked to SR Ca2+ cycling and, thus, to the LCR period and size. PKA-dependent phosphorylation and Ca2+-dependent allosteric effects on NCX resulting from crosstalk between L-type Ca2+ channel and NCX has also been noted in ventricular cells.19,20 This supports the idea that NCX may generate a larger inward current in response to LCRs.
Correlation Between the PKA-Dependent Modulation of LCR Characteristics and Spontaneous Beating
A fourth novel finding of the present study is that PKA dependence of basal LCRs is tightly linked to the PKA dependence of basal spontaneous beating. That spontaneous beating of SANC and LCR cease in response to PKI, and that this effect is reversible, suggest that a threshold level of PKA activity and protein phosphorylation in the basal state is required for spontaneous beating of these pacemaker cells. The present results demonstrate that the graded reduction of basal PKA-dependent protein phosphorylation, indexed by phospholamban phosphorylation at serine 16, is highly correlated with the concurrent prolongation of the basal SANC cycle length (Figure 2). But, as noted, the high basal phosphorylation of the PKA dependent proteins not included in the present study are likely implicated in the effect of PKI. In particular, the L-type Ca2+ channel, which resets the LCR clock and replenishes the SR Ca2+ load, is crucial for LCR characteristics. In addition to its impact on LCR characteristics, high basal PKA-dependent phosphorylation of L-type Ca2+ channels affects the removal of inactivation or the activation threshold, thus modulating response to a given membrane depolarization that results from NCX generated inward current activated by LCR occurrence just before the AP upstroke. Further, our prior studies have demonstrated that Ca2+/calmodulin-dependent protein kinase II plays a vital role in regulating cardiac pacemaker activity via modulating ICa,L inactivation and recovery from inactivation.8
When PKA-dependent protein phosphorylation increases above the basal level in response to β-AR stimulation, the LCR period is reduced, and this reduction is tightly correlated with the concomitant reduction in the spontaneous cycle length. The prolongation of basal LCR period and basal cycle length by PKA inhibition and reduction in LCR period and cycle length resulting from β-AR stimulation form a continuum (Figure 7d). We interpret this to indicate that reserve beating rate is elevated by an extension of the range of the same function that controls basal beating, ie, PKA-dependent protein phosphorylation.
Our numerical model simulations incorporating PKA-dependent changes in both LCRs and ion channels reproduce the full range of experimentally determined PKA dependence of spontaneous beating (Figure 7d, open symbols). However, model simulations not incorporating changes in LCRs fail to do so (Figure 2, online data supplement). The inability of cAMP (Figure 3, online data supplement) or β-AR stimulation to induce a robust increase in the spontaneous beating rate of isolated SANC in the presence of ryanodine, even when the β-AR stimulation induced increase in ICa,L is intact,13 supports the idea that SR Ca2+ cycling to generate LCRs is a crucial link between PKA-dependent phosphorylation and spontaneous beating. This result and model simulations including PKA-dependent changes in ICa,L, in the absence of changes in LCRs indicate that an increase in ICa,L following β-AR stimulation is essential but not sufficient to account for the full chronotropic response. The final novel finding of the present study is that the PKA activity and SR Ca2+ cycling regulate the heart rate in vivo, evident from the inability of β-AR stimulation to effect a heart rate in the presence of local application of ryanodine to the intact SA node.
In summary, the extent of spatiotemporal synchronization and phase of PKA-dependent spontaneous SR Ca2+ cycling within SANC is regulated by phosphorylation of multiple proteins including L-type Ca2+ channels, PLB, RyRs, and likely others, not measured in the present study. Thus, shifts in the LCR phase and size cause concomitant shifts in the phase and amplitude of the inward NCX current during diastolic depolarization and are linked to regulation of spontaneous beating on this basis. The scheme in Figure 8c integrates the present findings with those of prior studies1,2,8,13 and illustrates points at which 4 distinct maneuvers, each of which directly or indirectly markedly inhibits SR Ca2+ cycling to produce LCRs and blocks rabbit SANC pacemaker function. Intracellular BAPTA abolishes spontaneous beating8 by chelating intracellular Ca2+, thus affecting a variety of Ca2+-dependent functions, including inhibition of SR Ca2+ cycling by RyR while leaving PKA-dependent phosphorylation intact. Specific inhibition of L-type Ca2+ channels depletes the SR Ca2+ load and abolishes LCRs. Ryanodine, which locks RyRs in a subconductance open state, depleting the SR Ca2+ load, abolishes LCRs (ie, an effect opposite to that of PLB phosphorylation) but without reducing ICaL or the PKA-dependent increase in ICaL.13 Inhibition of basal PKA-dependent phosphorylation (present results) affects multiple proteins, among which are SR Ca2+ cycling proteins and L-type Ca2+ channels. Additionally, acute inhibition of NCX, with ICa,L and LCRs intact, blocks the link between LCRs and membrane depolarization and abolishes SANC beating.1
Thus LCRs can be envisioned as integrators of multiple Ca2+-dependent functions to ensure the stability of basal beating and to facilitate coordinated responses of these functions in response when a change in beating rate is required.
This work was supported, in part, by the Intramural Research Program of the National Institutes of Health, National Institute on Aging.
Original received August 22, 2005; revision received December 28, 2005; accepted January 11, 2006.
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