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 beta-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 beta-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.     

A full range of mouse sinoatrial node AP firing rates requires protein kinase A-dependent calcium signaling

Recent perspectives on sinoatrial nodal cell (SANC)^? function indicate that spontaneous sarcoplasmic reticulum (SR) Ca²⁺ cycling, i.e. an intracellular “Ca²⁺ clock,” driven by cAMP-mediated, PKA-dependent phosphorylation, interacts with an ensemble of surface membrane electrogenic molecules (“surface membrane clock”) to drive SANC normal automaticity. The role of AC-cAMP-PKA-Ca²⁺ signaling cascade in mouse, the species most often utilized for genetic manipulations, however, has not been systematically tested. Here we show that Ca²⁺ cycling proteins (e.g. RyR2, NCX1, and SERCA2) are abundantly expressed in mouse SAN and that spontaneous, rhythmic SR generated local Ca²⁺ releases (LCRs) occur in skinned mouse SANC, clamped at constant physiologic [Ca²⁺]. Mouse SANC also exhibits a high basal level of phospholamban (PLB) phosphorylation at the PKA-dependent site, Serine16. Inhibition of intrinsic PKA activity or inhibition of PDE in SANC, respectively: reduces or increases PLB phosphorylation, and markedly prolongs or reduces the LCR period; and markedly reduces or accelerates SAN spontaneous firing rate. Additionally, the increase in AP firing rate by PKA-dependent phosphorylation by β-adrenergic receptor (β-AR) stimulation requires normal intracellular Ca²⁺ cycling, because the β-AR chronotropic effect is markedly blunted when SR Ca²⁺ cycling is disrupted. Thus, AC-cAMP-PKA-Ca²⁺ signaling cascade is a major mechanism of normal automaticity in mouse SANC.     

The role of Ca2+ release from sarcoplasmic reticulum in the regulation of sinoatrial node automaticity

Summary The role of Ca²⁺ release channels in the sarcoplasmic reticulum in modulating physiological automaticity of the sinoatrial (SA) node was studied by recording transmembrane action potentials and membrane ionic currents in small preparations of the rabbit SA node. Ryanodine, which modifies the conductance and gating behavior of the Ca²⁺ release channels, was used to block Ca²⁺ release from the sarcoplasmic reticulum. Superfusion of 1-mM ryanodine decreased the spontaneous firing frequency as well as the maximal rate of depolarization of the SA, and these reductions reached a steady state within approximately 5min. The action potential recordings revealed that the latter part of diastolic depolarization was depressed and that the take-off potential became less negative. This suggested that the negative chronotropic effect of ryanodine resulted from the blockade of physiological Ca²⁺ release from the sarcoplasmic reticulum. In voltage clamp experiments, using double-microelectrode techniques, ryanodine did not markedly reduce the Ca²⁺ current (I_Ca) but decreased the delayed rectifying K+ current (I_K), the steady-state inward current (I_ss), and the hyperpolarization-activated inward current (I_h). These observations suggest that, even when the function of Ca²⁺ channels in the cell membrane is normally maintained, depression of Ca²⁺ release channels in the sarcoplasmic reticulum would prevent sufficient elevation of the Ca²⁺ concentration in SA node cells for the activation of various ionic currents, and, thus adversely affect the physiological automaticity of this primary cardiac pacemaker.     

Beat-to-beat Ca(2+)-dependent regulation of sinoatrial nodal pacemaker cell rate and rhythm

Whether intracellular Ca²⁺ regulates sinoatrial node cell (SANC) action potential (AP) firing rate on a beat-to-beat basis is controversial. To directly test the hypothesis of beat-to-beat intracellular Ca²⁺ regulation of the rate and rhythm of SANC we loaded single isolated SANC with a caged Ca²⁺ buffer, NP-EGTA, and simultaneously recorded membrane potential and intracellular Ca²⁺. Prior to introduction of the caged Ca²⁺ buffer, spontaneous local Ca²⁺ releases (LCRs) during diastolic depolarization were tightly coupled to rhythmic APs (r² = 0.9). The buffer markedly prolonged the decay time (T₅₀) and moderately reduced the amplitude of the AP-induced Ca²⁺ transient and partially depleted the SR load, suppressed spontaneous diastolic LCRs and uncoupled them from AP generation, and caused AP firing to become markedly slower and dysrhythmic. When Ca²⁺ was acutely released from the caged compound by flash photolysis, intracellular Ca²⁺ dynamics were acutely restored and rhythmic APs resumed immediately at a normal rate. After a few rhythmic cycles, however, these effects of the flash waned as interference with Ca²⁺ dynamics by the caged buffer was reestablished. Our results directly support the hypothesis that intracellular Ca²⁺ regulates normal SANC automaticity on a beat-to-beat basis.     

Ca2+-regulated-cAMP/PKA signaling in cardiac pacemaker cells links ATP supply to demand

Rationale

In sinoatrial node cells (SANC), Ca²⁺ activates adenylate cyclase (AC) to generate a high basal level of cAMP-mediated/protein kinase A (PKA)-dependent phosphorylation of Ca²⁺ cycling proteins. These result in spontaneous sarcoplasmic-reticulum (SR) generated rhythmic Ca²⁺ oscillations during diastolic depolarization, that not only trigger the surface membrane to generate rhythmic action potentials (APs), but, in a feed-forward manner, also activate AC/PKA signaling. ATP is consumed to pump Ca²⁺ to the SR, to produce cAMP, to support contraction and to maintain cell ionic homeostasis.

Objective

Since feedback mechanisms link ATP-demand to ATP production, we hypothesized that (1) both basal ATP supply and demand in SANC would be Ca²⁺-cAMP/PKA dependent; and (2) due to its feed-forward nature, a decrease in flux through the Ca²⁺-cAMP/PKA signaling axis will reduce the basal ATP production rate.

Methods and results

O₂ consumption in spontaneous beating SANC was comparable to ventricular myocytes (VM) stimulated at 3 Hz. Graded reduction of basal Ca²⁺-cAMP/PKA signaling to reduce ATP demand in rabbit SANC produced graded ATP depletion (r² = 0.96), and reduced O₂ consumption and flavoprotein fluorescence. Neither inhibition of glycolysis, selectively blocking contraction nor specific inhibition of mitochondrial Ca²⁺ flux reduced the ATP level.

Conclusions

Feed-forward basal Ca²⁺-cAMP/PKA signaling both consumes ATP to drive spontaneous APs in SANC and is tightly linked to mitochondrial ATP production. Interfering with Ca²⁺-cAMP/PKA signaling not only slows the firing rate and reduces ATP consumption, but also appears to reduce ATP production so that ATP levels fall. This distinctly differs from VM, which lack this feed-forward basal cAMP/PKA signaling, and in which ATP level remains constant when the demand changes.     

Beat-to-beat Ca2+-dependent regulation of sinoatrial nodal pacemaker cell rate and rhythm

Whether intracellular Ca²⁺ regulates sinoatrial node cell (SANC) action potential (AP) firing rate on a beat-to-beat basis is controversial. To directly test the hypothesis of beat-to-beat intracellular Ca²⁺ regulation of the rate and rhythm of SANC we loaded single isolated SANC with a caged Ca²⁺ buffer, NP-EGTA, and simultaneously recorded membrane potential and intracellular Ca²⁺. Prior to introduction of the caged Ca²⁺ buffer, spontaneous local Ca²⁺ releases (LCRs) during diastolic depolarization were tightly coupled to rhythmic APs (r² = 0.9). The buffer markedly prolonged the decay time (T₅₀) and moderately reduced the amplitude of the AP-induced Ca²⁺ transient and partially depleted the SR load, suppressed spontaneous diastolic LCRs and uncoupled them from AP generation, and caused AP firing to become markedly slower and dysrhythmic. When Ca²⁺ was acutely released from the caged compound by flash photolysis, intracellular Ca²⁺ dynamics were acutely restored and rhythmic APs resumed immediately at a normal rate. After a few rhythmic cycles, however, these effects of the flash waned as interference with Ca²⁺ dynamics by the caged buffer was reestablished. Our results directly support the hypothesis that intracellular Ca²⁺ regulates normal SANC automaticity on a beat-to-beat basis.     

Rhythmic ryanodine receptor Ca2+ releases during diastolic depolarization of sinoatrial pacemaker cells do not require membrane depolarization

Localized, subsarcolemmal Ca2+ release (LCR) via ryanodine receptors (RyRs) during diastolic depolarization of sinoatrial nodal cells augments the terminal depolarization rate. We determined whether LCRs in rabbit sinoatrial nodal cells require the concurrent membrane depolarization, or are intrinsically rhythmic, and whether rhythmicity is linked to the spontaneous cycle length. Confocal linescan images revealed persistent LCRs both in saponin-permeabilized cells and in spontaneously beating cells acutely voltage-clamped at the maximum diastolic potential. During the initial stage of voltage clamp, the LCR spatiotemporal characteristics did not differ from those in spontaneously beating cells, or in permeabilized cells bathed in 150 nmol/L Ca2+. The period of persistent rhythmic LCRs during voltage clamp was slightly less than the spontaneous cycle length before voltage clamp. In spontaneously beating cells, in both transient and steady states, LCR period was highly correlated with the spontaneous cycle length; and regardless of the cycle length, LCRs occurred predominantly at a constant time, ie, 80% to 90% of the cycle length. Numerical model simulations incorporating LCRs reproduce the experimental results. We conclude that diastolic LCRs reflect rhythmic intracellular Ca2+ cycling that does not require the concomitant membrane depolarization, and that LCR periodicity is closely linked to the spontaneous cycle length. Thus, the biological clock of sinoatrial nodal pacemaker cells, like that of many other rhythmic functions occurring throughout nature, involves an intracellular Ca2+ rhythm.     

Local control of Ca-induced Ca release in mouse sinoatrial node cells

Emerging evidence from large animal models implicates Ca²⁺ regulation, particularly intracellular sarcoplasmic reticulum (SR) Ca²⁺ release, as essential for sinoatrial node (SAN) automaticity. However, despite the apparent importance of SR Ca²⁺ release to SAN cell function it is uncertain how SR Ca²⁺ release is controlled in SAN cells from mouse. Understanding mouse SAN SR Ca²⁺ release mechanism will allow improved understanding of results in studies on SAN from genetic mouse models of Ca²⁺ homeostatic proteins. Here we investigated the functional relationship between sarcolemmal Ca²⁺ influx and SR Ca²⁺ release at the level of single SAN cell, using simultaneous patch-clamp current recording and high resolution confocal Ca²⁺ imaging techniques. In mouse SAN cells, both Ca²⁺ channel currents and triggered SR Ca²⁺ transients displayed bell-shaped, graded function with the membrane potential. Moreover, the gain function for Ca²⁺-induced Ca²⁺ release (CICR) displayed a monotonically decreasing function with strong voltage dependence, consistent with a “local control” mechanism for CICR. In addition, we observed numerous discrete Ca²⁺ sparks at the voltage range of diastolic depolarization, in sharp contrast to the much lower frequency of sparks observed at resting potentials. We concluded that the “local control” mechanism of CICR is responsible for both local Ca²⁺ release during diastolic depolarization and the synchronized Ca²⁺ transients observed during action potential in SAN cells.     

SR Ca store refill—a key factor in cardiac pacemaking

This study presents a theoretical analysis of the role of store Ca²⁺ uptake on sinoatrial node (SAN) cell pacemaking. Two mechanisms have been shown to be involved in SAN pacemaking, these being: 1) the membrane oscillator model where rhythm generation is based on the interaction of voltage-dependent membrane ion channels and, 2) the store oscillator model where cyclical release of Ca²⁺ from intracellular Ca²⁺ stores depolarizes the membrane through activation of the sodium-calcium exchanger (NCX). The relative roles of these oscillators in generation and modulation of pacemaker rate have been vigorously debated and have many consequences. The main new outcomes of our study are: 1) uptake of Ca²⁺ by intracellular Ca²⁺ stores increases the maximum diastolic potential (MDP) by reducing the cytosolic Ca²⁺ concentration [Ca²⁺]_c and hence decreasing the NCX current; 2) this hyperpolarization enhances recruitment of key pacemaker currents (e.g. the hyperpolarization-activated HCN current (I_f) and T-type Ca²⁺ current (I_T-Ca)); 3) the resultant enhanced Ca²⁺ entry during the pacemaker depolarization increases [Ca²⁺]_c causing advancement of the store Ca²⁺ release cycle and increased NCX current. In overview, the novel feature of our study is an investigation of the role of store Ca²⁺ uptake on SAN pacemaking. This occurs during the early diastolic period and causes enhanced I_f, I_T-Ca and store release (and hence I_NCX) during the later diastolic period. There is thus a symbiotic interaction between the two pacemaker “clocks” over the entire diastolic period, this providing robust and highly malleable SAN pacemaking. Accounting for store Ca²⁺ uptake also provides insight into hitherto unexplained SAN behaviour, as we exemplify for the sinus bradycardia exhibited in catecholaminergic polymorphic ventricular tachycardia (CPVT).     

Dynamic interactions of an intracellular Ca2+ clock and membrane ion channel clock underlie robust initiation and regulation of cardiac pacemaker function

For almost half a century it has been thought that the initiation of each heartbeat is driven by surface membrane voltage-gated ion channels (M clocks) within sinoatrial nodal cells. It has also been assumed that pacemaker cell automaticity is initiated at the maximum diastolic potential (MDP). Recent experimental evidence based on confocal cell imaging and supported by numerical modelling, however, shows that initiation of cardiac impulse is a more complex phenomenon and involves yet another clock that resides under the sarcolemma. This clock is the sarcoplasmic reticulum (SR): it generates spontaneous, but precisely timed, rhythmic, submembrane, local Ca(2+) releases (LCR) that appear not at the MDP but during the late, diastolic depolarization (DD). The Ca(2+) clock and M clock dynamically interact, defining a novel paradigm of robust cardiac pacemaker function and regulation. Rhythmic LCRs during the late DD activate inward Na(+)/Ca(2+) exchanger currents and ignite action potentials, which in turn induceCa(2+) transients and SR depletions, resetting the Ca(2+) clock. Both basal and reserve protein kinaseA-dependent phosphorylation of Ca(2+) cycling proteins control the speed and amplitude of SR Ca(2+) cycling to regulate the beating rate by strongly coupled Ca(2+) and M clocks.     

Diastolic transient inward current in long QT syndrome type 3 is caused by Ca overload and inhibited by ranolazine

Long QT syndrome variant 3 (LQT-3) is a channelopathy in which mutations in SCN5A, the gene coding for the primary heart Na⁺ channel alpha subunit, disrupt inactivation to elevate the risk of mutation carriers for arrhythmias that are thought to be calcium (Ca²⁺)-dependent. Spontaneous arrhythmogenic diastolic activity has been reported in myocytes isolated from mice harboring the well-characterized ΔKPQ LQT-3 mutation but the link to altered Ca²⁺ cycling related to mutant Na⁺ channel activity has not previously been demonstrated. Here we have investigated the relationship between elevated sarcoplasmic reticulum (SR) Ca²⁺ load and induction of spontaneous diastolic inward current (I_TI) in myocytes expressing ΔKPQ Na⁺ channels, and tested the sensitivity of both to the antianginal compound ranolazine. We combined whole-cell patch clamp measurements, imaging of intracellular Ca²⁺, and measurement of SR Ca²⁺ content using a caffeine dump methodology. We compared the Ca²⁺ content of ΔKPQ^+/− myocytes displaying I_TI to those without spontaneous diastolic activity and found that I_TI induction correlates with higher sarcoplasmic reticulum (SR) Ca²⁺. Both spontaneous diastolic I_TI and underlying Ca²⁺ waves are inhibited by ranolazine at concentrations that preferentially target I_NaL during prolonged depolarization. Furthermore, ranolazine I_TI inhibition is accompanied by a small but significant decrease in SR Ca²⁺ content. Our results provide the first direct evidence that induction of diastolic transient inward current (I_TI) in ΔKPQ^+/− myocytes occurs under conditions of elevated SR Ca²⁺ load.     

If and SR Ca release both contribute to pacemaker activity in canine sinoatrial node cells

Increasing evidence suggests that cardiac pacemaking is the result of two sinoatrial node (SAN) cell mechanisms: a ‘voltage clock’ and a Ca²⁺ dependent process, or ‘Ca²⁺ clock.’ The voltage clock initiates action potentials (APs) by SAN cell membrane potential depolarization from inward currents, of which the pacemaker current (I_f) is thought to be particularly important. A Ca²⁺ dependent process triggers APs when sarcoplasmic reticulum (SR) Ca²⁺ release activates inward current carried by the forward mode of the electrogenic Na⁺/Ca²⁺ exchanger (NCX). However, these mechanisms have mostly been defined in rodents or rabbits, but are unexplored in single SAN cells from larger animals. Here, we used patch-clamp and confocal microscope techniques to explore the roles of the voltage and Ca²⁺ clock mechanisms in canine SAN pacemaker cells. We found that ZD7288, a selective I_f antagonist, significantly reduced basal automaticity and induced irregular, arrhythmia-like activity in canine SAN cells. In addition, ZD7288 impaired but did not eliminate the SAN cell rate acceleration by isoproterenol. In contrast, ryanodine significantly reduced the SAN cell acceleration by isoproterenol, while ryanodine reduction of basal automaticity was modest (∼ 14%) and did not reach statistical significance. Importantly, pretreatment with ryanodine eliminated SR Ca²⁺ release, but did not affect basal or isoproterenol-enhanced I_f. Taken together, these results indicate that voltage and Ca²⁺ dependent automaticity mechanisms coexist in canine SAN cells, and suggest that I_f and SR Ca²⁺ release cooperate to determine baseline and catecholamine-dependent automaticity in isolated dog SAN cells.     

Differential effects of ivabradine and ryanodine on pacemaker activity in canine sinus node and purkinje fibers

Effects of Ivabradine and Ryanodine on Cardiac Pacemakers . Introduction: It is generally accepted that at least 2 major mechanisms contribute to sinus node (SN) pacemaking: a membrane voltage (mainly I_f) clock and a calcium (Ca) clock (localized submembrane sarcoplasmic reticulum Ca²⁺ release during late diastolic depolarization). The aim of this study was to compare the contributions of each mechanism to pacemaker activity in SN and Purkinje fibers (PFs) exhibiting normal or abnormal automaticity. Methods and Results: Conventional microelectrodes were used to record action potentials in isolated spontaneously beating canine SN and free running PF in control and in the presence of 0.1 μM isoproterenol. Ryanodine (0.1–3 μM) and ivabradine (3 μM) were used to inhibit sarcoplasmic reticulum Ca²⁺ release or I_f, respectively. To induce automaticity at low membrane potentials, PFs were superfused with BaCl₂. In SN, ivabradine reduced the rate whereas ryanodine had no effect. Isoproterenol significantly accelerated automatic rate, which was decreased by ivabradine and ryanodine. In normally polarized PFs, ryanodine had no effects on the automatic rate in the absence or presence of isoproterenol, whereas ivabradine inhibited both control and isoproterenol‐accelerated automaticity. In PF depolarized with BaCl₂, ivabradine decreased BaCl₂‐induced automatic rate while ryanodine had no effect. Conclusion: In canine SN, I_f contributes to both basal automaticity and β‐adrenergic‐induced rate acceleration while the ryanodine‐inhibited Ca clock appears more involved in β‐adrenergic regulation of pacemaker rate. In PF, normal automaticity depends mainly on I_f. Inhibition of basal potassium conductance results in high automatic rates at depolarized membrane potentials with SN‐like responses to inhibition of membrane and Ca clocks. (J Cardiovasc Electrophysiol, Vol. 23, pp. 650–655, June 2012)     

If at first you don't succeed try ... a new target in the treatment of angina.

Tardiff et al.¹ describe a promising new treatment forangina, ivabradine, that appears to selectively reduce heartrate by blocking the cardiac pacemaker ‘funny’ current,I_f. In contrast to the existing agents that slow heart rate,there is no accompanying intrinsic negative inotropic effect.Understanding the basis of this unique property requires a briefoverview of the underlying physiology. The cells of the sinoatrial node are the primary pacemaker ofthe heart. This function is the result of an upward, positive,depolarizing drift in their resting membrane potential duringthe diastolic phase of the action potential, known as the pacemakercurrent. Once this drift reaches a threshold potential, thenext action potential is triggered by the opening of slow calciumchannels and consequent influx of Ca²⁺ ions. Calcium antagonistsreduce Ca²⁺ influx through slow (L-type) calcium channels andthis leads to prolongation of the depolarization     

Calcium cycling protein density and functional importance to automaticity of isolated sinoatrial nodal cells are independent of cell size

Spontaneous, localized, rhythmic ryanodine receptor (RyRs) Ca(2+) releases occur beneath the cell membrane during late diastolic depolarization in cardiac sinoatrial nodal cells (SANCs). These activate the Na(+)/Ca(2+) exchanger (NCX1) to generate inward current and membrane excitation that drives normal spontaneous beating. The morphological background for the proposed functional of RyR and NCX crosstalk, however, has not been demonstrated. Here we show that the average isolated SANC whole cell labeling density of RyRs and SERCA2 is similar to atrial and ventricle myocytes, and is similar among SANCs of all sizes. Labeling of NCX1 is also similar among SANCs of all sizes and exceeds that in atrial and ventricle myocytes. Submembrane colocalization of NCX1 and cardiac RyR (cRyR) in all SANCs exceeds that in the other cell types. Further, the Cx43 negative primary pacemaker area of the intact rabbit sinoatrial node (SAN) exhibits robust positive labeling for cRyR, NCX1, and SERCA2. Functional studies in isolated SANCs show that neither the average action potential (AP) characteristics, nor those of intracellular Ca(2+) releases, nor the spontaneous cycle length vary with cell size. Chelation of intracellular [Ca(2+)], or disabling RyRs or NCX1, markedly attenuates or abolishes spontaneous SANC beating in all SANCs. Thus, there is dense labeling of SERCA2, RyRs, and NCX1 in small-sized SANCs, thought to reside within the SAN center, the site of impulse initiation. Because normal automaticity of these cells requires intact Ca(2+) cycling, interactions of SERCA, RyR2 and NCX molecules are implicated in the initiation of the SAN impulse.     

The human CASQ2 mutation K206N is associated with hyperglycosylation and altered cellular calcium handling

Mutations in the human cardiac calsequestrin gene (CASQ2) are linked to catecholaminergic polymorphic ventricular tachycardia (CPVT-2). This inherited disorder is characterized by life-threatening arrhythmias induced by physical and emotional stress in young patients. Here we identified a novel heterozygous missense mutation (K206N) in the CASQ2 gene in a symptomatic family in which one member died of cardiac arrest. The functional properties of CSQ^K206N were investigated in comparison to the wild-type form of CASQ2 (CSQ^WT) by expression in eukaryotic cell lines and neonatal mouse myocytes. The mutation created an additional N-glycosylation site resulting in a higher molecular weight form of the recombinant protein on immunoblots. The mutation reduced the Ca²⁺ binding capacity of the protein and exhibited an altered aggregation state. Consistently, CSQ^K206N-expressing myocytes exhibited an impaired response to caffeine administration, suggesting a lower Ca²⁺ load of the sarcoplasmic reticulum (SR). The interaction of the mutated CSQ with triadin and the protein levels of the ryanodine receptor were unchanged but the maximal specific [³H]ryanodine binding was increased in CSQ^K206N-expressing myocytes, suggesting a higher opening state of the SR Ca²⁺ release channel. Myocytes with expression of CSQ^K206N showed a higher rate of spontaneous SR Ca²⁺ releases under basal conditions and after β-adrenergic stimulation. We conclude that CSQ^K206N caused a reduced Ca²⁺ binding leading to an abnormal regulation of intracellular Ca²⁺ in myocytes. This may then contribute to the increased propensity to trigger spontaneous Ca²⁺ transients in CSQ^K206N-expressing myocytes.     

Sustained inward current during pacemaker depolarization in mammalian sinoatrial node cells

Several time- and voltage-dependent ionic currents have been identified in cardiac pacemaker cells, including Na(+) current, L- and T-type Ca(2+) currents, hyperpolarization-activated cation current, and various types of delayed rectifier K(+) currents. Mathematical models have demonstrated that spontaneous action potentials can be reconstructed by incorporating these currents, but relative contributions of individual currents vary widely between different models. In 1995, the presence of a novel inward current that was activated by depolarization to the potential range of the slow diastolic depolarization in rabbit sinoatrial (SA) node cells was reported. Because the current showed little inactivation during depolarizing pulses, it was called the sustained inward current (I(st)). A similar current is also found in SA node cells of the guinea pig and rat and in subsidiary pacemaker atrioventricular node cells. Recently, single-channel analysis has revealed a nicardipine-sensitive, 13-pS Na(+) current, which is activated by depolarization to the diastolic potential range in guinea pig SA node cells. This channel differs from rapid voltage-gated Na(+) or L-type Ca(2+) channels both in unitary conductance and gating kinetics. Because I(st) was observed only in spontaneously beating SA node cells, ie, it was absent in quiescent cells dissociated from the same SA or atrioventricular node, an important role of I(st) for generation of intrinsic cardiac automaticity was suggested.     

Normal heart rhythm is initiated and regulated by an intracellular calcium clock within pacemaker cells

For almost half a century it has been thought that the heart rhythm originates on the surface membrane of the cardiac pacemaker cells and is driven by voltage-gated ion channels (membrane clocks). Data from several recent studies, however, conclusively show that the rhythm is initiated, sustained, and regulated by oscillatory Ca(2+) releases (Ca(2+) clock) from the sarcoplasmic reticulum, a major Ca(2+) store within sinoatrial node cells, the primary heart's pacemakers. Activation of the local oscillatory Ca(2+) releases is independent of membrane depolarisation and driven by a high level of basal state phosphorylation of Ca(2+) cycling proteins. The releases produce Ca(2+) wavelets under the cell surface membrane during the later phase of diastolic depolarisation and activate the forward mode of Na(+)/Ca(2+) exchanger resulting in inward membrane current, which ignites an action potential. Phosphorylation-dependent gradation of speed at which Ca(2+) clock cycles is the essential regulatory mechanism of normal pacemaker rate and rhythm. The robust regulation of pacemaker function is insured by tight integration of Ca(2+) and membrane clocks: the action potential shape and ion fluxes are tuned by membrane clocks to sustain operation of the Ca(2+) clock which produces timely and powerful ignition of the membrane clocks to effect action potentials.     

Cyclic variation of intracellular calcium: a critical factor for cardiac pacemaker cell dominance

While a diversity of cell types and distribution within the sinoatrial node and cell-cell interactions add complexity to a complete elucidation of the heart's pacemaker function, it has become clear that cyclic variation of submembrane [Ca2+] and activation of the Na+-Ca2+ exchanger during diastolic depolarization (DD) act in concert with ion channels to confer on sinoatrial node cells (SANCs) their status of dominance with respect to pacemaker function. Studies using confocal microscopy indicate that subsarcolemmal Ca2+ release via ryanodine receptors occurs not only in response to the action potential (AP) upstroke, but also during the DD, and this is augmented by beta-adrenergic receptor (beta-AR) stimulation. Spontaneous APs simulated by mathematical SANC models beat at a faster rate when this subsarcolemmal Ca2+ waveform measured under beta-AR stimulation is introduced into the modeling scheme. Thus, in future investigation of pacemaker functioning in health, disease, and disease therapies the "bar ought to be raised" to embrace the impact of cyclic variation in submembrane [Ca2+] on pacemaker function. The full text of this article is available at http://www.circresaha.org.