Spontaneous, submembrane local Ca(2+) releases (LCRs) generated by the sarcoplasmic reticulum in sinoatrial nodal cells, the cells of the primary cardiac pacemaker, activate inward ...Na(+)/Ca(2+)-exchange current to accelerate the diastolic depolarization rate, and therefore to impact on cycle length. Since LCRs are generated by Ca(2+) release channel (i.e. ryanodine receptor) openings, they exhibit a degree of stochastic behavior, manifested as notable cycle-to-cycle variations in the time of their occurrence.
The present study tested whether variation in LCR periodicity contributes to intrinsic (beat-to-beat) cycle length variability in single sinoatrial nodal cells.
We imaged single rabbit sinoatrial nodal cells using a 2D-camera to capture LCRs over the entire cell, and, in selected cells, simultaneously measured action potentials by perforated patch clamp.
LCRs begin to occur on the descending part of the action potential-induced whole-cell Ca(2+) transient, at about the time of the maximum diastolic potential. Shortly after the maximum diastolic potential (mean 54±7.7 ms, n = 14), the ensemble of waxing LCR activity converts the decay of the global Ca(2+) transient into a rise, resulting in a late, whole-cell diastolic Ca(2+) elevation, accompanied by a notable acceleration in diastolic depolarization rate. On average, cells (n = 9) generate 13.2±3.7 LCRs per cycle (mean±SEM), varying in size (7.1±4.2 µm) and duration (44.2±27.1 ms), with both size and duration being greater for later-occurring LCRs. While the timing of each LCR occurrence also varies, the LCR period (i.e. the time from the preceding Ca(2+) transient peak to an LCR's subsequent occurrence) averaged for all LCRs in a given cycle closely predicts the time of occurrence of the next action potential, i.e. the cycle length.
Intrinsic cycle length variability in single sinoatrial nodal cells is linked to beat-to-beat variations in the average period of individual LCRs each cycle.
Uptake and release calcium from the sarcoplasmic reticulum (SR) (dubbed "calcium clock"), in the form of spontaneous, rhythmic, local diastolic calcium releases (LCRs), together with ...voltage-sensitive ion channels (membrane clock) form a coupled system that regulates the action potential (AP) firing rate. LCRs activate Sodium/Calcium exchanger (NCX) that accelerates diastolic depolarization and thus participating in regulation of the time at which the next AP will occur. Previous studies in rabbit SA node cells (SANC) demonstrated that the basal AP cycle length (APCL) is tightly coupled to the basal LCR period (time from the prior AP-induced Ca2+ transient to the diastolic LCR occurrence), and that this coupling is further modulated by autonomic receptor stimulation. Although spontaneous LCRs during diastolic depolarization have been reported in SANC of various species (rabbit, cat, mouse, toad), prior studies have failed to detect LCRs in spontaneously beating SANC of guinea-pig, a species that has been traditionally used in studies of cardiac pacemaker cell function. We performed a detailed investigation of whether guinea-pig SANC generate LCRs and whether they play a similar key role in regulation of the AP firing rate. We used two different approaches, 2D high-speed camera and classical line-scan confocal imaging. Positioning the scan-line beneath sarcolemma, parallel to the long axis of the cell, we found that rhythmically beating guinea-pig SANC do, indeed, generate spontaneous, diastolic LCRs beneath the surface membrane. The average key LCR characteristics measured in confocal images in guinea-pig SANC were comparable to rabbit SANC, both in the basal state and in the presence of β-adrenergic receptor stimulation. Moreover, the relationship between the LCR period and APCL was subtended by the same linear function. Thus, LCRs in guinea-pig SANC contribute to the diastolic depolarization and APCL regulation. Our findings indicate that coupled-clock system regulation of APCL is a general, species-independent, mechanism of pacemaker cell normal automaticity. Lack of LCRs in prior studies is likely explained by technical issues, as individual LCRs are small stochastic events occurring mainly near the cell border.
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•Cells isolated from SA node include dormant, dysrhythmic, and rhythmic cells.•All cells generate Local Ca Releases.•Local Ca releases can be increased and synchronized by ...β-adrenergic stimulation.•Dormant & dysrhythmic cells can fire rhythmically on β-adrenergic stimulation.
Current understanding of how cardiac pacemaker cells operate is based mainly on studies in isolated single sinoatrial node cells (SANC), specifically those that rhythmically fire action potentials similar to the in vivo behavior of the intact sinoatrial node. However, only a small fraction of SANC exhibit rhythmic firing after isolation. Other SANC behaviors have not been studied.
Here, for the first time, we studied all single cells isolated from the sinoatrial node of the guinea pig, including traditionally studied rhythmically firing cells (‘rhythmic SANC’), dysrhythmically firing cells (‘dysrhythmic SANC’) and cells without any apparent spontaneous firing activity (‘dormant SANC’). Action potential-induced cytosolic Ca2+ transients and spontaneous local Ca2+ releases (LCRs) were measured with a 2D camera.
LCRs were present not only in rhythmically firing SANC, but also in dormant and dysrhythmic SANC. While rhythmic SANC were characterized by large LCRs synchronized in space and time towards late diastole, dysrhythmic and dormant SANC exhibited smaller LCRs that appeared stochastically and were widely distributed in time. β-adrenergic receptor (βAR) stimulation increased LCR size and synchronized LCR occurrences in all dysrhythmic and a third of dormant cells (25 of 75 cells tested). In response to βAR stimulation, these dormant SANC developed automaticity, and LCRs became coupled to spontaneous action potential-induced cytosolic Ca2+ transients. Conversely, dormant SANC that did not develop automaticity showed no significant change in average LCR characteristics. The majority of dysrhythmic cells became rhythmic in response to βAR stimulation, with the rate of action potential-induced cytosolic Ca2+ transients substantially increasing. In summary, isolated SANC can be broadly categorized into three major populations: dormant, dysrhythmic, and rhythmic. We interpret our results based on simulations of a numerical model of SANC operating as a coupled-clock system. On this basis, the two previously unstudied dysrhythmic and dormant cell populations have intrinsically partially or completely uncoupled clocks. Such cells can be recruited to fire rhythmically in response to βAR stimulation via increased rhythmic LCR activity and ameliorated coupling between the Ca2+ and membrane clocks.
The sinoatrial node, whose cells (sinoatrial node cells SANCs) generate rhythmic action potentials, is the primary pacemaker of the heart. During diastole, calcium released from the sarcoplasmic ...reticulum (SR) via ryanodine receptors (RyRs) interacts with membrane currents to control the rate of the heartbeat. This "calcium clock" takes the form of stochastic, partially periodic, localized calcium release (LCR) events that propagate, wave-like, for limited distances. The detailed mechanisms controlling the calcium clock are not understood. We constructed a computational model of SANCs, including three-dimensional diffusion and buffering of calcium in the cytosol and SR; explicit, stochastic gating of individual RyRs and L-type calcium channels; and a full complement of voltage- and calcium-dependent membrane currents. We did not include an anatomical submembrane space or inactivation of RyRs, the two heuristic components that have been used in prior models but are not observed experimentally. When RyRs were distributed in discrete clusters separated by >1 µm, only isolated sparks were produced in this model and LCR events did not form. However, immunofluorescent staining of SANCs for RyR revealed the presence of bridging RyR groups between large clusters, forming an irregular network. Incorporation of this architecture into the model led to the generation of propagating LCR events. Partial periodicity emerged from the interaction of LCR events, as observed experimentally. This calcium clock becomes entrained with membrane currents to accelerate the beating rate, which therefore was controlled by the activity of the SERCA pump, RyR sensitivity, and L-type current amplitude, all of which are targets of β-adrenergic-mediated phosphorylation. Unexpectedly, simulations revealed the existence of a pathological mode at high RyR sensitivity to calcium, in which the calcium clock loses synchronization with the membrane, resulting in a paradoxical decrease in beating rate in response to β-adrenergic stimulation. The model indicates that the hierarchical clustering of surface RyRs in SANCs may be a crucial adaptive mechanism. Pathological desynchronization of the clocks may explain sinus node dysfunction in heart failure and RyR mutations.
The heartbeat is initiated by pacemaker cells residing in the sinoatrial node (SAN). SAN cells generate spontaneous action potentials (APs), i.e., normal automaticity. The sympathetic nervous system ...increases the heart rate commensurate with the cardiac output demand via stimulation of SAN β-adrenergic receptors (βAR). While SAN cells reportedly represent a highly heterogeneous cell population, the current dogma is that, in response to βAR stimulation, all cells increase their spontaneous AP firing rate in a similar fashion. The aim of the present study was to investigate the cell-to-cell variability in the responses of a large population of SAN cells. We measured the βAR responses among 166 single SAN cells isolated from 33 guinea pig hearts. In contrast to the current dogma, the SAN cell responses to βAR stimulation substantially varied. In each cell, changes in the AP cycle length were highly correlated (R
= 0.97) with the AP cycle length before βAR stimulation. While, as expected, on average, the cells increased their pacemaker rate, greater responses were observed in cells with slower basal rates, and vice versa: cells with higher basal rates showed smaller responses, no responses, or even decreased their rate. Thus, βAR stimulation synchronized the operation of the SAN cell population toward a higher average rate, rather than uniformly shifting the rate in each cell, creating a new paradigm of βAR-driven fight-or-flight responses among individual pacemaker cells.
In sinoatrial node cells of the heart, beating rate is controlled, in part, by local Ca²⁺ releases (LCRs) from the sarcoplasmic reticulum, which couple to the action potential via electrogenic ...Na⁺/Ca²⁺ exchange. We observed persisting, roughly periodic LCRs in depolarized rabbit sinoatrial node cells (SANCs). The features of these LCRs were reproduced by a numerical model consisting of a two-dimensional array of stochastic, diffusively coupled Ca²⁺ release units (CRUs) with fixed refractory period. Because previous experimental studies showed that β-adrenergic receptor stimulation increases the rate of Ca²⁺ release through each CRU (dubbed Iₛₚₐᵣₖ), we explored the link between LCRs and Iₛₚₐᵣₖ in our model. Increasing the CRU release current Iₛₚₐᵣₖ facilitated Ca²⁺-induced-Ca²⁺ release and local recruitment of neighboring CRUs to fire more synchronously. This resulted in a progression in simulated LCR size (from sparks to wavelets to global waves), LCR rhythmicity, and decrease of LCR period that parallels the changes observed experimentally with β-adrenergic receptor stimulation. The transition in LCR characteristics was steeply nonlinear over a narrow range of Iₛₚₐᵣₖ, resembling a phase transition. We conclude that the (partial) periodicity and rate regulation of the “Calcium clock” in SANCs are emergent properties of the diffusive coupling of an ensemble of interacting stochastic CRUs. The variation in LCR period and size with Iₛₚₐᵣₖ is sufficient to account for β-adrenergic regulation of SANC beating rate.
Basal phosphorylation of sarcoplasmic reticulum (SR) Ca2+ proteins is high in sinoatrial nodal cells (SANC), which generate partially synchronized, spontaneous, rhythmic, diastolic local Ca2+ ...releases (LCRs), but low in ventricular myocytes (VM), which exhibit rare diastolic, stochastic SR-generated Ca2+ sparks. We tested the hypothesis that in a physiologic Ca2+ milieu, and independent of increased Ca2+ influx, an increase in basal phosphorylation of SR Ca2+ cycling proteins will convert stochastic Ca2+ sparks into periodic, high-power Ca2+ signals of the type that drives SANC normal automaticity. We measured phosphorylation of SR-associated proteins, phospholamban (PLB) and ryanodine receptors (RyR), and spontaneous local Ca2+ release characteristics (LCR) in permeabilized single, rabbit VM in physiologic Ca2+, prior to and during inhibition of protein phosphatase (PP) and phosphodiesterase (PDE), or addition of exogenous cAMP, or in the presence of an antibody (2D12), that specifically inhibits binding of the PLB to SERCA-2. In the absence of the aforementioned perturbations, VM could only generate stochastic local Ca2+ releases of low power and low amplitude, as assessed by confocal Ca2+ imaging and spectral analysis. When the kinetics of Ca2+ pumping into the SR were increased by an increase in PLB phosphorylation (via PDE and PP inhibition or addition of cAMP) or by 2D12, self-organized, “clock-like” local Ca2+ releases, partially synchronized in space and time (Ca2+ wavelets), emerged, and the ensemble of these rhythmic local Ca2+ wavelets generated a periodic high-amplitude Ca2+ signal. Thus, a Ca2+ clock is not specific to pacemaker cells, but can also be unleashed in VM when SR Ca2+ cycling increases and spontaneous local Ca2+ release becomes partially synchronized. This unleashed Ca2+ clock that emerges in a physiological Ca2+ milieu in VM has two faces, however: it can provoke ventricular arrhythmias; or if harnessed, can be an important feature of novel bio-pacemaker designs.
•A Ca2+ clock emerges in permeabilized ventricular myocytes at normal Ca2+i.•The Ca2+ clock has phosphorylation-dependent mechanism.•The Ca2+ clock is manifested by rhythmic local Ca2+ releases as in pacemaker cells.•The Ca2+ clock can be activated by PP and PDE inhibition.•The Ca2+ clock can be arrhythmogenic and also a candidate for biopacemaker design.
Abstract There is an intense interest in differentiating embryonic stem cells to engineer biological pacemakers as an alternative to electronic pacemakers for patients with cardiac pacemaker function ...deficiency. Embryonic stem cell-derived cardiocytes (ESCs), however, often exhibit dysrhythmic excitations. Using Ca2+ imaging and patch-clamp techniques, we studied requirements for generation of spontaneous rhythmic action potentials (APs) in late-stage mouse ESCs. Sarcoplasmic reticulum (SR) of ESCs generates spontaneous, rhythmic, wavelet-like L ocal C a2+ R eleases (LCRs) (inhibited by ryanodine, tetracaine, or thapsigargin). L-type Ca2+ current ( ICaL ) induces a global Ca2+ release (CICR), depleting the Ca2+ content SR which resets the phases of LCR oscillators. Following a delay, SR then generates a highly synchronized spontaneous Ca2+ release of multiple LCRs throughout the cell. The LCRs generate an inward Na+ / Ca2+ exchanger (NCX) current (absent in Na+ -free solution) that ignites the next AP. Interfering with SR Ca2+ cycling (ryanodine, caffeine, thapsigargin, cyclopiazonic acid, BAPTA-AM), NCX (Na+ -free solution), or ICaL (nifedipine) results in dysrhythmic excitations or cessation of automaticity. Inhibition of cAMP/PKA signaling by a specific PKA inhibitor, PKI, decreases SR Ca2+ loading, substantially reducing both spontaneous LCRs (number, size, and amplitude) and rhythmic AP firing. In contrast, enhancing PKA signaling by cAMP increases the LCRs (number, size, duration) and converts irregularly beating ESCs to rhythmic “pacemaker-like” cells. SR Ca2+ loading and LCR activity could be also increased with a selective activation of SR Ca2+ pumping by a phospholamban antibody. We conclude that SR Ca2+ loading and spontaneous rhythmic LCRs are driven by inherent cAMP/PKA activity. ICaL synchronizes multiple LCR oscillators resulting in strong, partially synchronized diastolic Ca2+ release and NCX current. Rhythmic ESC automaticity can be achieved by boosting “coupling” factors, such as cAMP/PKA signaling, that enhance interactions between SR and sarcolemma.
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