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Objectives This study sought to identify subcellular Ca2+ signals within and among cells comprising the sinoatrial node (SAN) tissue. Background The current paradigm of SAN impulse generation: 1) is that full-scale action potentials (APs) of a common frequency are initiated at 1 site and are conducted within the SAN along smooth isochrones; and 2) does not feature fine details of Ca2+ signaling present in isolated SAN cells, in which small subcellular, subthreshold local Ca2+ releases (LCRs) self-organize to generate cell-wide APs. Methods Immunolabeling was combined with a novel technique to detect the occurrence of LCRs and AP-induced Ca2+ transients (APCTs) in individual pixels (chronopix) across the entire mouse SAN images. Results At high magnification, Ca2+ signals appeared markedly heterogeneous in space, amplitude, frequency, and phase among cells comprising an HCN4+/CX43− cell meshwork. The signaling exhibited several distinguishable patterns of LCR/APCT interactions within and among cells. Rhythmic APCTs that were apparently conducted within the meshwork were transferred to a truly conducting HCN4−/CX43+ network of striated cells via narrow functional interfaces where different cell types intertwine, that is, the SAN anatomic/functional unit. At low magnification, the earliest APCT of each cycle occurred within a small area of the HCN4 meshwork, and subsequent APCT appearance throughout SAN pixels was discontinuous and asynchronous. Conclusions The study has discovered a novel, microscopic Ca2+ signaling paradigm of SAN operation that has escaped detection using low-resolution, macroscopic tissue isochrones employed in prior studies: synchronized APs emerge from heterogeneous subcellular subthreshold Ca2+ signals, resembling multiscale complex processes of impulse generation within clusters of neurons in neuronal networks.

Excitation–contraction coupling kinetics is dictated by the action potential rate of sinoatrial-nodal cells. These cells generate local Ca releases (LCRs) that activate Na/Ca exchanger current, which accelerates diastolic depolarization and determines the pace. LCRs are generated by clusters of ryanodine receptors, Ca release units (CRUs), residing in the sarcoplasmic reticulum. While CRU distribution exhibits substantial heterogeneity, its functional importance remains unknown. Using numerical modeling, here we show that with a square lattice distribution of CRUs, Ca-induced-Ca-release propagation during diastolic depolarization is insufficient for pacemaking within a broad range of realistic ICaL densities. Allowing each CRU to deviate randomly from its lattice position allows sparks to propagate, as observed experimentally. As disorder increases, the CRU distribution exhibits larger empty spaces and simultaneously CRU clusters, as in Poisson clumping. Propagating within the clusters, Ca release becomes synchronized, increasing action potential rate and reviving pacemaker function of dormant/nonfiring cells. However, cells with fully disordered CRU positions could not reach low firing rates and their β-adrenergic–receptor stimulation effect was substantially decreased. Inclusion of Cav1.3, a low-voltage activation L-type Ca channel isoform into ICaL, strongly increases recruitment of CRUs to fire during diastolic depolarization, increasing robustness of pacemaking and complementing effects of CRU distribution. Thus, order/disorder in CRU locations along with Cav1.3 expression regulates pacemaker function via synchronization of CRU firing. Excessive CRU disorder and/or overexpression of Cav1.3 boosts pacemaker function in the basal state, but limits the rate range, which may contribute to heart rate range decline with age and disease.

Spontaneous AP (action potential) firing of sinoatrial nodal cells (SANC) is critically dependent on protein kinase A (PKA) and Ca2+/calmodulin-dependent protein kinase II (CaMKII)-dependent protein phosphorylation, which are required for the generation of spontaneous, diastolic local Ca2+ releases (LCRs). Although phosphoprotein phosphatases (PP) regulate protein phosphorylation, the expression level of PPs and phosphatase inhibitors in SANC and the impact of phosphatase inhibition on the spontaneous LCRs and other players of the oscillatory coupled-clock system is unknown. Here, we show that rabbit SANC express both PP1, PP2A, and endogenous PP inhibitors I-1 (PPI-1), dopamine and cyclic adenosine 3′,5′-monophosphate (cAMP)-regulated phosphoprotein (DARPP-32), kinase C-enhanced PP1 inhibitor (KEPI). Application of Calyculin A, (CyA), a PPs inhibitor, to intact, freshly isolated single SANC: (1) significantly increased phospholamban (PLB) phosphorylation (by 2–3-fold) at both CaMKII-dependent Thr17 and PKA-dependent Ser16 sites, in a time and concentration dependent manner, (2) increased ryanodine receptor (RyR) phosphorylation at the Ser2809 site, (3) substantially increased sarcoplasmic reticulum (SR) Ca2+ load, (4) augmented L-type Ca2+ current amplitude, (5) augmented LCR’s characteristics and decreased LCR period in intact and permeabilized SANC, and (6) increased the spontaneous basal AP firing rate. In contrast, the selective PP2A inhibitor okadaic acid (100 nmol/L) had no significant effect on spontaneous AP firing, LCR parameters, or PLB phosphorylation. Application of purified PP1 to permeabilized SANC suppressed LCR, whereas purified PP2A had no effect on LCR characteristics. Our numerical model simulations demonstrated that PP inhibition increases AP firing rate via a coupled-clock mechanism, including respective increases in the SR Ca2+ pumping rate, L-type Ca2+ current, and Na+/Ca2+-exchanger current. Thus, PP1 and its endogenous inhibitors modulate the basal spontaneous firing rate of cardiac pacemaker cells by suppressing SR Ca2+ cycling protein phosphorylation, the SR Ca2+ load and LCRs, and L-type Ca2+ current.

OBJECTIVES: The purpose of this study was to discover regulatory universal mechanisms of normal automaticity in sinoatrial nodal (SAN) pacemaker cells that are self-similar across species. BACKGROUND: Translation of knowledge of SAN automaticity gleaned from animal studies to human dysrhythmias (e.g., “sick sinus” syndrome [SSS]) requiring electronic pacemaker insertion has been suboptimal, largely because heart rate varies widely across species. METHODS: Subcellular Ca(2+) releases, whole cell action potential (AP)–induced Ca(2+) transients, and APs were recorded in isolated mouse, guinea pig, rabbit, and human SAN cells. Ca(2+)-Vm kinetic parameters during phases of AP cycles from their ignition to recovery were quantified. RESULTS: Although both AP cycle lengths (APCLs) and Ca(2+)-Vm kinetic parameters during AP cycles differed across species by 10-fold, trans-species scaling of these during AP cycles and scaling of these to APCL in cells in vitro, electrocardiogram RR intervals in vivo, and body mass (BM) were self-similar (obeyed power laws) across species. Thus, APCL in vitro, heart rate in vivo, and BM of any species can be predicted by Ca(2+)-Vm kinetics during AP cycles in SAN cells measured in any single species in vitro. CONCLUSIONS: In designing optimal heart rate to match widely different BM and energy requirements from mice to humans, nature did not “reinvent pacemaker cell wheels,” but differentially scaled kinetics of gears that regulate the rates at which the “wheels spin.” This discovery will facilitate the development of novel pharmacological and biological pacemakers featuring a normal, wide-range rate regulation in animal models and the translation of these to humans to target recalcitrant human SSS. (J Am Coll Cardiol EP 2021;7:1331–1344) Published by Elsevier on behalf of the American College of Cardiology Foundation.

BackgroundTranslation of knowledge of sinoatrial nodal “SAN” automaticity gleaned from animal studies to human dysrhythmias, e.g. “Sick Sinus” Syndrome (SSS) requiring electronic pacemaker insertion has been sub-optimal, largely because heart rate (HR) varies widely across species.ObjectivesTo discover regulatory universal mechanisms of normal automaticity in SAN pacemaker cells that are self-similar across species.MethodSub-cellular Ca2+ releases, whole cell AP-induced Ca2+ transients and APs were recorded in isolated mouse, guinea-pig, rabbit and human SAN cells. Parametric Ca2+ and Vm Kinetic Transitions (PCVKT) during phases of AP cycles from their ignition to recovery were quantified.ResultsAlthough both action potential cycle lengths (APCL) and PCVKT during AP cycles differed across species by ten-fold, trans-species scaling of PCVKT during AP cycles and scaling, of PCVKT to APCL in cells in vitro, EKG RR intervals in vivo, and BM were self-similar (obeyed power laws) across species. Thus, APCL in vitro, HR in vivo, and BM of any species can be predicted by PCVKT during AP cycles in SAN cells measured in any single species in vitro.ConclusionsIn designing optimal HR to match widely different BM and energy requirements from mice to humans, nature did not “reinvent pacemaker cell wheels”, but differentially scaled kinetics of gears that regulate the rates at which the “wheels spin”. This discovery will facilitate the development of novel pharmalogic therapies and biologic pacemakers featuring a normal, wide-range rate regulation in animal models and the translation of these to humans to target recalcitrant human SSS.Condensed AbstractStudies in animal models are an important facet of cardiac arrhythmia research. Because HR differs by over ten-fold between some animals and humans, translation of knowledge about regulatory mechanisms of SAN normal automaticity gleaned from studies in animal models to target human SSS has been sub-optimal. Our findings demonstrating that trans-species self-similarity of sub-cellular and cellular mechanisms that couple Ca2+ to Vm during AP cycles can predict heart rate in vivo from mice to humans will inform on the design of novel studies in animal models and facilitate translation of this knowledge to target human disease.

BackgroundThe current paradigm of Sinoatrial Node (SAN) impulse generation: (i) is that full-scale action potentials (APs) of a common frequency are initiated at one site and are conducted within the SAN along smooth isochrones; and (ii) does not feature fine details of Ca2+ signalling present in isolated SAN cells, in which small subcellular, subthreshold local Ca2+ releases (LCRs) self-organize to generate cell-wide APs.ObjectivesTo study subcellular Ca2+ signals within and among cells comprising the SAN tissue.MethodsWe combined immunolabeling with a novel technique to detect the occurrence of LCRs and AP-induced Ca2+ transients (APCTs) in individual pixels (chonopix) across the entire mouse SAN images.ResultsAt high magnification, Ca2+ signals appeared markedly heterogeneous in space, amplitude, frequency, and phase among cells comprising an HCN4+/CX43- cell meshwork. The signalling exhibited several distinguishable patterns of LCR/APCT interactions within and among cells. Apparently conducting rhythmic APCTs of the meshwork were transferred to a truly conducting HCN4-/CX43+ network of straited cells via narrow functional interfaces where different cell types intertwine, i.e. the SAN anatomical/functional unit. At low magnification, the earliest APCT of each cycle occurred within a small area of the HCN4 meshwork and subsequent APCT appearance throughout SAN pixels was discontinuous.ConclusionsWe have discovered a novel, microscopic Ca2+ signalling paradigm of SAN operation that has escaped detection using low-resolution, macroscopic tissue isochrones employed in prior studies: APs emerge from heterogeneous subcellular subthreshold Ca2+ signals, resembling multiscale complex processes of impulse generation within clusters of neurons in neuronal networks.Condensed abstractBy combining immunolabeling with a novel optical technique we detected markedly heterogenous Ca2+signals within and among cell clusters of an HCN4+/CX43- meshwork in mouse sinoatrial node. These Ca2+ signals self-organized and transferred, throughout the node, to projections from an HCN4-/CX43+ network connected to a highly organized, rapidly conducting part of the CX43+ network. Thus, APs emerge from heterogeneous, subthreshold Ca2+ signaling not detected in low-resolution macroscopic isochrones. Our discovery requires a fundamental paradigm shift from concentric impulse propagation initiated within a leading site, to a multiscale/complex process, resembling the emergence of organized signals from heterogeneous local signals within neuronal networks.

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 Ca(2+) transients and spontaneous local Ca(2+) 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, dys-rhythmic 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 Ca(2+) 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 Ca(2+) 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 Ca(2+) and membrane clocks.

Coupling of an intracellular Ca2+ clock to surface membrane ion channels, i.e., a “membrane clock, ” via coupling of electrochemical Na+ and Ca2+ gradients ( ENa and ECa, respectively) has been theorized to regulate sinoatrial nodal cell (SANC) normal automaticity. To test this hypothesis, we measured responses of [Na+]i, [Ca2+]i, membrane potential, action potential cycle length (APCL), and rhythm in rabbit SANCs to Na+/K+ pump inhibition by the digitalis glycoside, digoxigenin (DG, 10–20 μmol/l). Initial small but significant increases in [Na+]i and [Ca2+]i and reductions in ENa and ECa in response to DG led to a small reduction in maximum diastolic potential (MDP), significantly enhanced local diastolic Ca2+ releases (LCRs), and reduced the average APCL. As [Na+]i and [Ca2+]i continued to increase at longer times following DG exposure, further significant reductions in MDP, ENa, and ECa occurred; LCRs became significantly reduced, and APCL became progressively and significantly prolonged. This was accompanied by increased APCL variability. We also employed a coupled-clock numerical model to simulate changes in ENa and ECa simultaneously with ion currents not measured experimentally. Numerical modeling predicted that, as the ENa and ECa monotonically reduced over time in response to DG, ion currents ( ICaL, ICaT, If, IKr, and IbNa) monotonically decreased. In parallel with the biphasic APCL, diastolic INCX manifested biphasic changes; initial INCX increase attributable to enhanced LCR ensemble Ca2+ signal was followed by INCX reduction as ENCX ( ENCX = 3 ENa − 2 ECa) decreased. Thus SANC automaticity is tightly regulated by ENa, ECa, and ENCX via a complex interplay of numerous key clock components that regulate SANC clock coupling.

Heart muscle contraction is normally activated by a synchronized Ca release from sarcoplasmic reticulum (SR), a major intracellular Ca store. However, under abnormal conditions Ca leaks from the SR, decreasing heart contraction amplitude and increasing risk of life-threatening arrhythmia. The mechanisms and regimes of SR operation generating the abnormal Ca leak remain unclear. Here we employed both numerical and analytical modeling to get mechanistic insights into the emergent Ca leak phenomenon. Our numerical simulations using a detailed realistic model of Ca release unit (CRU) reveal sharp transitions resulting in Ca leak. The emergence of leak is closely mapped mathematically to the Ising model from statistical mechanics. The system steady-state behavior is determined by two aggregate parameters: the analogues of magnetic field ($h$) and the inverse temperature ($\beta$) in the Ising model, for which we have explicit formulas in terms of SR Ca and release channel opening/closing rates. The classification of leak regimes takes the shape of a phase $\beta$-$h$ diagram, with the regime boundaries occurring at $h$=0 and a critical value of $\beta$ ($\beta*$) which we estimate using a classical Ising model and mean field theory. Our theory predicts that a synchronized Ca leak will occur when $h$>0 and $\beta>\beta*$ and a disordered leak occurs when $\beta, Comment: 20 pages, 6 figures, supplemental material

The spontaneous rhythmic action potentials generated by the sinoatrial node (SAN), the primary pacemaker in the heart, dictate the regular and optimal cardiac contractions that pump blood around the body. Although the heart rate of humans is substantially slower than that of smaller experimental animals, current perspectives on the biophysical mechanisms underlying the automaticity of sinoatrial nodal pacemaker cells (SANCs) have been gleaned largely from studies of animal hearts. Using human SANCs, we demonstrated that spontaneous rhythmic local Ca2+ releases generated by a Ca2+ clock were coupled to electrogenic surface membrane molecules (the M clock) to trigger rhythmic action potentials, and that Ca2+-cAMP-protein kinase A (PKA) signaling regulated clock coupling. When these clocks became uncoupled, SANCs failed to generate spontaneous action potentials, showing a depolarized membrane potential and disorganized local Ca2+ releases that failed to activate the M clock. β-Adrenergic receptor (β-AR) stimulation, which increases cAMP concentrations and clock coupling in other species, restored spontaneous, rhythmic action potentials in some nonbeating "arrested" human SANCs by increasing intracellular Ca2+ concentrations and synchronizing diastolic local Ca2+ releases. When β-AR stimulation was withdrawn, the clocks again became uncoupled, and SANCs reverted to a nonbeating arrested state. Thus, automaticity of human pacemaker cells is driven by a coupled-clock system driven by Ca2+-cAMP-PKA signaling. Extreme clock uncoupling led to failure of spontaneous action potential generation, which was restored by recoupling of the clocks. Clock coupling and action potential firing in some of these arrested cells can be restored by β-AR stimulation-induced augmentation of Ca2+-cAMP-PKA signaling.

Recent data suggest that cardiac pacemaker cell function is determined by numerous time-, voltage-, and Ca-dependent interactions of cell membrane electrogenic proteins (M-clock) and intracellular Ca cycling proteins (Ca-clock), forming a coupled-clock system. Many aspects of the coupled-clock system, however, remain underexplored. The key players of the system are Ca release channels (ryanodine receptors), generating local Ca releases (LCRs) from sarcoplasmic reticulum, electrogenic Na/Ca exchanger (NCX) current, and L-type Ca current (I CaL ). We combined numerical model simulations with experimental simultaneous recordings of action potentials (APs) and Ca to gain further insight into the complex interactions within the system. Our simulations revealed a positive feedback mechanism, dubbed AP ignition, which accelerates the diastolic depolarization (DD) to reach AP threshold. The ignition phase begins when LCRs begin to occur and the magnitude of inward NCX current begins to increase. The NCX current, together with funny current and T-type Ca current accelerates DD, bringing the membrane potential to I CaL activation threshold. During the ignition phase, I CaL -mediated Ca influx generates more LCRs via Ca-induced Ca release that further activates inward NCX current, creating a positive feedback. Simultaneous recordings of membrane potential and confocal Ca images support the model prediction of the positive feedback among LCRs and I CaL , as diastolic LCRs begin to occur below and continue within the voltage range of I CaL activation. The ignition phase onset (identified within the fine DD structure) begins when DD starts to notably accelerate (∼0.15 V/s) above the recording noise. Moreover, the timing of the ignition onset closely predicted the duration of each AP cycle in the basal state, in the presence of autonomic receptor stimulation, and in response to specific inhibition of either the M-clock or Ca-clock, thus indicating general importance of the new coupling mechanism for regulation of the pacemaker cell cycle duration, and ultimately the heart rate.

Intracellular Ca signals represent a universal mechanism of cell function. Messages carried by Ca are local, rapid, and powerful enough to be delivered over the thermal noise. A higher signal to noise ratio is achieved by a cooperative action of Ca release channels such as IP3 receptors or ryanodine receptors arranged in clusters or release units containing a few to several hundred release channels. The release channels synchronize their openings via Ca-induced-Ca-release, generating high-amplitude local Ca signals known as puffs in neurons or sparks in muscle cells. Despite the high release amplitude and positive feedback nature of the activation, Ca signals are strictly confined in time and space by an unexplained termination mechanism. Here we show that the collective transition of release channels from an open to a closed state is identical to the phase transition associated with the reversal of magnetic field in an Ising ferromagnet. We demonstrate this mechanism using numerical model simulations of Ca sparks over a wide range of cluster sizes from 25 to 169 release channels. While prior studies suggested contributions of stochastic attrition and Ca store depletion, our new simple quantitative criterion closely predicts the depletion level required for spark termination for each cluster size. We further formulate exact requirements for a cluster of release channels to follow the Ising model in any cell type. Thus we describe deterministically the behaviour of a system on a coarser scale (release unit) which is random on a finer scale (release channels), bridging the gap between scales. Our results provide the first exact mapping of a nanoscale biological signalling model to an interacting particle system in statistical physics, making the extensive mathematical apparatus available to quantitative biology., Comment: 22 pages, 4 figures, 4 videos. Published in PNAS 2017

Numerical modeling indicates that hierarchical clustering of ryanodine receptors in cells of the sinoatrial node is crucial to the calcium clock and thereby to regulation of heart rate., 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.

Local Ca Releases (LCRs) are crucial events involved in cardiac pacemaker cell function. However, specific algorithms for automatic LCR detection and analysis have not been developed in live, spontaneously beating pacemaker cells. Here we measured LCRs using a high-speed 2D-camera in spontaneously contracting sinoatrial (SA) node cells isolated from rabbit and guinea pig and developed a new algorithm capable of detecting and analyzing the LCRs spatially in two-dimensions, and in time. Our algorithm tracks points along the midline of the contracting cell. It uses these points as a coordinate system for affine transform, producing a transformed image series where the cell does not contract. Action potential-induced Ca transients and LCRs were thereafter isolated from recording noise by applying a series of spatial filters. The LCR birth and death events were detected by a differential (frame-to-frame) sensitivity algorithm. An LCR was detected when its signal changes sufficiently quickly within a sufficiently large area. The LCR is considered to have died when its amplitude decays substantially, or when it merges into the rising whole cell Ca transient. Our algorithm provides major LCR parameters such as period, signal mass, duration, and path area. As LCRs propagate within cells, the algorithm identifies splitting and merging behaviors, indicating the importance of Ca-induced-Ca-release for the fate of LCRs and for generating a powerful ensemble Ca signal. Thus, our new computer algorithms eliminate motion artifacts and detect 2D local spatiotemporal Ca release events from recording noise and global signals. While the algorithms detect LCRs in sinoatrial nodal cells, they have the potential to be used in other applications in biophysics and cell physiology, for example, to detect Ca wavelets (abortive waves), sparks and embers in muscle cells and Ca puffs and syntillas in neurons., Comment: 43 pages, 7 figures, 2 tables, 5 videos (with web links)

Calcium sparks in cardiac myocytes are brief, localized calcium releases from the sarcoplasmic reticulum (SR) believed to be caused by locally regenerative calcium-induced calcium release (CICR) via couplons, clusters of ryanodine receptors (RyRs). How such regeneration is terminated is uncertain. We performed numerical simulations of an idealized stochastic model of spark production, assuming a RyR gating scheme with only two states (open and closed). Local depletion of calcium in the SR was inevitable during a spark, and this could terminate sparks by interrupting CICR, with or without assumed modulation of RyR gating by SR lumenal calcium. Spark termination by local SR depletion was not robust: under some conditions, sparks could be greatly and variably prolonged, terminating by stochastic attrition–a phenomenon we dub “spark metastability.” Spark fluorescence rise time was not a good surrogate for the duration of calcium release. Using a highly simplified, deterministic model of the dynamics of a couplon, we show that spark metastability depends on the kinetic relationship of RyR gating and junctional SR refilling rates. The conditions for spark metastability resemble those produced by known mutations of RyR2 and CASQ2 that cause life-threatening triggered arrhythmias, and spark metastability may be mitigated by altering the kinetics of the RyR in a manner similar to the effects of drugs known to prevent those arrhythmias. The model was unable to explain the distributions of spark amplitudes and rise times seen in chemically skinned cat atrial myocytes, suggesting that such sparks may be more complex events involving heterogeneity of couplons or local propagation among sub-clusters of RyRs.

Intracellular Local Ca releases (LCRs) from sarcoplasmic reticulum (SR) regulate cardiac pacemaker cell function by activation of electrogenic Na/Ca exchanger (NCX) during diastole. Prior studies demonstrated the existence of powerful compensatory mechanisms of LCR regulation via a complex local cross-talk of Ca pump, release and NCX. One major obstacle to study these mechanisms is that LCR exhibit complex Ca release propagation patterns (including merges and separations) that have not been characterized. Here we developed new terminology, classification, and computer algorithms for automatic detection of numerically simulated LCRs and examined LCR regulation by SR Ca pumping rate (Pup) that provides a major contribution to fight-or-flight response. In our simulations the faster SR Ca pumping accelerates action potential-induced Ca transient decay and quickly clears Ca under the cell membrane in diastole, preventing premature releases. Then the SR generates an earlier, more synchronized, and stronger diastolic LCR signal activating an earlier and larger inward NCX current. LCRs at higher Pup exhibit larger amplitudes and faster propagation with more collisions to each other. The LCRs overlap with Ca transient decay, causing an elevation of the average diastolic [Ca] nadir to ~200 nM (at Pup = 24 mM/s). Background Ca (in locations lacking LCRs) quickly decays to resting Ca levels (, Author summary Life’s vital rhythm, the heartbeat is guaranteed by specialized, cardiac pacemaker cells. Based upon the Hodgkin-Huxley membrane excitation theory and its application to cardiac cells, the cardiac pacemaker clock has been, for a long time, to be considered essentially a surface membrane oscillator, i.e. a membrane clock, driven by an ensemble of time- and voltage-dependent ion channels. More recent studies, however, discovered a tight integration of the membrane clock with an intracellular Ca oscillator (dubbed Ca clock). The Ca clock generates rhythmic, diastolic locally propagating Ca releases from sarcoplasmic reticulum, a cell major Ca store, that is refilled with Ca via a Ca pump. The released Ca activates Na/Ca exchanger that generates an inward current and accelerates the diastolic depolarization. Despite their importance, the local releases have not been systematically studied. Here we developed a new computer algorithm for automatic detection, classification, and analysis of local Ca releases generated by a recent computational model of a sinoatrial node cell. Then we investigated how the releases are regulated by the Ca pump, a major functional component of Ca clock. We discovered counterintuitive behaviors stabilizing diastolic Ca signal mass and ensuring fail-safe pacemaker operation at various pumping rates.

Whether intracellular Ca(2+) 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 Ca(2+) regulation of the rate and rhythm of SANC we loaded single isolated SANC with a caged Ca(2+) buffer, NP-EGTA, and simultaneously recorded membrane potential and intracellular Ca(2+). Prior to introduction of the caged Ca(2+) buffer, spontaneous local Ca(2+) 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 Ca(2+) 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 Ca(2+) was acutely released from the caged compound by flash photolysis, intracellular Ca(2+) 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 Ca(2+) dynamics by the caged buffer was reestablished. Our results directly support the hypothesis that intracellular Ca(2+) regulates normal SANC automaticity on a beat-to-beat basis.

Recent perspectives on sinoatrial nodal cell (SANC)(*) function indicate that spontaneous sarcoplasmic reticulum (SR) Ca(2+) cycling, i.e. an intracellular "Ca(2+) 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-Ca(2+) signaling cascade in mouse, the species most often utilized for genetic manipulations, however, has not been systematically tested. Here we show that Ca(2+) cycling proteins (e.g. RyR2, NCX1, and SERCA2) are abundantly expressed in mouse SAN and that spontaneous, rhythmic SR generated local Ca(2+) releases (LCRs) occur in skinned mouse SANC, clamped at constant physiologic [Ca(2+)]. 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 Ca(2+) cycling, because the β-AR chronotropic effect is markedly blunted when SR Ca(2+) cycling is disrupted. Thus, AC-cAMP-PKA-Ca(2+) signaling cascade is a major mechanism of normal automaticity in mouse SANC.

Prior studies indicate that cholinergic receptor (ChR) activation is linked to beating rate reduction (BRR) in sinoatrial nodal cells (SANC) via 1) a Gi-coupled reduction in adenylyl cyclase (AC) activity, leading to a reduction of cAMP or protein kinase A (PKA) modulation of hyperpolarization-activated current ( If) or L-type Ca2+ currents ( ICa,L), respectively; and 2) direct Gi-coupled activation of ACh-activated potassium current ( IKACh). More recent studies, however, have indicated that Ca2+ cycling by the sarcoplasmic reticulum within SANC (referred to as a Ca2+ clock) generates rhythmic, spontaneous local Ca2+ releases (LCR) that are AC-PKA dependent. LCRs activate Na+-Ca2+ exchange (NCX) current, which ignites the surface membrane ion channels to effect an AP. The purpose of the present study was to determine how ChR signaling initiated by a cholinergic agonist, carbachol (CCh), affects AC, cAMP, and PKA or sarcolemmal ion channels and LCRs and how these effects become integrated to generate the net response to a given intensity of ChR stimulation in single, isolated rabbit SANC. The threshold CCh concentration ([CCh]) for BRR was ∼10 nM, half maximal inhibition (IC50) was achieved at 100 nM, and 1,000 nM stopped spontaneous beating. Gi inhibition by pertussis toxin blocked all CCh effects on BRR. Using specific ion channel blockers, we established that If blockade did not affect BRR at any [CCh] and that IKACh activation, evidenced by hyperpolarization, first became apparent at [CCh] > 30 nM. At IC50, CCh reduced cAMP and reduced PKA-dependent phospholamban (PLB) phosphorylation by ∼50%. The dose response of BRR to CCh in the presence of IKACh blockade by a specific inhibitor, tertiapin Q, mirrored that of CCh to reduced PLB phosphorylation. At IC50, CCh caused a time-dependent reduction in the number and size of LCRs and a time dependent increase in LCR period that paralleled coincident BRR. The phosphatase inhibitor calyculin A reversed the effect of IC50 CCh on SANC LCRs and BRR. Numerical model simulations demonstrated that Ca2+ cycling is integrated into the cholinergic modulation of BRR via LCR-induced activation of NCX current, providing theoretical support for the experimental findings. Thus ChR stimulation-induced BRR is entirely dependent on Gi activation and the extent of Gi coupling to Ca2+ cycling via PKA signaling or to IKACh: at low [CCh], IKACh activation is not evident and BRR is attributable to a suppression of cAMP-mediated, PKA-dependent Ca2+ signaling; as [CCh] increases beyond 30 nM, a tight coupling between suppression of PKA-dependent Ca2+ signaling and IKACh activation underlies a more pronounced BRR.

Most cardiac Na+ channels open transiently within milliseconds upon membrane depolarization and are responsible for the excitation propagation. However, some channels remain active during hundreds of milliseconds, carrying the so-called persistent or late Na+ current (I(NaL)) throughout the action potential plateau. I(NaL) is produced by special gating modes of the cardiac-specific Na+ channel isoform. Experimental data accumulated over the past decade show the emerging importance of this late current component for the function of both normal and especially failing myocardium, where I(NaL) is reportedly increased. Na+ channels represent a multi-protein complex and its activity is determined not only by the pore-forming alpha subunit but also by its auxiliary beta subunits, cytoskeleton, and by Ca2+ signaling and trafficking proteins. Remodeling of this protein complex and intracellular signaling pathways may lead to alterations of I(NaL) in pathological conditions. Increased I(NaL) and the corresponding Na+ influx in failing myocardium contribute to abnormal repolarization and an increased cell Ca2+ load. Interventions designed to correct I(NaL) rescue normal repolarization and improve Ca2+ handling and contractility of the failing cardiomyocytes. New therapeutic strategies to target both arrhythmias and deficient contractility in HF may not be limited to the selective inhibition of I(NaL) but also include multiple indirect, modulatory (e.g. Ca(2+)- or cytoskeleton- dependent) mechanisms of I(NaL) function.

Augmented and slowed late Na+ current ( INaL) is implicated in action potential duration variability, early afterdepolarizations, and abnormal Ca2+ handling in human and canine failing myocardium. Our objective was to study INaL modulation by cytosolic Ca2+ concentration ([Ca2+]i) in normal and failing ventricular myocytes. Chronic heart failure was produced in 10 dogs by multiple sequential coronary artery microembolizations; 6 normal dogs served as a control. INaL fine structure was measured by whole cell patch clamp in ventricular myocytes and approximated by a sum of fast and slow exponentials produced by burst and late scattered modes of Na+ channel gating, respectively. INaL greatly enhanced as [Ca2+]i increased from “Ca2+ free” to 1 μM: its maximum density increased, decay of both exponentials slowed, and the steady-state inactivation (SSI) curve shifted toward more positive potentials. Testing the inhibition of CaMKII and CaM revealed similarities and differences of INaL modulation in failing vs. normal myocytes. Similarities include the following: 1) CaMKII slows INaL decay and decreases the amplitude of fast exponentials, and 2) Ca2+ shifts SSI rightward. Differences include the following: 1) slowing of INaL by CaMKII is greater, 2) CaM shifts SSI leftward, and 3) Ca2+ increases the amplitude of slow exponentials. We conclude that Ca2+/CaM/CaMKII signaling increases INaL and Na+ influx in both normal and failing myocytes by slowing inactivation kinetics and shifting SSI. This Na+ influx provides a novel Ca2+ positive feedback mechanism (via Na+/Ca2+ exchanger), enhancing contractions at higher beating rates but worsening cardiomyocyte contractile and electrical performance in conditions of poor Ca2+ handling in heart failure.

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.

Stochastic but roughly periodic LCRs ( L ocal subsarcolemmal ryanodine receptor–mediated C a 2+ R eleases) during the late phase of diastolic depolarization (DD) in rabbit sinoatrial nodal pacemaker cells (SANCs) generate an inward current ( I NCX ) via the Na + /Ca 2+ exchanger. Although LCR characteristics have been correlated with spontaneous beating, the specific link between LCR characteristics and SANC spontaneous beating rate, ie, impact of LCRs on the fine structure of the DD, have not been explicitly defined. Here we determined how LCRs and resultant I NCX impact on the DD fine structure to control the spontaneous SANC firing rate. Membrane potential ( V m ) recordings combined with confocal Ca 2+ measurements showed that LCRs impart a nonlinear, exponentially rising phase to the DD later part, which exhibited beat-to-beat V m fluctuations with an amplitude of approximately 2 mV. Maneuvers that altered LCR timing or amplitude of the nonlinear DD (ryanodine, BAPTA, nifedipine or isoproterenol) produced corresponding changes in V m fluctuations during the nonlinear DD component, and the V m fluctuation response evoked by these maneuvers was tightly correlated with the concurrent changes in spontaneous beating rate induced by these perturbations. Numerical modeling, using measured LCR characteristics under these perturbations, predicted a family of local I NCX that reproduced V m fluctuations measured experimentally and determined the onset and amplitude of the nonlinear DD component and the beating rate. Thus, beat-to-beat V m fluctuations during late DD phase reflect the underlying LCR/ I NCX events, and the ensemble of these events forms the nonlinear DD component that ultimately controls the SANC chronotropic state in tight cooperation with surface membrane ion channels.

Although the ensemble of voltage- and time-dependent rhythms of surface membrane ion channels, the membrane "Clock", is the immediate cause of a sinoatrial nodal cell (SANC) action potential (AP), it does not necessarily follow that this ion channel ensemble is the formal cause of spontaneous, rhythmic APs. SANC also generates intracellular oscillatory spontaneous Ca(2+) releases that ignite excitation (SCaRIE) of the surface membrane via Na(+)/Ca(2+) exchanger activation. The idea that a rhythmic intracellular Ca(2+) Clock might keep time for normal automaticity of SANC, however, has not been assimilated into mainstream pacemaker dogma. Recent experimental evidence, derived from simultaneous, confocal imaging of submembrane Ca(2+) and membrane potential of SANC, and supported by numerical modeling, indicates that normal automaticity of SANC is entrained and stabilized by the tight integration of the SR Ca(2+) Clock that generates rhythmic SCaRIE, and the surface membrane Clock that responds to SCaRIE to immediately produce APs of an adequate shape. Thus, tightly controlled, rhythmic SCaRIE does not merely fine tune SANC AP firing, but is the formal cause of the basal and reserve rhythms, insuring pacemaker stability by rhythmically integrating multiple Ca(2+)-dependent functions, and effects normal automaticity by rhythmic ignition of the surface membrane Clock.

Local, rhythmic, subsarcolemmal Ca 2+ 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 + -Ca 2+ 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 Ca 2+ 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 Ca 2+ cycling is disrupted by ryanodine. Thus, PKA-dependent phosphorylation of proteins that regulate cell Ca 2+ balance and spontaneous SR Ca 2+ cycling, ie, phospholamban and L-type Ca 2+ channels (and likely others not measured in this study), controls the phase and size of LCRs and the resultant Na + -Ca 2+ exchanger current and is crucial for both basal and reserve cardiac pacemaker function.

Recent studies employing Ca2+ indicators and confocal microscopy demonstrate substantial local Ca2+ release beneath the cell plasma membrane (subspace) of sinoatrial node cells (SANCs) occurring during diastolic depolarization. Pharmacological and biophysical experiments have suggested that the released Ca2+ interacts with the plasma membrane via the ion current (INaCa) produced by the Na+/Ca2+ exchanger and constitutes an important determinant of the pacemaker rate. This study provides a numerical validation of the functional importance of diastolic Ca2+ release for rate control. The subspace Ca2+ signals in rabbit SANCs were measured by laser confocal microscopy, averaged, and calibrated. The time course of the subspace [Ca2+] displayed both diastolic and systolic components. The diastolic component was mainly due to the local Ca2+ releases; it was numerically approximated and incorporated into a SANC cellular electrophysiology model. The model predicts that the diastolic Ca2+ release strongly interacts with plasma membrane via INaCa and thus controls the phase of the action potential upstroke and ultimately the final action potential rate.

Evidence has accumulated recently about the importance of alterations in Na+ channel function and slow myocardial conduction for arrhythmias in the infarcted and failing heart. The present study tested a hypothesis that Na+ current (INa/C) density decreases in chronic heart failure (HF) and that Na+ channel (NaCh) functional density can be restored by long-term therapy with carvedilol, a mixed alpha- and beta-adrenergic blocker. Studies were performed using a canine model of chronic HF produced in dogs by sequential intracoronary embolizations with microspheres. HF developed approximately 3 months after the last embolization (left ventricle, LV, ejection fraction = 28 +/- 1%). Ventricular cardiomyocytes (VCs) were isolated enzymatically from LV mid-myocardium, and INa was measured by whole-cell patch-clamp. The maximum INa/C was decreased in failing (n = 19) compared to normal (n = 12) hearts (33.1 +/- 1.6 vs 48.5 +/- 5.1 pA/pF, mean +/- SE, p0.001). The steady-state inactivation and activation of INa remained unchanged in failing compared to normal hearts. Long-term treatment with carvedilol (1 mg/kg, twice daily for 3 months) normalized INa/C in dogs with HF. INa/C in HF dogs (n = 6) treated with carvedilol was higher compared to that of non-treated HF dogs (n = 6) (49.4 +/- 0.9 vs 29 +/- 4.8 pA/pF, p0.007). In vitro culture of VCs of failing hearts for 24 h did not restore INa/C. However, INa/C was partially restored when VCs were incubated for 24 h with BAPTA-AM, an intracellular Ca2+ buffer. Thus, we conclude that experimental chronic HF in dogs results in down-regulation of the functional density of NaCh that can be restored by long-term therapy with carvedilol. The mechanism of NaCh down-regulation in HF may be linked to poor Ca2+ handling in this stage of disease.

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.

Sinoatrial node (SAN) is the primary heart pacemaker which initiates each heartbeat under normal conditions. Numerous experimental data have demonstrated that Ca(2+-) and CaMKII-dependent processes are crucially important for regulation of SAN cells. However, specific mechanisms of this regulation and their relative contribution to pacemaker function remain mainly unknown. Our review summarizes available data and existing numerical modeling approaches to understand Ca(2+) and CaMKII effects on the SAN. Data interpretation and future directions to address the problem are given within the coupled-clock theory, i.e., a modern view on the cardiac pacemaker cell function generated by a system of sarcolemmal and intracellular proteins.

We previously showed that a canine model of chronic heart failure (HF) produced by multiple coronary microembolizations manifests ventricular arrhythmias similar to those observed in patients with chronic HF. In the present study, we used single canine cardiomyocytes isolated from the left ventricle (LV) of normal dogs (n = 13) and dogs with HF (n = 15) to examine the cellular substrate of these arrhythmias. Action potentials (APs) and ion currents were measured by perforated and whole cell patch clamp, respectively. We found prolonged APs and alterations of AP duration resulting in early afterdepolarizations (EADs) at the low pacing rates of 0.5 Hz and 0.2 Hz. Na+ channel blockers saxitoxin (STX, 100 nM) and lidocaine (90 microM) reduced AP duration dispersion and abolished EADs in HF cardiomyocytes. The steady-state current (Iss)-voltage relation, in the voltage range from -25 mV to 25 mV analogous to the AP plateau level, was significantly shifted inward in HF cardiomyocytes. STX and lidocaine shifted the Iss-voltage relationship in an outward direction. The shifts produced by both drugs was significantly greater in cardiomyocytes of dogs with HF, indicating an increase in inward current. In the experimental configuration in which K+ currents were blocked, the density of the steady-state Ca2+ current (ICa) was found to decrease in HF cardiomyocytes by approximately 33%. In contrast, the density of the steady-state Na+ current (INa) significantly (P < 0.01) increased in HF cardiomyocytes (0.17 +/- 0.06 pA/pF) compared with normal cells (0.08 +/- 0.02 pA/pF). The relative contribution of INa to the net inward current was greater in HF cardiomyocytes, as evident from the increased ratio of INa/ICa (from 0.22 to 0.68). These observation support a hypothesis that anomalous repolarization of HF cardiomyocytes is due, at least in part, to an increased steady-state inward Na+ current.

Spontaneous, rhythmic LCRs are an important factor of pacemaker function of SA node cells (SANC). LCRs operate within the context of a coupled-clock system involving interactions of plasma membrane and sarcoplasmic reticulum (SR)-based calcium clocks. To date, the functional importance of this coupled-clock system has been demonstrated in rabbit SANC that beat spontaneously and rhythmically. The contribution of LCRs to action potential firing in guinea pig SANC, however, is controversial.We quantified the entire ensemble of LCRs in healthy appearing, single guinea pig SANC prior to and in the presence of beta-adrenergic stimulation (isoproterenol, 1µM). LCRs were measured by a high-speed 2D-camera and analyzed by a novel computer program capable of identifying LCRs in video-recordings.Although nine of the 22 SANC examined did not exhibit Ca2+ transients, LCRs were present in all cells. LCRs were smaller and more intense in cells without Ca2+ transients than in those with Ca2+ transients (rhythmic or dysrhythmic). Isoproterenol increased LCR size and induced rhythmic Ca2+ transients in majority of cells (6/9) without Ca2+ transients prior to isoproterenol. SANC that exhibited Ca2+ transients prior to isoproterenol generated approximately 80 LCRs/s. Cells in which Ca2+ transients were rhythmic had greater LCR size and amplitude than those with dysrhythmic Ca2+ transients. In cells with dysrhythmic Ca2+ transients, isoproterenol increased LCR size and amplitude and decreased LCR period, concurrently increasing the Ca2+ transient rhythmicity and frequency via presenting a larger and earlier occurring net Ca2+ signal to electrogenic Na+/Ca2+exchanger. Thus, all guinea pig SANC do indeed exhibit LCRs that contribute to generation of rhythmic action potential-induced Ca2+ transients.

The effects of cytochalasin D, a specific F-actin depolymerizing agent, on Ca2+ transients in rat ventricular cardiomyocytes were investigated. Cytochalasin D (20 microM) significantly slowed decay of Ca2+ transients (tau decay control cells=28.1+/-1.3, n=28tau decay=47.3+/-2.8 ms, n=20, P

We constructed a three dimensional stochastic numerical model of propagated spontaneous diastolic calcium release in sino-atrial node cells, using explicit gating of individual calcium channels, without assuming either a discrete sub-membrane compartment or an inactivated state of the RyR. Immunofluorescence staining of SA node cells for RyRs revealed a dense, variable network of clusters, with small clusters intermediate between major ones, located immediately below the surface membrane. The presence of such intermediate “bridging” clusters was crucial to allow propagation of local calcium releases in the presence of 3D diffusion and buffering. When this RyR network was simulated, local calcium releases propagated as partially synchronized wavelets localized near the surface membrane. Calcium in the junctional and free sarcoplasmic reticulum followed a complex pattern of recycling through the interior of the cell, with areas of both depletion and enhancement. These calcium wavelets acted as a partially periodic “calcium clock” that entrained with classical membrane currents to regulate beating rate in response to simulated beta-adrenergic stimulation, as demonstrated experimentally. When the RyR sensitivity was very high or NCX density was low, there was a paradoxical regime in which synchronization was lost, causing sympathetic stimulation to reduce rather than increase beating rate. This regime may be important in heart failure or with RyR mutations.

Whether intracellular Ca(2+) cycling dynamics regulate cardiac pacemaker cell function on a beat-to-beat basis remains unknown. Here we show that under physiological conditions, application of low concentrations of caffeine (2-4 mM) to isolated single rabbit sinoatrial node cells acutely reduces their spontaneous action potential cycle length (CL) and increases Ca(2+) transient amplitude for several cycles. Numerical simulations, using a modified Maltsev-Lakatta coupled-clock model, faithfully reproduced these effects, and also the effects of CL prolongation and dysrhythmic spontaneous beating (produced by cytosolic Ca(2+) buffering) and an acute CL reduction (produced by flash-induced Ca(2+) release from a caged Ca(2+) buffer), which we had reported previously. Three contemporary numerical models (including the original Maltsev-Lakatta model) failed to reproduce the experimental results. In our proposed new model, Ca(2+) releases acutely change the CL via activation of the Na(+)/Ca(2+) exchanger current. Time-dependent CL reductions after flash-induced Ca(2+) releases (the memory effect) are linked to changes in Ca(2+) available for pumping into sarcoplasmic reticulum which, in turn, changes the sarcoplasmic reticulum Ca(2+) load, diastolic Ca(2+) releases, and Na(+)/Ca(2+) exchanger current. These results support the idea that Ca(2+) regulates CL in cardiac pacemaker cells on a beat-to-beat basis, and suggest a more realistic numerical mechanism of this regulation.

UNLABELLED: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. AIM:The present study tested whether variation in LCR periodicity contributes to intrinsic (beat-to-beat) cycle length variability in single sinoatrial nodal cells. METHODS: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. RESULTS: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. CONCLUSION: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.

Beneficial clinical bradycardic effects of ivabradine (IVA) have been interpreted solely on the basis of If inhibition, because IVA specifically inhibits If in sinoatrial nodal pacemaker cells (SANC). However, it has been recently hypothesized that SANC normal automaticity is regulated by crosstalk between an "M clock," the ensemble of surface membrane ion channels, and a "Ca(2+) clock," the sarcoplasmic reticulum (SR). We tested the hypothesis that crosstalk between the two clocks regulates SANC automaticity, and that indirect suppression of the Ca(2+) clock further contributes to IVA-induced bradycardia. IVA (3 μM) not only reduced If amplitude by 45 ± 6% in isolated rabbit SANC, but the IVA-induced slowing of the action potential (AP) firing rate was accompanied by reduced SR Ca(2+) load, slowed intracellular Ca(2+) cycling kinetics, and prolonged the period of spontaneous local Ca(2+) releases (LCRs) occurring during diastolic depolarization. Direct and specific inhibition of SERCA2 by cyclopiazonic acid (CPA) had effects similar to IVA on LCR period and AP cycle length. Specifically, the LCR period and AP cycle length shift toward longer times almost equally by either direct perturbations of the M clock (IVA) or the Ca(2+) clock (CPA), indicating that the LCR period reports the crosstalk between the clocks. Our numerical model simulations predict that entrainment between the two clocks that involves a reduction in INCX during diastolic depolarization is required to explain the experimentally AP firing rate reduction by IVA. In summary, our study provides new evidence that a coupled-clock system regulates normal cardiac pacemaker cell automaticity. Thus, IVA-induced bradycardia includes a suppression of both clocks within this system.

Recent evidence supports the idea that robust and, importantly, FLEXIBLE automaticity of cardiac pacemaker cells is conferred by a coupled system of membrane ion currents (an “M-clock”) and a sarcoplasmic reticulum (SR)-based Ca2 + oscillator (“Ca2 +clock”) that generates spontaneous diastolic Ca2 + releases. This study identified numerical models of a human biological pacemaker that features robust and flexible automaticity generated by a minimal set of electrogenic proteins and a Ca2 +clock. Following the Occam's razor principle (principle of parsimony), M-clock components of unknown molecular origin were excluded from Maltsev–Lakatta pacemaker cell model and thirteen different model types of only 4 or 5 components were derived and explored by a parametric sensitivity analysis. The extended ranges of SR Ca2 + pumping (i.e. Ca2 +clock performance) and conductance of ion currents were sampled, yielding a large variety of parameter combination, i.e. specific model sets. We tested each set's ability to simulate autonomic modulation of human heart rate (minimum rate of 50 to 70 bpm; maximum rate of 140 to 210 bpm) in response to stimulation of cholinergic and β-adrenergic receptors. We found that only those models that include a Ca2 +clock (including the minimal 4-parameter model “ICaL + IKr + INCX + Ca2 +clock”) were able to reproduce the full range of autonomic modulation. Inclusion of If or ICaT decreased the flexibility, but increased the robustness of the models (a relatively larger number of sets did not fail during testing). The new models comprised of components with clear molecular identity (i.e. lacking IbNa & Ist) portray a more realistic pacemaking: A smaller Na+ influx is expected to demand less energy for Na+ extrusion. The new large database of the reduced coupled-clock numerical models may serve as a useful tool for the design of biological pacemakers. It will also provide a conceptual basis for a general theory of robust, flexible, and energy-efficient pacemaking based on realistic components.

Mitochondria dynamically buffer cytosolic Ca(2+) in cardiac ventricular cells and this affects the Ca(2+) load of the sarcoplasmic reticulum (SR). In sinoatrial-node cells (SANC) the SR generates periodic local, subsarcolemmal Ca(2+) releases (LCRs) that depend upon the SR load and are involved in SANC automaticity: LCRs activate an inward Na(+)-Ca(2+) exchange current to accelerate the diastolic depolarization, prompting the ensemble of surface membrane ion channels to generate the next action potential (AP).To determine if mitochondrial Ca(2+) (Ca(2+) (m)), cytosolic Ca(2+) (Ca(2+) (c))-SR-Ca(2+) crosstalk occurs in single rabbit SANC, and how this may relate to SANC normal automaticity.Inhibition of mitochondrial Ca(2+) influx into (Ru360) or Ca(2+) efflux from (CGP-37157) decreased [Ca(2+)](m) to 80 ± 8% control or increased [Ca(2+)](m) to 119 ± 7% control, respectively. Concurrent with inhibition of mitochondrial Ca(2+) influx or efflux, the SR Ca(2+) load, and LCR size, duration, amplitude and period (imaged via confocal linescan) significantly increased or decreased, respectively. Changes in total ensemble LCR Ca(2+) signal were highly correlated with the change in the SR Ca(2+) load (r(2) = 0.97). Changes in the spontaneous AP cycle length (Ru360, 111 ± 1% control; CGP-37157, 89 ± 2% control) in response to changes in [Ca(2+)](m) were predicted by concurrent changes in LCR period (r(2) = 0.84).A change in SANC Ca(2+) (m) flux translates into a change in the AP firing rate by effecting changes in Ca(2+) (c) and SR Ca(2+) loading, which affects the characteristics of spontaneous SR Ca(2+) release.

A modern view on regulation of cardiac pacemaker cell function postulates that local calcium releases (LCRs) contribute to diastolic depolarization via electrogenic sodium/calcium exchanger. An unresolved problem, however, remains: how intrinsically stochastic and heterogeneously distributed release channels (RyR) generate a strong, synchronized ensemble LCR signal and how this signal is effectively regulated by autonomic system to insure pacemaker rate flexibility.We measured calcium dynamics by a high-speed camera in isolated, single rabbit sinoatrial node cells (SANC) and assessed the kinetics of local calcium-pumping synchronization by examining the distributions of time constants (τ) of calcium-transient decay among cell neighborhoods at baseline and during stimulation of either beta-adrenergic receptors or cholinergic receptors.Beta-adrenergic receptor stimulation (isoproterenol) not only decreased cycle length (CL) and average τ vs. baseline, but also decreased the standard deviation (SD) of all τ's across all neighborhoods, suggesting a shift into a less heterogeneous, i.e. more synchronized calcium pumping throughout SANC. Conversely, cholinergic receptor stimulation (carbachol) not only increased CL and average τ vs. baseline, but also increased the local SD(τ), suggesting a shift into a more heterogeneous, i.e. less synchronized calcium pumping within SANC. Furthermore, on a beat-to-beat basis, the relationship between either τ or SD(τ) and CL was linear under both baseline conditions and autonomic stimulation.Conclusions: The degree of heterogeneity of local calcium pumping is a new universal factor that affects the CL and insures effective rate and rhythm regulation of the coupled-clock pacemaker system via autonomic modulation. More synchronized and faster calcium pumping is presumably achieved via phospholamban phosphorylation and allows cell neighborhoods to reach the calcium release threshold quicker and more synchronously, thereby synchronizing LCRs and amplifying their ensemble diastolic signal, accelerating pacemaker rate.

The question of the extent to which cytosolic Ca(2+) affects sinoatrial node pacemaker activity has been discussed for decades. We examined this issue by analyzing two mathematical pacemaker models, based on the "Ca(2+) clock" (C) and "membrane clock" (M) hypotheses, together with patch-clamp experiments in isolated guinea pig sinoatrial node cells. By applying lead potential analysis to the models, the C mechanism, which is dependent on potentiation of Na(+)/Ca(2+) exchange current via spontaneous Ca(2+) release from the sarcoplasmic reticulum (SR) during diastole, was found to overlap M mechanisms in the C model. Rapid suppression of pacemaker rhythm was observed in the C model by chelating intracellular Ca(2+), whereas the M model was unaffected. Experimental rupturing of the perforated-patch membrane to allow rapid equilibration of the cytosol with 10 mM BAPTA pipette solution, however, failed to decrease the rate of spontaneous action potential within ∼30 s, whereas contraction ceased within ∼3 s. The spontaneous rhythm also remained intact within a few minutes when SR Ca(2+) dynamics were acutely disrupted using high doses of SR blockers. These experimental results suggested that rapid disruption of normal Ca(2+) dynamics would not markedly affect spontaneous activity. Experimental prolongation of the action potentials, as well as slowing of the Ca(2+)-mediated inactivation of the L-type Ca(2+) currents induced by BAPTA, were well explained by assuming Ca(2+) chelation, even in the proximity of the channel pore in addition to the bulk cytosol in the M model. Taken together, the experimental and model findings strongly suggest that the C mechanism explicitly described by the C model can hardly be applied to guinea pig sinoatrial node cells. The possible involvement of L-type Ca(2+) current rundown induced secondarily through inhibition of Ca(2+)/calmodulin kinase II and/or Ca(2+)-stimulated adenylyl cyclase was discussed as underlying the disruption of spontaneous activity after prolonged intracellular Ca(2+) concentration reduction for5 min.

We elucidate the role of late Na+ current (INaL) for diastolic intracellular Ca2+ (DCa) accumulation in chronic heart failure (HF). HF was induced in 19 dogs by multiple coronary artery microembolizations; 6 normal dogs served as control. Ca2+ transients were recorded in field-paced (0.25 or 1.5 Hz) fluo-4-loaded ventricular myocytes (VM). INaL and action potentials were recorded by patch-clamp. Failing VM, but not normal VM, exhibited (1) prolonged action potentials and Ca2+ transients at 0.25 Hz, (2) substantial DCa accumulation at 1.5 Hz, and (3) spontaneous Ca2+ releases, which occurred after 1.5 Hz stimulation trains in ~31% cases. Selective INaL blocker ranolazine (10 microM) or the prototypical Na+ channel blocker tetrodotoxin (2 microM) reversibly improved function of failing VM. The DCa accumulation and the beneficial effect of INaL blockade were reproduced in silico using an excitation-contraction coupling model. We conclude that INaL contributes to diastolic Ca2+ accumulation and spontaneous Ca2+ release in HF.

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.

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.

Sarcoplasmic reticulum (SR) Ca2+ cycling, that is, the Ca2+ clock, entrained by externally delivered action potentials has been a major focus in ventricular myocyte research for the past 5 decades. In contrast, the focus of pacemaker cell research has largely been limited to membrane-delimited pacemaker mechanisms (membrane clock) driven by ion channels, as the immediate cause for excitation. Recent robust experimental evidence, based on confocal cell imaging, and supported by numerical modeling suggests a novel concept: the normal rhythmic heart beat is governed by the tight integration of both intracellular Ca2+ and membrane clocks. In pacemaker cells the intracellular Ca2+ clock is manifested by spontaneous, rhythmic submembrane local Ca2+ releases from SR, which are tightly controlled by a high degree of basal and reserve PKA-dependent protein phosphorylation. The Ca2+ releases rhythmically activate Na+/Ca2+ exchange inward currents that ignite action potentials, whose shape and ion fluxes are tuned by the membrane clock which, in turn, sustains operation of the intracellular Ca2+ clock. The idea that spontaneous SR Ca2+ releases initiate and regulate normal automaticity provides the key that reunites pacemaker and ventricular cell research, thus evolving a general theory of the initiation and strength of the heartbeat. Keywords:: cardiac excitation, contractility, pacemaker mechanism, calcium cycling, heart rate regulation

Voltage-gated Ca(v)1.2 channels are composed of the pore-forming alpha1C and auxiliary beta and alpha2delta subunits. Voltage-dependent conformational rearrangements of the alpha1C subunit C-tail have been implicated in Ca2+ signal transduction. In contrast, the alpha1C N-tail demonstrates limited voltage-gated mobility. We have asked whether these properties are critical for the channel function. Here we report that transient anchoring of the alpha1C subunit C-tail in the plasma membrane inhibits Ca2+-dependent and slow voltage-dependent inactivation. Both alpha2delta and beta subunits remain essential for the functional channel. In contrast, if alpha1C subunits with are expressed alpha2delta but in the absence of a beta subunit, plasma membrane anchoring of the alpha1C N terminus or its deletion inhibit both voltage- and Ca2+-dependent inactivation of the current. The following findings all corroborate the importance of the alpha1C N-tail/beta interaction: (i) co-expression of beta restores inactivation properties, (ii) release of the alpha1C N terminus inhibits the beta-deficient channel, and (iii) voltage-gated mobility of the alpha1C N-tail vis a vis the plasma membrane is increased in the beta-deficient (silent) channel. Together, these data argue that both the alpha1C N- and C-tails have important but different roles in the voltage- and Ca2+-dependent inactivation, as well as beta subunit modulation of the channel. The alpha1C N-tail may have a role in the channel trafficking and is a target of the beta subunit modulation. The beta subunit facilitates voltage gating by competing with the N-tail and constraining its voltage-dependent rearrangements. Thus, cross-talk between the alpha1C C and N termini, beta subunit, and the cytoplasmic pore region confers the multifactorial regulation of Ca(v)1.2 channels.

The heartbeat originates within the sinoatrial node (SAN), a small structure containing10,000 genuine pacemaker cells. If the SAN fails, the ∼5 billion working cardiomyocytes downstream of it become quiescent, leading to circulatory collapse in the absence of electronic pacemaker therapy. Here we demonstrate conversion of rodent cardiomyocytes to SAN cells in vitro and in vivo by expression of Tbx18, a gene critical for early SAN specification. Within days of in vivo Tbx18 transduction, 9.2% of transduced, ventricular cardiomyocytes develop spontaneous electrical firing physiologically indistinguishable from that of SAN cells, along with morphological and epigenetic features characteristic of SAN cells. In vivo, focal Tbx18 gene transfer in the guinea-pig ventricle yields ectopic pacemaker activity, correcting a bradycardic disease phenotype. Myocytes transduced in vivo acquire the cardinal tapering morphology and physiological automaticity of native SAN pacemaker cells. The creation of induced SAN pacemaker (iSAN) cells opens new prospects for bioengineered pacemakers.