Action Potential Cycle Length Research Articles

Computational modelling of mouse atrio ventricular node action potential and automaticity.

The atrioventricular node (AVN) is a crucial component of the cardiac conduction system. Despite its pivotal role in regulating the transmission of electrical signals between atria and ventricles, a comprehensive understanding of the cellular electrophysiological mechanisms governing AVN function has remained elusive. This paper presents a detailed computational model of mouse AVN cell action potential (AP). Our model builds upon previous work and introduces several key refinements, including accurate representation of membrane currents and exchangers, calcium handling, cellular compartmentalization, dynamic update of intracellular ion concentrations, and calcium buffering. We recalibrated and validated the model against existing and unpublished experimental data. In control conditions, our model reproduces the AVN AP experimental features, (e.g. rate=175bpm, experimental range [121, 191]bpm). Notably, our study sheds light on the contribution of L-type calcium currents, through both Cav1.2 and Cav1.3 channels, in AVN cells. The model replicates several experimental observations, including the cessation of firing upon block of Cav1.3 or INa,r current. If block induces a reduction in beating rate of 11%. In summary, this work presents a comprehensive computational model of mouse AVN cell AP, offering a valuable tool for investigating pacemaking mechanisms and simulating the impact of ionic current blockades. By integrating calcium handling and refining formulation of ionic currents, our model advances understanding of this critical component of the cardiac conduction system, providing a platform for future developments in cardiac electrophysiology. KEY POINTS: This paper introduces a comprehensive computational model of mouse atrioventricular node (AVN) cell action potentials (APs). Our model is based on the electrophysiological data from isolated mouse AVN cells and exhibits an action potential and calcium transient that closely match the experimental records. By simulating the effects of blocking specific ionic currents, the model effectively predicts the roles of L-type Cav1.2 and Cav1.3 channels, T-type calcium channels, sodium currents (TTX-sensitive and TTX-resistant), and the funny current (If) in AVN pacemaking. The study also emphasizes the significance of other ionic currents, including IKr, Ito, IKur, in regulating AP characteristics and cycle length in AVN cells. The model faithfully reproduces the rate dependence of action potentials under pacing, opening the possibility of use in impulse propagation models. The population-of-models approach showed the robustness of this new AP model in simulating a wide spectrum of cellular pacemaking in AVN.

Glucagon-like peptide-1 increases heart rate by a direct action on the sinus node.

Glucagon-like peptide-1 receptor agonists (GLP-1 RAs) are increasingly used to treat type 2 diabetes and obesity. Albeit cardiovascular outcomes generally improve, treatment with GLP-1 RAs is associated with increased heart rate, the mechanism of which is unclear. We employed a large animal model, the female landrace pig, and used multiple in vivo and ex vivo approaches including pharmacological challenges, electrophysiology, and high-resolution mass spectrometry to explore how GLP-1 elicits an increase in heart rate. In anaesthetized pigs, neither cervical vagotomy, adrenergic blockers (alpha, beta, or combined alpha-beta blockade), ganglionic blockade (hexamethonium), nor inhibition of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels (ivabradine) abolished the marked chronotropic effect of GLP-1. GLP-1 administration to isolated perfused pig hearts also increased heart rate, which was abolished by GLP-1 receptor blockade. Electrophysiological characterization of GLP-1 effects in vivo and in isolated perfused hearts localized electrical modulation to the atria and conduction system. In isolated sinus nodes, GLP-1 administration shortened the action potential cycle length of pacemaker cells and shifted the site of earliest activation. The effect was independent of HCN blockade. Collectively, these data support a direct effect of GLP-1 on GLP-1 receptors within the heart. Consistently, single nucleus RNA sequencing showed GLP-1 receptor expression in porcine pacemaker cells. Quantitative phosphoproteomics analyses of sinus node samples revealed that GLP-1 administration leads to phosphorylation changes of calcium cycling proteins of the sarcoplasmic reticulum, known to regulate heart rate. GLP-1 has direct chronotropic effects on the heart mediated by GLP-1 receptors in pacemaker cells of the sinus node, inducing changes in action potential morphology and the leading pacemaker site through a calcium signalling response characterized by PKA-dependent phosphorylation of Ca2+ cycling proteins involved in pacemaking. Targeting the pacemaker calcium clock may be a strategy to lower heart rate in people treated with GLP-1 RAs.

β-Adrenergic Stimulation Synchronizes a Broad Spectrum of Action Potential Firing Rates of Cardiac Pacemaker Cells toward a Higher Population Average.

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 (R2 = 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.

A 3-D human model of complex cardiac arrhythmias

Cardiac arrhythmias impact over 12 million people globally, with an increasing incidence of acquired arrhythmias. Although animal models have shed light onto fundamental arrhythmic mechanisms, species-specific differences and ethical concerns remain. Current human models using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) either lack the higher order tissue organization of the heart or implement unreliable arrhythmia induction techniques. Our goal was to develop a robust model of acquired arrhythmia by disrupting cardiomyocyte cell-cell signaling – one of the hallmarks of complex arrhythmias. Human 3D microtissues were generated by seeding hydrogel-embedded hiPSC-CMs and cardiac fibroblasts into an established microwell system designed to enable active and passive force assessment. Cell-cell signaling was disrupted using methyl-beta cyclodextrin (MBCD), previously shown to disassemble cardiac gap junctions. We demonstrate that arrhythmias were progressive and present in all microtissues within 5 days of treatment. Arrhythmic tissues exhibited reduced conduction velocity, an increased number of distinct action potentials, and reduced action potential cycle length. Arrhythmic tissues also showed significant reduction in contractile force generation, increased beating frequency, and increased passive tension and collagen deposition, in line with fibrosis. A subset of tissues with more complex arrhythmias exhibited 3D spatial differences in action potential propagation. Pharmacological and electrical defibrillation was successful. Transcriptomic data indicated an enrichment of genes consistent with cardiac arrhythmias. MBCD removal reversed the arrhythmic phenotype, resulting in synchronicity despite not resolving fibrosis. This innovative & reliable human-relevant 3D acquired arrhythmia model shows potential for improving our understanding of arrhythmic action potential conduction and furthering therapeutic development. Statement of significanceThis work describes a 3D human model of cardiac arrhythmia-on-a-chip with high reproducibility, fidelity, and extensive functional applicability. To mimic in vivo conditions, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and cardiac fibroblasts from healthy controls were combined in a biocompatible fibrin hydrogel and seeded between two deflectable polymeric rods. Using the innate functional properties of this 3D model as well as advanced optical imaging techniques we demonstrated dramatic changes in contraction rate, synchronicity, and electrophysiological conduction in arrhythmic tissues relative to controls. Taken together, these data demonstrate the distinctive potential of this new model for pathophysiological studies, and for arrhythmia drug testing applications.

Abstract 145: Isolation & Characterization of Heart-field Specific Cardiomyocytes

Cardiovascular disease (CVD) is the leading cause of death worldwide. Human embryonic stem cell (hESC)-derived cardiovascular progenitors (CVPs) or cardiomyocytes (CMs) represent a promising candidate for cell-based therapies to treat CVD. Myocardial infarction leads to extensive CM death mainly within the left ventricle, which is predominantly derived from the first heart field (FHF) during embryonic development. We postulate that the generation of chamber-specific CMs may play a key role in the development of safe and efficacious regenerative therapy. As a first step, we generated and characterized a FHF-specific TBX5-TdTomato +/W hESC reporter line. We show that TBX5 + cells represent an enriched population of FHF CVPs that can give rise to CMs, endothelial, and smooth muscle cells in vitro . Interestingly, we observed that TBX5 - cells can also generate contractile CMs. Bulk RNA-sequencing analysis at different stages of development suggested that TBX5 - cells are enriched for second heart field (SHF) CMs. To enable prospective isolation of FHF and SHF CMs, we generated a double transgenic TBX5-TdTomato +/W /NKX2-5 eGFP/W hESC reporter line. We performed detailed electrophysiological, functional, and transcriptional studies to characterize the heart-field specificity of these hESC-derived CMs. Electrophysiological studies revealed that, despite the presence of atrial and ventricular action potentials (APs) in both FHF and SHF, there are significant differences in their AP duration and cycle length. In addition, both FHF and SHF CMs responded appropriately to adrenergic stimuli. Single-cell RNA sequencing analysis confirmed the absence of nodal genes within these populations and provided evidence of unique molecular signatures for isolating FHF- and SHF-like CMs. Finally, we identified CORIN as a novel cell surface marker for FHF CMs. Our studies provide a platform for investigating in vitro cardiovascular development, drug screening, and may facilitate a safe approach for cell therapy in heart disease.

The Periodicity of a Ca2+ Clock Intrinsic to Individual Cardiac Sinoatrial Nodal Pacemaker Cells Universally Scales to Body Mass from Mice to Humans

Prior studies showed allometric scaling relationships between body mass (BM) and EKG of mammal's heart, including heart rate (HR), atrioventricular conduction (PR interval),etc., as a ¼-power of BM. But can ¼-power scaling that regulates HR in vivo relate to BM when downscaled to the subcellular level? Our prior studies revealed that coupling of spontaneous local intracellular calcium releases (LCRs), generated by a Ca2+ clock (sarcoplasmic reticulum-SR) to a membrane clock sarcolemmal electrogenic molecules regulates spontaneous sinoatrial nodal cell (SANC) action potential cycle length (APCL) via activating NCX inward current, the larger amplitude and early occurrence (period) of the spontaneous LCR Ca2+ signal, the earlier and larger NCX, the faster AP (shorter APCL). Relationships among LCR periods and APCL conforms to a unique linear function. We hypothesized that the LCR periods, generated within SANC from hearts of different species that vary in BM and HR scale non-linearly as ¼-power of BM of the donor species. Methods: Confocal-line-scan microscopy was used to record AP-induced-Ca2+ transients and spontaneous LCRs in spontaneously beating, single SANC, isolated from mouse-(M), guinea-pig-(GP), rabbit-(R) and human-(H) hearts. The APCL and other LCR characteristics and variations of BM across species were analyzed via double-logarithmic plots. Results: Species with larger BM exhibited longer APCL and LCR period, and more LCR events per cycle versus that in species with smaller BM. The average APCL and LCR period linearly scaled with BM with scaling exponent close to ¼. Conclusions: LCR period and APCL in single spontaneously beating SANC obey the universal law for allometric scaling for BM as does in vivo HR, therefore species differences in LCR characteristics of subcellular LCRs predict species differences in EKG parameters in vivo.

Coupling of Calcium- and Membrane Clocks Ignites De Novo Spontaneous Action Potential in Dormant Guinea Pig Sinoatrial Nodal Cells via Camp-PKA Signaling

Firing rate and rhythm of spontaneous action potentials (APs) in sinoatrial nodal cells (SANC) are controlled via the fidelity of clock coupling: rhythmic, spontaneous, local Ca2+ releases (LCRs) that emerges beneath the cell membrane (‘Ca2+ clock’) during diastole couples to sarcolemmal electrogenic mechanisms (‘membrane clock’). We hypothesized that dormant guinea pig SANC, i.e. cells that don’t fire spontaneous APs, represent an extreme form of clock uncoupling, and increased cAMP-mediated, PKA-dependent phosphorylation of the coupled-clock system proteins ignite rhythmic AP firing in dormant SANC. Membrane potential of dormant GP SANC averaged −38±1mV(n=46), ICaL, If, and IK current densities were comparable to those that fire spontaneous APs, and LCRs were small and random. Isoproterenol or CPT-cAMP led to de novo spontaneous AP generation in ∼50% of dormant SANC. The initial response involved increases in LCR size and rhythmicity and small amplitude, spontaneous APs and whole-cell Ca2+ transients. Average LCR size continued to increase, average LCR period shortened, partially synchronizing LCRs to late diastole, paralleled by MDP hyperpolarization to ∼-60 mV. AP cycle length shortened similar to LCR period. β-AR stimulation increased availability of ICaL and IK, and If. Inhibition of Ca2+ clock by CPA precluded the isoproterenol-induced AP generation in 91% of dormant SANC, but If block by ivabradine did not inhibit the activation. Ivabradine, however, prolonged the transition between dormant and steady firing state following the initiation of β-AR stimulation. When β-AR stimulation/CPT-cAMP was removed, all changes reversed in order and SANC again became dormant. At least in some SANC, dormancy represents membrane & Ca2+ clock uncoupling that is rescued via cAMP-mediated, PKA-induced increased function of both membrane and Ca2+ clock proteins.

Spontaneous, local diastolic subsarcolemmal calcium releases in single, isolated guinea-pig sinoatrial nodal cells.

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.

Electrically Dormant Sinoatrial Nodal Cells (SANC) are Awakened by Increased Camp-Dependent Phosphorylation of Coupled-Clock Proteins

SANC firing rate and rhythm is controlled via the degree of coupling between rhythmic local Ca2+ releases (LCRs) emerging beneath the cell membrane (‘Ca2+clock’) and sarcolemmal electrogenic mechanisms (‘membrane clock’). Dormant SANC (ones that don’t fire spontaneous action potentials (APs)) may therefore represent the extreme of clock uncoupling. We hypothesized that increased cAMP-mediated, PKA-dependent phosphorylation of the coupled-clock system proteins would restore rhythmic AP firing in dormant SANC. We recorded membrane potential (Vm) and intracellular Ca2+ kinetics in enzymatically isolated single, dormant guinea pig SANC prior to and during β-adrenoceptor stimulation (BARs, isoproterenol 0.1-1 µM) or during exposure to cell-permeant cAMP (CPT-cAMP, 300 µM). Dormant SANC that did not fire still maintained a resting membrane potential of −35+/-3mV (n=17), and exhibited small, random LCRs. ∼40% responded to BARs/CPT-cAMP. This initial response involved increases in LCR size and rhythmicity, and hyperpolarization of Vm, accompanied by small amplitude, spontaneous APs (rate ∼1Hz) triggering whole-cell Ca2+ transients. During the subsequent transition to steady-state AP firing, average LCR size increased while average LCR period shortened, partially synchronizing LCRs to late diastole, (rate ∼2Hz); this was paralleled by MDP hyperpolarization to ∼-60 mV. AP amplitude increased, and average AP cycle length shortened similar to LCR period. When BARs/CPT-cAMP was washed out, all changes reversed in order, and SANC again exhibited small random LCRs, with Vm ∼-40 mV. In conclusion, at least in some SANC, dormancy represents membrane & Ca2+ clock uncoupling that is rescuable through increased cAMP and PKA-dependent phosphorylation of both membrane and Ca2+ clock proteins. Specifically, rescuing impaired clock coupling occurs through: (1) increased spatial LCR synchronization, yielding larger LCRs; (2) increased temporal LCR synchronization, decreasing average LCR period and shifting larger LCR occurrence to late diastole.

Electrochemical Na+ and Ca2+ gradients drive coupled-clock regulation of automaticity of isolated rabbit sinoatrial nodal pacemaker cells.

Coupling of an intracellular Ca(2+) clock to surface membrane ion channels, i.e., a "membrane clock, " via coupling of electrochemical Na(+) and Ca(2+) 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, [Ca(2+)]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 [Ca(2+)]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 Ca(2+) releases (LCRs), and reduced the average APCL. As [Na(+)]i and [Ca(2+)]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 Ca(2+) signal was followed by INCX reduction as ENCX (ENCX = 3ENa - 2ECa) 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.

Dynamics of PKA phosphorylation and gain of function in cardiac pacemaker cells: a computational model analysis.

Cardiac pacemaker cell function is regulated by a coupled-clock system that integrates molecular cues on the cell-membrane surface (i.e., membrane clock) and on the sarcoplasmic reticulum (SR) (i.e., Ca(2+) clock). A recent study has shown that cotransfection of spontaneous beating cells (HEK293 cells and neonatal rat myocytes) with R524Q-mutant human hyperpolarization-activated cyclic nucleotide-gated molecules (the dominant component of funny channels) increases the funny channel's sensitivity to cAMP and leads to a decrease in spontaneous action potential (AP) cycle length (i.e., tachycardia). We hypothesize that in rabbit pacemaker cells, the same behavior is expected, and because of the coupled-clock system, the resultant steady-state decrease in AP cycle length will embody contributions from both clocks: the initial decrease in the spontaneous AP beating interval, arising from increased sensitivity of the f-channel to cAMP, will be accompanied by an increase in the adenylyl cyclase (AC)-cAMP-PKA-dependent phosphorylation activity, which will further decrease this interval. To test our hypothesis, we used the recently developed Yaniv-Lakatta pacemaker cell numerical model. This model predicts the cAMP signaling dynamics, as well as the kinetics and magnitude of protein phosphorylation in both normal and mutant pacemaker cells. We found that R524Q-mutant pacemaker cells have a shorter AP firing rate than that of wild-type cells and that gain in pacemaker function is the net effect of the R514Q mutation on the functioning of the coupled-clock system. Specifically, our results directly support the hypothesis that changes in Ca(2+)-activated AC-cAMP-PKA signaling are involved in the development of tachycardia in R524Q-mutant pacemaker cells.

The Coupled-Pacemaker Clock System of Sinoatrial Nodal Cells Regulates Both the Action Potential Rate and Rhythm

Normal automaticity of the sinatrial-node cells (SANC) is regulated by integrated molecular functions within a system of two clock-like oscillators: the sarcoplasmic reticulum, acting as a “Ca2+ clock,” rhythmically discharges diastolic local-Ca2+ releases (LCRs) beneath the cell surface membrane; LCRs activate an inward Na+/Ca2+ exchanger current (INCX) that prompts the “Membrane clock,” the ensemble of other sarcolemmal-electrogenic molecules, to generate action potentials (APs). Crosstalk between the two clocks regulates SANC spontaneous AP cycle length (CL). We determine whether clock crosstalk also regulates the rhythm of AP CL in response to perturbation that induce bradycardia via specific inhibition of either membrane or Ca2+ clock functions.We employed ivabradine (IVA, 3, 10 and 30µM) which directly inhibits membrane clock ion channel functions, but has no direct effect on Ca2+ clock proteins functions. The IVA-induced increase in the AP CL, however, lowers Ca2+ influx, which reduces Ca2+-activated calmodulin-AC-cAMP/PKA signaling, affecting Ca2+ and phosphorylation-dependent functions that deive the coupled clock system. Direct and specific inhibition of SERCA2 by cyclopiazonic acid (CPA, 0.5 and 5µM) reduces the magnitude and delays the occurrence of the LCR-Ca2+ signal, leading to a reduction in Ca2+-activated calmodulin-AC-cAMP/PKA signaling, and delayed and reduced Ca2+ activation of INCX and a prolongation of the AP CL. Importantly, prolongation of LCR period (IVA by 16±2 to 46±5%, CPA by 17±2 to 53±5%), AP CL (IVA by 13±4 to 36±8%, CPA by 16±3 to 44±8%) and the increase in AP CL variability (IVA by 38±10 to 190±50%, CPA by 36±17 to 183±50%) are proportional to each other. The tight inter-relationships among these variables in response to specific perturbation of either the M or the Ca2+ clock, therefore, reflect clock crosstalk.

CA2+ Cycling Proteins Phosphorylation in Isolated Sinoatrial Nodal Cells Modulates the Stochasticity of Spontaneous Diastolic Local CA2+ Releases which Regulate the Action Potential Cycle Length and its Beat-To-Beat Variation

Spontaneous local ryanodine receptor (RyR) activation and resultant local Ca2+ sparks are intrinsically stochastic. But in freshly isolated adult rabbit sinoatrial nodal cells (f-SANC), the basal phosphorylation of RyR and phospholamban (PLB) organizes spontaneous diastolic local Ca2+ releases (LCR) to occur (measured in confocal image) as roughly periodic Ca2+ wavelets that activate an inward NCX current to regulate the Action Potential beat interval (APBI). In cultured SANC (c-SANC), RyR and PLB phosphorylation become reduced, the average APBI is prolonged; these effects, however, are reversed by β-Adrenergic Receptor stimulation (β-ARs, fig1.AB 2) that the relationship of LCR period to APBI in c-SANC, f-SANC and c-SANC+ISO would be subtended by a unique linear function. These hypotheses are verified (fig1.B∼D).View Large Image | View Hi-Res Image | Download PowerPoint Slide

Mechanisms of Beat-to-Beat Regulation of Cardiac Pacemaker Cell Function by Ca2+ Cycling Dynamics

Whether intracellular Ca2+ 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 Ca2+ 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 Ca2+ buffering) and an acute CL reduction (produced by flash-induced Ca2+ release from a caged Ca2+ 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, Ca2+ releases acutely change the CL via activation of the Na+/Ca2+ exchanger current. Time-dependent CL reductions after flash-induced Ca2+ releases (the memory effect) are linked to changes in Ca2+ available for pumping into sarcoplasmic reticulum which, in turn, changes the sarcoplasmic reticulum Ca2+ load, diastolic Ca2+ releases, and Na+/Ca2+ exchanger current. These results support the idea that Ca2+ regulates CL in cardiac pacemaker cells on a beat-to-beat basis, and suggest a more realistic numerical mechanism of this regulation.

Pacemaker Gene Mutations, Bradycardia, Arrhythmias and the Coupled Clock Theory

To the Editor: In a recent paper, Nawathe et al.1 showed that M54T LQTS6 mutation in neonatal rat ventricular myoctes that were co-transfected with human HCN4, the dominate component of f-channels, is associated with bradycardia. The bradycardiac effects that were associated with these mutations, as claimed by the authors, are attributable soley to direct If inhibition. It is well established that normal automaticity of sinoatrial node cell (SANC) is regulated by integrated functions within a system of coupled-clocks. The sarcoplasmic reticulum (SR), traditionally defined as “Ca2+ clock”, spontaneously generates diastolic Ca2+ releases that activate an inward current (specifically Na+-Ca2+ exchanger current (INCX)), that accelerates the rate of diastolic membrane depolarization. The f-channel current (If) another member of the “Membrane clock” (i.e., the ensemble of sarcolemmal electrogenic molecules), concurrently drives the diastolic membrane depolarization with INCX.2 The two clocks are tightly coupled by a common chemical axes: Ca2+-calmodulin activated adenylyl cyclases that generates the second messenger cAMP, which activates protein kinase-A (PKA), and Ca2+ activated calmodulin-dependent kinase II (CaMKII). Both kinases phosphorylate different proteins of the M clock (e.g., L-type and K+ currents) and of the Ca2+ clock (phospholamban and ryanodine receptors). Additionally, cAMP positively shifts the f-channel activation curve. According to the coupled-clock theory, the membrane and Ca2+ clocks should crosstalk. Thus, a change in action potential (AP) firing rate in response to any disturbance signal that directly perturbs either clock entrains the function of the other clock. Consequently, the resultant steady-state AP cycle length change embodies contributions of both clocks. Therefore, the interpretation that bradycardia associated with mutation in HCN4 or with pharmacological blockade of If, occurs solely on the basis of direct inhibition of If function may not be correct. In this regard, a recent study demonstrated that ivabradine, at a concentration that specifically inhibits If, but does not directly suppress L-type current, SR Ca2+ cycling and other surface membrane ion channels, indirectly suppresses intracellular Ca2+ cycling.3 Similar clock coupling effects should occur in the presence of HCN4 mutations. A plausible, indirect effect on SR Ca2+ in response to HCN4 mutations or pharmacological blockade of If can be explained on the basis of this net reduction in Ca2+ influx associated with the bradycardic effect (fewer APs per unit time). A reduction in If initiates a small reduction in number of APs per unit time, which reduces the Ca2+ influx per unit time. This reduction in net Ca2+ influx results in a reduction in intracellular Ca2+, and Ca2+ available for pumping, reducing the SR Ca2+ load and prolonging the restitution time required for spontaneous diastolic Ca2+ releases to occur, i.e., the period of local Ca2+ release becomes prolonged. Consequently, the Ca2+ activation of INCX to a later time during diastolic depolarization, which further reduces the heart rate, leading to further reduction in Ca2+ influx per unit time. Moreover, as a result of reduced intracellular Ca2+, Ca2+ activated CaMKII and adenylyl cyclases-cAMP/PKA signaling axes become damped. The resultant reduction in phosphorylation of Ca2+ cycling proteins that further reduces the net Ca2+ influx, further reduces SR Ca2+ loading that further reduces AP rate and, in parallel, the resultant reduction in cAMP further shifts the If activation curve. Thus, the steady-state bradycardia measured experimentally in response to HCN4 mutations or pharmacological blockade of If is achieved when the induced reduced Ca2+ influx is balanced by a reduction in Ca2+ efflux via INCX. Similar to mutations in the f-channel within the membrane clock, mutations in the intracellular Ca2+ clock proteins are associated with bradicardia. A mutation in ryanodine receptor, axon-3, carried by patients with catecholaminergic polymorphic ventricular tachycardia, is associated with arrhythmias.4 Isolated SANC from mice that express this mutation have a slower spontaneous AP firing rate.5 Patients with a mutation in CACNA1D, which encodes the pore-forming α1 subunit of Cav1.3 (Cav1.3 is largely absent from the ventricles, but is highly expressed in atria, atrioventricular node and sinoatrial node6) experience pronounced bradycardia in 12–24-h ECG recordings, and their heart rate variability is increased.7 Similar to HCN4 mutations, the steady-state bradycardia associated with mutations in Ca2+ clock proteins may be mediated only in part by a reduction in available Ca2+ that inhibits Ca2+ cycling, and in part, by the resultant changes in cross-talk induced change in the membrane clock. In summary, crosstalk occurs between the Membrane and Ca2+ clocks whenever either the membrane or Ca2+ clock is directly perturbed the other clock is indirectly perturbed. Therefore, the steady-state bradycardia associated with different HCN4 mutations, ivabradine or Ca2+ regulatory protein is likely mediated, only in part by If inhibition, and in part, by change in Ca2+ clock due to clock cross-talk. Only an understanding of the full profile underling mechanisms of bradycardia that is associated with HCN4 mutations will promote the discovery of optionally effective treatments for bradycardia.

New evidence for coupled clock regulation of the normal automaticity of sinoatrial nodal pacemaker cells: Bradycardic effects of ivabradine are linked to suppression of intracellular Ca2 + cycling

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 “Ca2+ clock,” the sarcoplasmic reticulum (SR). We tested the hypothesis that crosstalk between the two clocks regulates SANC automaticity, and that indirect suppression of the Ca2+ clock further contributes to IVA-induced bradycardia. IVA (3μM) 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 Ca2+ load, slowed intracellular Ca2+ cycling kinetics, and prolonged the period of spontaneous local Ca2+ 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 Ca2+ 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.

Crosstalk between Mitochondrial and Sarcoplasmic Reticulum Ca2+ Cycling Modulates Cardiac Pacemaker Cell Automaticity

BackgroundMitochondria dynamically buffer cytosolic Ca2+ in cardiac ventricular cells and this affects the Ca2+ load of the sarcoplasmic reticulum (SR). In sinoatrial-node cells (SANC) the SR generates periodic local, subsarcolemmal Ca2+ releases (LCRs) that depend upon the SR load and are involved in SANC automaticity: LCRs activate an inward Na+-Ca2+ exchange current to accelerate the diastolic depolarization, prompting the ensemble of surface membrane ion channels to generate the next action potential (AP).ObjectiveTo determine if mitochondrial Ca2+ (Ca2+ m), cytosolic Ca2+ (Ca2+ c)-SR-Ca2+ crosstalk occurs in single rabbit SANC, and how this may relate to SANC normal automaticity.ResultsInhibition of mitochondrial Ca2+ influx into (Ru360) or Ca2+ efflux from (CGP-37157) decreased [Ca2+]m to 80±8% control or increased [Ca2+]m to 119±7% control, respectively. Concurrent with inhibition of mitochondrial Ca2+ influx or efflux, the SR Ca2+ load, and LCR size, duration, amplitude and period (imaged via confocal linescan) significantly increased or decreased, respectively. Changes in total ensemble LCR Ca2+ signal were highly correlated with the change in the SR Ca2+ load (r2 = 0.97). Changes in the spontaneous AP cycle length (Ru360, 111±1% control; CGP-37157, 89±2% control) in response to changes in [Ca2+]m were predicted by concurrent changes in LCR period (r2 = 0.84).ConclusionA change in SANC Ca2+ m flux translates into a change in the AP firing rate by effecting changes in Ca2+ c and SR Ca2+ loading, which affects the characteristics of spontaneous SR Ca2+ release.

Action Potential (AP) Cycle Length in Connexin-43-Negative and Positive Single Isolated Sinoatrial Node Cells (SANC) does not Differ in the Basal State or during High-Level Beta-AR Stimulation

SANC generate rhythmic APs that drive the heartbeat. Perspectives gleaned from the sinoatrial node(SAN) have been interpreted to indicate that smaller cells from the central SAN area control the SAN beating rate, while data from isolated SANC argue that spontaneous AP cycle length is independent of cell size, and that calcium cycling protein density is independent of cell area and size. It's well documented that the gap junction protein, connexin-43(Cx43), is highly expressed in peripheral vs. central SAN area. Since it is possible that cell size doesn’t completely discriminate between Cx43-negative and positive cells, we measured Cx43-immunolabeling, electrophysiological and Ca2+ cycling properties of single isolated SANC.Freshly isolated adult rabbit Cx43-negative SANC are, on average, smaller (592.3±9.2μm2, n=579) than Cx43-positive SANC (747.8±12.2μm2, n=571, p<0.001), but there is no difference in the spontaneous AP cycle length based on Cx43 expression (340.2±13.6ms n=30 in Cx43-negative cells, vs. 326.5±9.0ms n=50 in Cx43-positive cells, p=0.39). AP parameters also do not differ between Cx43-negative and positive cells, but the AP of later has a shorter repolarization time (APD90: 136.9±8.6ms vs. 110.2±4.8ms, p<0.01), which is not related to cell size. No significant differences are detected in the major characteristics of basal AP-triggered Ca2+-transient or spontaneous Local-Ca2+-Releases during diastolic depolarization between Cx43-negative and positive SANC. Acute β-AR stimulation (1μM isoproterenol) reduces the AP cycle length to the same level in both Cx43-negative and positive SANC (257.3±9.0ms n=9 vs. 271.6±7.7ms n=14, respectively, p=0.25), and no differences of APD90 are detected (p=0.17).Our results indicate that although different in size, there is no statistical difference between single isolated Cx43-negative and positive SANC of spontaneous AP cycle length or during maximal β-AR stimulation.

Numerical Modeling of Flash-Induced Ca2+ Release in Sa Node Cells (SANC) Supports the “Coupled-Clock” Hypothesis of Cardiac Pacemaker Cell Function

Our recent experiments in SA node cells using flash-induced Ca2+ release from a caged buffer (NP-EGTA) demonstrated that intracellular Ca2+ regulates normal SANC automaticity on a beat-to-beat basis. It cannot be directly envisioned, however, to what extent the reduction in action potential (AP) cycle length upon acute Ca2+ photo-release is attributable to rapid augmentation of Ca2+ pumping into sarcoplasmic reticulum (SR) followed by local Ca2+ releases (LCRs) vs. a direct effect of the flash-released Ca2+ on sarcolemmal proteins. To gain mechanistic insight into the problem, we numerically modeled the effects of flash-released Ca2+ using the 2009-Maltsev-Lakatta model of rabbit SANC that portrays pacemaker cell function as “coupled-clock” system, i.e. coupled intracellular Ca2+ oscillator of the SR and a cell membrane voltage oscillator. Knowing (from prior studies) Ca2+ “on” and “off” rates of NP-EGTA and experimentally measured AP cycle length increase following incubation with the buffer, by varying [NP-EGTA]i in model simulations we estimated [NP-EGTA]i (∼8 mM) required to reproduce experimental results. With this estimated [NP-EGTA]i, we further estimated the rate of Ca2+ release during flash photolysis (a function of laser intensity) to be 1 μM/ms in order to reproduce experimental results on acute AP cycle length shortening. The model also faithfully reproduced diastolic Ca2+ release increase (assessed experimentally as LCR signal mass) following the flash-photolysis. Other numerical models based on a dominance of the membrane voltage oscillator, including the most recent and advanced Kurata et al. model and “Kyoto” model (developed by the Noma's group), could not reproduce the experimental results with any reasonable [NP-EGTA]i (up to 10 mM) and flash-induced Ca2+ release flux (i.e. laser intensity). Our numerical model simulations support the “coupled-clock” hypothesis of cardiac pacemaker cell function.

Synchronization of Local Calcium Releases by Beta-Adrenergic Stimulation in Cardiac Pacemaker Cells

Sympathetic heart rate acceleration is a complex orchestration of intercellular, subcellular and molecular events. According to a modern concepts, rhythmic local Ca2+releases (LCRs), generated by a subcellular “Ca-clock”, interact with a “membrane-voltage-clock”, forming a coupled-clock system that produces rhythmic action potentials (AP). We imaged LCRs using a fast-2D-camera with simultaneous AP recording via perforated-patch-clamping to explore how β-AR stimulation modulates the phase and synchronization of the ensemble of individual LCRs within a given diastole to accelerate spontaneous beating rate. In single rabbit SA-node cells (36oC) isoproterenol (100nM): (i) synchronized LCR occurrence [the standard deviation of individual LCR periods (time from Ca2+transient peak to LCR occurrence) decreased (−30%; baseline,71.5ms vs. ISO,50ms)], (ii) shifted the LCR phases to earlier time-point, (iii) shifted “Ca-nadir”, i.e. time-point when AP-induced Ca2+-transient decay ceases and [Ca2+] begins to increase due to the occurrence of LCRs (time from maximum-diastolic-potential to Ca-nadir: baseline, 103ms vs. ISO,86ms); (iv) accelerated diastolic depolarization (baseline,0.09mV/ms vs. ISO,0.15mV/ms); (v) shortened spontaneous cycle length (baseline,420ms vs. ISO,345ms). We previously had demonstrated a close relationship between the decay of the global AP-induced Ca2+ transient and the LCR phase (within a given cycle) and the AP cycle length; we now found that the decays of each LOCAL Ca2+Transient (LCaT) induced by the prior AP vary in different subcellular locations/neighborhoods and that isoproterenol not only accelerated LCaT-decays (average time constant τ: baseline, 104 vs. ISO,87ms) but also synchronized LCaT-decays among neighborhoods (τ's SD/mean: baseline,40.5% vs. ISO,33.7%). More synchronous LCaT-decay mirrors more synchronous local SR Ca2+-pumping and therefore more synchronous achievement of the threshold SR Ca load required for spontaneous LCR occurrence. Thus, earlier and more synchronized LCR occurrence under β-AR stimulation insures an earlier and larger Na+/Ca2+-exchanger current that ultimately accelerates diastolic depolarization rate.