[Show abstract][Hide abstract] ABSTRACT: At the beginning of this century, debates regarding "what are the main control mechanisms that ignite the action potential (AP) in heart pacemaker cells" dominated the electrophysiology field. The original theory which prevailed for over 50 years had advocated that the ensemble of surface membrane ion channels (i.e., "M-clock") is sufficient to ignite rhythmic APs. However, more recent experimental evidence in a variety of mammals has shown that the sarcoplasmic reticulum (SR) acts as a "Ca(2+)-clock" rhythmically discharges diastolic local Ca(2+) releases (LCRs) beneath the cell surface membrane. LCRs activate an inward current (likely that of the Na(+)/Ca(2+) exchanger) that prompts the surface membrane "M-clock" to ignite an AP. Theoretical and experimental evidence has mounted to indicate that this clock "crosstalk" operates on a beat-to-beat basis and determines both the AP firing rate and rhythm. Our review is focused on the evolution of experimental definition and numerical modeling of the coupled-clock concept, on how mechanisms intrinsic to pacemaker cell determine both the heart rate and rhythm, and on future directions to develop further the coupled-clock pacemaker cell concept.
Frontiers in Physiology 02/2015; 6:28. DOI:10.3389/fphys.2015.00028 · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Key points:
Late Na(+) current (INaL) contributes to action potential remodelling and Ca(2+)/Na(+) changes in heart failure. The molecular identity of INaL remains unclear. The contributions of different Na(+) channel isoforms, apart from the cardiac isoform, remain unknown. We discovered and characterized a substantial contribution of neuronal isoform Nav1.1 to INaL. This new component is physiologically relevant to the control of action potential shape and duration, as well as to cell Ca(2+) dynamics, especially in heart failure.
Late Na(+) current (INaL) contributes to action potential (AP) duration and Ca(2+) handling in cardiac cells. Augmented INaL was implicated in delayed repolarization and impaired Ca(2+) handling in heart failure (HF). We tested if Na(+) channel (Nav) neuronal isoforms contribute to INaL and Ca(2+) cycling defects in HF in 17 dogs in which HF was achieved via sequential coronary artery embolizations. Six normal dogs served as control. Transient Na(+) current (INaT ) and INaL in left ventricular cardiomyocytes (VCMs) were recorded by patch clamp while Ca(2+) dynamics was monitored using Fluo-4. Virally delivered short interfering RNA (siRNA) ensured Nav1.1 and Nav1.5 post-transcriptional silencing. The expression of six Navs was observed in failing VCMs as follows: Nav1.5 (57.3%) > Nav1.2 (15.3%) > Nav1.1 (11.6%) > Nav2.1 (10.7%) > Nav1.3 (4.6%) > Nav1.6 (0.5%). Failing VCMs showed up-regulation of Nav1.1 expression, but reduction of Nav1.6 mRNA. A similar Nav expression pattern was found in samples from human hearts with ischaemic HF. VCMs with silenced Nav1.5 exhibited residual INaT and INaL (∼30% of control) with rightwardly shifted steady-state activation and inactivation. These currents were tetrodotoxin sensitive but resistant to MTSEA, a specific Nav1.5 blocker. The amplitude of the tetrodotoxin-sensitive INaL was 0.1709 ± 0.0299 pA pF(-1) (n = 7 cells) and the decay time constant was τ = 790 ± 76 ms (n = 5). This INaL component was lacking in VCMs with a silenced Nav1.1 gene, indicating that, among neuronal isoforms, Nav1.1 provides the largest contribution to INaL. At -10 mV this contribution is ∼60% of total INaL. Our further experimental and in silico examinations showed that this new Nav1.1 INaL component contributes to Ca(2+) accumulation in failing VCMs and modulates AP shape and duration. In conclusion, we have discovered an Nav1.1-originated INaL component in dog heart ventricular cells. This component is physiologically relevant to controlling AP shape and duration, as well as to cell Ca(2+) dynamics.
The Journal of Physiology 10/2014; 593(6). DOI:10.1113/jphysiol.2014.278259 · 5.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Heart rate (HR) variability (HRV; beat-to-beat changes in the R-wave to R-wave interval) has attracted considerable attention during the past 30+ years (PubMed currently lists >17 000 publications). Clinically, a decrease in HRV is correlated to higher morbidity and mortality in diverse conditions, from heart disease to fetal distress. It is usually attributed to fluctuation in cardiac autonomic nerve activity. We calculated HRV parameters from a variety of cardiac preparations (including humans, living animals, Langendorff-perfused heart, and single sinoatrial nodal cell) in diverse species, combining this with data from previously published articles. We show that regardless of conditions, there is a universal exponential decay-like relationship between HRV and HR. Using 2 biophysical models, we develop a theory for this and confirm that HRV is primarily dependent on HR and cannot be used in any simple way to assess autonomic nerve activity to the heart. We suggest that the correlation between a change in HRV and altered morbidity and mortality is substantially attributable to the concurrent change in HR. This calls for re-evaluation of the findings from many articles that have not adjusted properly or at all for HR differences when comparing HRV in multiple circumstances.
[Show abstract][Hide abstract] ABSTRACT: The sinoatrial node, whose cells (sinoatrial node cells [SANCs]) generate rhythmic action potentials, is the primary pacemaker of the heart. During diastole, calcium released from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyRs) interacts with membrane currents to control the rate of the heartbeat. This "calcium clock" takes the form of stochastic, partially periodic, localized calcium release (LCR) events that propagate, wave-like, for limited distances. The detailed mechanisms controlling the calcium clock are not understood. We constructed a computational model of SANCs, including three-dimensional diffusion and buffering of calcium in the cytosol and SR; explicit, stochastic gating of individual RyRs and L-type calcium channels; and a full complement of voltage- and calcium-dependent membrane currents. We did not include an anatomical submembrane space or inactivation of RyRs, the two heuristic components that have been used in prior models but are not observed experimentally. When RyRs were distributed in discrete clusters separated by >1 µm, only isolated sparks were produced in this model and LCR events did not form. However, immunofluorescent staining of SANCs for RyR revealed the presence of bridging RyR groups between large clusters, forming an irregular network. Incorporation of this architecture into the model led to the generation of propagating LCR events. Partial periodicity emerged from the interaction of LCR events, as observed experimentally. This calcium clock becomes entrained with membrane currents to accelerate the beating rate, which therefore was controlled by the activity of the SERCA pump, RyR sensitivity, and L-type current amplitude, all of which are targets of β-adrenergic-mediated phosphorylation. Unexpectedly, simulations revealed the existence of a pathological mode at high RyR sensitivity to calcium, in which the calcium clock loses synchronization with the membrane, resulting in a paradoxical decrease in beating rate in response to β-adrenergic stimulation. The model indicates that the hierarchical clustering of surface RyRs in SANCs may be a crucial adaptive mechanism. Pathological desynchronization of the clocks may explain sinus node dysfunction in heart failure and RyR mutations.
The Journal of General Physiology 05/2014; 143(5):577-604. DOI:10.1085/jgp.201311123 · 4.79 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cardiac pacemaking is a complex phenomenon that is still not completely understood. Together with experimental studies, numerical modeling has been traditionally used to acquire mechanistic insights in this research area. This review summarizes the present state of numerical modeling of the cardiac pacemaker, including approaches to resolve present paradoxes and controversies. Specifically we discuss the requirement for realistic modeling to consider symmetrical importance of both intracellular and cell membrane processes (within a recent "coupled-clock" theory). Promising future developments of the complex pacemaker system models include the introduction of local calcium control, mitochondria function, and biochemical regulation of protein phosphorylation and cAMP production. Modern numerical and theoretical methods such as multi-parameter sensitivity analyses within extended populations of models and bifurcation analyses are also important for the definition of the most realistic parameters that describe a robust, yet simultaneously flexible operation of the coupled-clock pacemaker cell system. The systems approach to exploring cardiac pacemaker function will guide development of new therapies such as biological pacemakers for treating insufficient cardiac pacemaker function that becomes especially prevalent with advancing age.
[Show abstract][Hide abstract] ABSTRACT: 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.
Frontiers in Pharmacology 04/2014; 5:58. DOI:10.3389/fphar.2014.00058 · 3.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Rationale:
A recent study published in Circulation Research by Gao et al used sinoatrial node (SAN)-targeted, incomplete Ncx1 knockout in mice to explore the role of the Na(+)/Ca(2+) exchanger (NCX) in cardiac pacemaker. The authors concluded that NCX is required for increasing sinus rates, but not for maintaining resting heart rate. This conclusion was based, in part, on numeric model simulations performed by Gao et al that reproduced their experimental results of unchanged action potentials in the knockout SAN cells. The authors, however, did not simulate the NCX current (INCX), that is, the subject of the study.
We extended numeric examinations to simulate INCX in their incomplete knockout SAN cells that is crucial to interpret the study results.
Methods and results:
INCX and Ca(2+) dynamics were simulated using different contemporary numeric models of SAN cells. We found that minimum diastolic Ca(2+) levels and INCX amplitudes generated by remaining NCX molecules (only 20% of control) remained almost unchanged. Simulations using a new local Ca(2+) control model indicate that these powerful compensatory mechanisms involve complex local cross-talk of Ca(2+) cycling proteins and NCX. Specifically, lower NCX expression facilitates Ca(2+)-induced Ca(2+) release and larger local Ca(2+) releases that stabilize diastolic INCX. Further reduction of NCX expression results in arrhythmia and halt of automaticity.
Remaining NCX molecules in the incomplete knockout model likely produce almost the same diastolic INCX as in wild-type cells. INCX contribution is crucially important for both basal automaticity of SAN cells and during the fight-or-flight reflex.
Circulation Research 10/2013; 113(10):e94-e100. DOI:10.1161/CIRCRESAHA.113.302465 · 11.02 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
The Journal of General Physiology 09/2013; 142(3):257-74. DOI:10.1085/jgp.201311034 · 4.79 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Spontaneous, submembrane local Ca(2+) releases (LCRs) generated by the sarcoplasmic reticulum in sinoatrial nodal cells, the cells of the primary cardiac pacemaker, activate inward Na(+)/Ca(2+)-exchange current to accelerate the diastolic depolarization rate, and therefore to impact on cycle length. Since LCRs are generated by Ca(2+) release channel (i.e. ryanodine receptor) openings, they exhibit a degree of stochastic behavior, manifested as notable cycle-to-cycle variations in the time of their occurrence.
The present study tested whether variation in LCR periodicity contributes to intrinsic (beat-to-beat) cycle length variability in single sinoatrial nodal cells.
We imaged single rabbit sinoatrial nodal cells using a 2D-camera to capture LCRs over the entire cell, and, in selected cells, simultaneously measured action potentials by perforated patch clamp.
LCRs begin to occur on the descending part of the action potential-induced whole-cell Ca(2+) transient, at about the time of the maximum diastolic potential. Shortly after the maximum diastolic potential (mean 54±7.7 ms, n = 14), the ensemble of waxing LCR activity converts the decay of the global Ca(2+) transient into a rise, resulting in a late, whole-cell diastolic Ca(2+) elevation, accompanied by a notable acceleration in diastolic depolarization rate. On average, cells (n = 9) generate 13.2±3.7 LCRs per cycle (mean±SEM), varying in size (7.1±4.2 µm) and duration (44.2±27.1 ms), with both size and duration being greater for later-occurring LCRs. While the timing of each LCR occurrence also varies, the LCR period (i.e. the time from the preceding Ca(2+) transient peak to an LCR's subsequent occurrence) averaged for all LCRs in a given cycle closely predicts the time of occurrence of the next action potential, i.e. the cycle length.
Intrinsic cycle length variability in single sinoatrial nodal cells is linked to beat-to-beat variations in the average period of individual LCRs each cycle.
PLoS ONE 06/2013; 8(6):e67247. DOI:10.1371/journal.pone.0067247 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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 (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 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.
Journal of Molecular and Cellular Cardiology 05/2013; 62. DOI:10.1016/j.yjmcc.2013.04.026 · 4.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Calpain is an intracellular Ca2+ -activated protease that is involved in numerous Ca2+ dependent regulation of protein function in many cell types. This paper tests a hypothesis that calpains are involved in Ca2+ -dependent increase of the late sodium current (INaL) in failing heart. Chronic heart failure (HF) was induced in 2 dogs by multiple coronary artery embolization. Using a conventional patch-clamp technique, the whole-cell INaL was recorded in enzymatically isolated ventricular cardiomyocytes (VCMs) in which INaL was activated by the presence of a higher (1μM) intracellular [Ca2+] in the patch pipette. Cell suspensions were exposed to a cell- permeant calpain inhibitor MDL-28170 for 1–2 h before INaL recordings. The numerical excitation-contraction coupling (ECC) model was used to evaluate electrophysiological effects of calpain inhibition in silico. MDL caused acceleration of INaL decay evaluated by the two-exponential fit (τ1 = 42±3.0 ms τ2 = 435±27 ms, n = 6, in MDL vs. τ1 = 52±2.1 ms τ2 = 605±26 control no vehicle, n = 11, and vs. τ1 = 52±2.8 ms τ2 = 583±37 ms n = 7, control with vehicle, P<0.05 ANOVA). MDL significantly reduced INaL density recorded at –30 mV (0.488±0.03, n = 12, in control no vehicle, 0.4502±0.0210, n = 9 in vehicle vs. 0.166±0.05pA/pF, n = 5, in MDL). Our measurements of current-voltage relationships demonstrated that the INaL density was decreased by MDL in a wide range of potentials, including that for the action potential plateau. At the same time the membrane potential dependency of the steady-state activation and inactivation remained unchanged in the MDL-treated VCMs. Our ECC model predicted that calpain inhibition greatly improves myocyte function by reducing the action potential duration and intracellular diastolic Ca2+ accumulation in the pulse train.
Calpain inhibition reverses INaL changes in failing dog ventricular cardiomyocytes in the presence of high intracellular Ca2+. Specifically it decreases INaL density and accelerates INaL kinetics resulting in improvement of myocyte electrical response and Ca2+ handling as predicted by our in silico simulations.
PLoS ONE 04/2013; 8(4):e54436. DOI:10.1371/journal.pone.0054436 · 3.23 Impact Factor