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.
Augmented and slowed late Na(+) current (I(NaL)) is implicated in action potential duration variability, early afterdepolarizations, and abnormal Ca(2+) handling in human and canine failing ...myocardium. Our objective was to study I(NaL) modulation by cytosolic Ca(2+) concentration (Ca(2+)(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. I(NaL) 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. I(NaL) greatly enhanced as Ca(2+)(i) increased from "Ca(2+) free" to 1 microM: 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 I(NaL) modulation in failing vs. normal myocytes. Similarities include the following: 1) CaMKII slows I(NaL) decay and decreases the amplitude of fast exponentials, and 2) Ca(2+) shifts SSI rightward. Differences include the following: 1) slowing of I(NaL) by CaMKII is greater, 2) CaM shifts SSI leftward, and 3) Ca(2+) increases the amplitude of slow exponentials. We conclude that Ca(2+)/CaM/CaMKII signaling increases I(NaL) and Na(+) influx in both normal and failing myocytes by slowing inactivation kinetics and shifting SSI. This Na(+) influx provides a novel Ca(2+) positive feedback mechanism (via Na(+)/Ca(2+) exchanger), enhancing contractions at higher beating rates but worsening cardiomyocyte contractile and electrical performance in conditions of poor Ca(2+) handling in heart failure.
Most cardiac Na
+ channels open transiently upon membrane depolarization and then are quickly inactivated. However, some channels remain active, carrying the so-called persistent or late Na
+ current ...(
I
NaL) throughout the action potential (AP) plateau. Experimental data and the results of numerical modeling accumulated over the past decade show the emerging importance of this late current component for the function of both normal and failing myocardium.
I
NaL is produced by special gating modes of the cardiac-specific Na
+ channel isoform. Heart failure (HF) slows channel gating and increases
I
NaL, but HF-specific Na
+ channel isoform underlying these changes has not been found. Na
+ channels represent a multi-protein complex and its activity is determined not only by the pore-forming
α subunit but also by its auxiliary
β subunits, cytoskeleton, calmodulin, regulatory kinases and phosphatases, and trafficking proteins. Disruption of the integrity of this protein complex may lead to alterations of
I
NaL in pathological conditions. Increased
I
NaL and the corresponding Na
+ flux in failing myocardium contribute to abnormal repolarization and an increased cell Ca
2+ load. Interventions designed to correct
I
NaL rescue normal repolarization and improve Ca
2+ handling and contractility of the failing cardiomyocytes. This review considers (1) quantitative integration of
I
NaL into the established electrophysiological and Ca
2+ regulatory mechanisms in normal and failing cardiomyocytes and (2) a new therapeutic strategy utilizing a selective inhibition of
I
NaL to target both arrhythmias and impaired contractility in HF.
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 (1 μM) 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.
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 70bpm; maximum rate of 140 to 210bpm) 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.
•We found a minimal set of molecular components for robust and flexible pacemaking.•Calcium clock is required for flexible pacemaker function.•Only three currents ICaL, IK, and INCX are sufficient to comprise the membrane clock.•The new model database is a useful tool for the design of biological pacemakers.
A delicate balance between quiescence and division of the radial glia-like stem cells (RGLs) ensures continuation of adult hippocampal neurogenesis (AHN) over the lifespan. Transient or persistent ...perturbations of this balance due to a brain pathology, drug administration, or therapy can lead to unfavorable long-term outcomes such as premature depletion of the RGLs, decreased AHN, and cognitive deficit. Memantine, a drug used for alleviating the symptoms of Alzheimer’s disease, and electroconvulsive seizure (ECS), a procedure used for treating drug-resistant major depression or bipolar disorder, are known strong AHN inducers; they were earlier demonstrated to increase numbers of dividing RGLs. Here, we demonstrated that 1-month stimulation of quiescent RGLs by either memantine or ECS leads to premature exhaustion of their pool and altered AHN at later stages of life and that aging of the brain modulates the ability of the quiescent RGLs to be recruited into the cell cycle by these AHN inducers. Our findings support the aging-related divergence of functional features of quiescent RGLs and have a number of implications for the practical assessment of drugs and treatments with respect to their action on quiescent RGLs at different stages of life in animal preclinical studies.
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.
The heartbeat is initiated by pacemaker cells residing in the sinoatrial node (SAN). SAN cells generate spontaneous action potentials (APs), i.e., normal automaticity. The sympathetic nervous system ...increases the heart rate commensurate with the cardiac output demand via stimulation of SAN β-adrenergic receptors (βAR). While SAN cells reportedly represent a highly heterogeneous cell population, the current dogma is that, in response to βAR stimulation, all cells increase their spontaneous AP firing rate in a similar fashion. The aim of the present study was to investigate the cell-to-cell variability in the responses of a large population of SAN cells. We measured the βAR responses among 166 single SAN cells isolated from 33 guinea pig hearts. In contrast to the current dogma, the SAN cell responses to βAR stimulation substantially varied. In each cell, changes in the AP cycle length were highly correlated (R
= 0.97) with the AP cycle length before βAR stimulation. While, as expected, on average, the cells increased their pacemaker rate, greater responses were observed in cells with slower basal rates, and vice versa: cells with higher basal rates showed smaller responses, no responses, or even decreased their rate. Thus, βAR stimulation synchronized the operation of the SAN cell population toward a higher average rate, rather than uniformly shifting the rate in each cell, creating a new paradigm of βAR-driven fight-or-flight responses among individual pacemaker cells.
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