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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; · 5.17 Impact Factor
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ABSTRACT: 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 Ca(2+) oscillator ("Ca(2+)clock") that generates spontaneous diastolic Ca(2+) 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 Ca(2+)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 Ca(2+) pumping (i.e. Ca(2+)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 Ca(2+)clock (including the minimal 4-parameter model "ICaL+IKr+INCX+Ca(2+)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.
Journal of Molecular and Cellular Cardiology 03/2013; · 5.17 Impact Factor
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AJP Cell Physiology 03/2013; · 3.54 Impact Factor
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ABSTRACT: Physiological processes governing the heart beat have been under investigation for several hundred years. Major advances have been made in the recent past. A review of the present paradigm is presented here, including a look back at important steps that led us to where we are today, alongside a glimpse into the exciting future of pacemaker research.
Physiology 03/2013; 28(2):74-92.
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Nature Biotechnology 01/2013; 31(1):31-2. · 29.50 Impact Factor
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ABSTRACT: Calpain is an intracellular Ca(2+) -activated protease that is involved in numerous Ca(2+) dependent regulation of protein function in many cell types. This paper tests a hypothesis that calpains are involved in Ca(2+) -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 [Ca(2+)] 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 Ca(2+) accumulation in the pulse train. CONCLUSIONS: Calpain inhibition reverses INaL changes in failing dog ventricular cardiomyocytes in the presence of high intracellular Ca(2+). Specifically it decreases INaL density and accelerates INaL kinetics resulting in improvement of myocyte electrical response and Ca(2+) handling as predicted by our in silico simulations.
PLoS ONE 01/2013; 8(4):e54436. · 4.09 Impact Factor
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ABSTRACT: Recent clinical trials have shown that ivabradine (IVA), a drug that inhibits the funny current (I(f)) in isolated sinoatrial nodal cells (SANC), decreases heart rate and reduces morbidity and mortality in patients with cardiovascular diseases. While IVA inhibits I(f), this effect has been reported at essentially unphysiological voltages, i.e., those more negative than the spontaneous diastolic depolarization (DD) between action potentials (APs). We tested the relative potency of IVA to block I(f) over a wide range of membrane potentials, including those that encompass DD governing to the SANC spontaneous firing rate. A clinically relevant IVA concentration of 3 μM to single, isolated rabbit SANC slowed the spontaneous AP firing rate by 15%. During voltage clamp the maximal I(f) was 18 ± 3 pA/pF (at -120 mV) and the maximal I(f) reduction by IVA was 60 ± 8% observed at -92 ± 4 mV. At the maximal diastolic depolarization (~-60 mV) I(f) amplitude was only -2.9 ± 0.4 pA/pF, and was reduced by only 41 ± 6% by IVA. Thus, I(f) amplitude and its inhibition by IVA at physiologically relevant membrane potentials are substantially less than that at unphysiological (hyperpolarized) membrane potentials. This novel finding more accurately describes how IVA affects SANC function and is of direct relevance to numerical modeling of SANC automaticity.
Molecules 01/2012; 17(7):8241-54. · 2.39 Impact Factor
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ABSTRACT: 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.
PLoS ONE 01/2012; 7(5):e37582. · 4.09 Impact Factor
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Heart rhythm: the official journal of the Heart Rhythm Society 09/2011; 9(2):302-7. · 4.56 Impact Factor
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Heart rhythm: the official journal of the Heart Rhythm Society 09/2011; 9(3):459-60. · 4.56 Impact Factor
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ABSTRACT: 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.
Journal of Molecular and Cellular Cardiology 09/2011; 51(6):902-5. · 5.17 Impact Factor
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Jie Liu,
Syevda Sirenko,
Magdalena Juhaszova,
Bruce Ziman,
Veena Shetty,
Silvia Rain,
Shweta Shukla,
Harold A Spurgeon,
Tatiana M Vinogradova, Victor A Maltsev,
Edward G Lakatta
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ABSTRACT: 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.
Journal of Molecular and Cellular Cardiology 08/2011; 51(5):730-9. · 5.17 Impact Factor
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ABSTRACT: The emerging paradigm for Na(+) current in heart failure (HF) is that its transient component (I(NaT)) responsible for the action potential (AP) upstroke is decreased, whereas the late component (I(NaL)) involved in AP plateau is augmented. Here we tested whether Na(v)β(1)- and Na(v)β(2)-subunits can modulate I(NaL) parameters in normal and failing ventricular cardiomyocytes (VCMs). Chronic HF was produced in nine dogs by multiple sequential coronary artery microembolizations, and six dogs served as a control. I(Na) and APs were measured by the whole cell and perforated patch-clamp in freshly isolated and cultured VCMs, respectively. I(NaL) was augmented with slower decay in HF VCMs compared with normal heart VCMs, and these properties remained unchanged within 5 days of culture. Post-transcriptional silencing SCN1B and SCN2B were achieved by virally delivered short interfering RNA (siRNA) specific to Na(v)β(1) and Na(v)β(2). The delivery and efficiency of siRNA were evaluated by green fluorescent protein expression, by the real-time RT-PCR, and Western blots, respectively. Five days after infection, the levels of mRNA and protein for Na(v)β(1) and Na(v)β(2) were reduced by >80%, but mRNA and protein of Na(v)1.5, as well as I(NaT), remained unchanged in HF VCMs. Na(v)β(1)-siRNA reduced I(NaL) density and accelerated I(NaL) two-exponential decay, whereas Na(v)β(2)-siRNA produced an opposite effect in VCMs from both normal and failing hearts. Physiological importance of the discovered I(NaL) modulation to affect AP shape and duration was illustrated both experimentally and by numerical simulations of a VCM excitation-contraction coupling model. We conclude that in myocytes of normal and failing dog hearts Na(v)β(1) and Na(v)β(2) exhibit oppositely directed modulation of I(NaL).
AJP Heart and Circulatory Physiology 06/2011; 301(4):H1596-605. · 3.71 Impact Factor
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AJP Heart and Circulatory Physiology 06/2011; 300(6):H2323-4; author reply H2325-6. · 3.71 Impact Factor
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ABSTRACT: In sinoatrial node cells of the heart, beating rate is controlled, in part, by local Ca²(+) releases (LCRs) from the sarcoplasmic reticulum, which couple to the action potential via electrogenic Na(+)/Ca²(+) exchange. We observed persisting, roughly periodic LCRs in depolarized rabbit sinoatrial node cells (SANCs). The features of these LCRs were reproduced by a numerical model consisting of a two-dimensional array of stochastic, diffusively coupled Ca²(+) release units (CRUs) with fixed refractory period. Because previous experimental studies showed that β-adrenergic receptor stimulation increases the rate of Ca²(+) release through each CRU (dubbed I(spark)), we explored the link between LCRs and I(spark) in our model. Increasing the CRU release current I(spark) facilitated Ca²(+)-induced-Ca²(+) release and local recruitment of neighboring CRUs to fire more synchronously. This resulted in a progression in simulated LCR size (from sparks to wavelets to global waves), LCR rhythmicity, and decrease of LCR period that parallels the changes observed experimentally with β-adrenergic receptor stimulation. The transition in LCR characteristics was steeply nonlinear over a narrow range of I(spark), resembling a phase transition. We conclude that the (partial) periodicity and rate regulation of the "Calcium clock" in SANCs are emergent properties of the diffusive coupling of an ensemble of interacting stochastic CRUs. The variation in LCR period and size with I(spark) is sufficient to account for β-adrenergic regulation of SANC beating rate.
Biophysical Journal 01/2011; 100(2):271-83. · 3.65 Impact Factor
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ABSTRACT: Recent experimental and theoretical studies demonstrate that the sinoatrial node cells (SANCs), the primary pacemaker cells
of heart, operate as a complex system of functionally coupled sarcolemmal and intracellular proteins. The proteins of this
system dynamically (beat-to-beat) interact throughout the entire pacemaker cycle via membrane voltage and local subsarcolemmal
Ca2+ changes. Furthermore, functions of the sarcolemmal (SL) electrogenic proteins and sarcoplasmic reticulum (SR) Ca2+ cycling proteins are coupled and regulated enzymatically via Ca2+-, PKA-, and CaMKII-dependent protein phosphorylation. The system is not only robust (i.e., fail-safe within wide parameter
range), but simultaneously flexible, because the autonomic neural modulation of the beating rate, via G protein-coupled receptor
(GPCR) signaling, acts upon the very same regulatory factors (i.e., the coupling factors, Ca2+, PKA, and CaMKII) that ensure and regulate robust system function in the basal state. This chapter summarizes the experimental
and theoretical evidences for this novel pacemaker concept.
12/2010: pages 83-99;
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ABSTRACT: There is an intense interest in differentiating embryonic stem cells to engineer biological pacemakers as an alternative to electronic pacemakers for patients with cardiac pacemaker function deficiency. Embryonic stem cell-derived cardiocytes (ESCs), however, often exhibit dysrhythmic excitations. Using Ca(2+) imaging and patch-clamp techniques, we studied requirements for generation of spontaneous rhythmic action potentials (APs) in late-stage mouse ESCs. Sarcoplasmic reticulum (SR) of ESCs generates spontaneous, rhythmic, wavelet-like Local Ca(2+)Releases (LCRs) (inhibited by ryanodine, tetracaine, or thapsigargin). L-type Ca(2+)current (I(CaL)) induces a global Ca(2+) release (CICR), depleting the Ca(2+) content SR which resets the phases of LCR oscillators. Following a delay, SR then generates a highly synchronized spontaneous Ca(2+)release of multiple LCRs throughout the cell. The LCRs generate an inward Na(+)/Ca(2+)exchanger (NCX) current (absent in Na(+)-free solution) that ignites the next AP. Interfering with SR Ca(2+) cycling (ryanodine, caffeine, thapsigargin, cyclopiazonic acid, BAPTA-AM), NCX (Na(+)-free solution), or I(CaL) (nifedipine) results in dysrhythmic excitations or cessation of automaticity. Inhibition of cAMP/PKA signaling by a specific PKA inhibitor, PKI, decreases SR Ca(2+) loading, substantially reducing both spontaneous LCRs (number, size, and amplitude) and rhythmic AP firing. In contrast, enhancing PKA signaling by cAMP increases the LCRs (number, size, duration) and converts irregularly beating ESCs to rhythmic "pacemaker-like" cells. SR Ca(2+) loading and LCR activity could be also increased with a selective activation of SR Ca(2+) pumping by a phospholamban antibody. We conclude that SR Ca(2+) loading and spontaneous rhythmic LCRs are driven by inherent cAMP/PKA activity. I(CaL) synchronizes multiple LCR oscillators resulting in strong, partially synchronized diastolic Ca(2+) release and NCX current. Rhythmic ESC automaticity can be achieved by boosting "coupling" factors, such as cAMP/PKA signaling, that enhance interactions between SR and sarcolemma.
Journal of Molecular and Cellular Cardiology 10/2010; 50(1):66-76. · 5.17 Impact Factor
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ABSTRACT: 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.
The Journal of Physiological Sciences 07/2010; 60(4):245-57. · 1.61 Impact Factor
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Journal of Molecular and Cellular Cardiology 04/2010; 48(4):804-6. · 5.17 Impact Factor
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ABSTRACT: Classical numerical models have attributed the regulation of normal cardiac automaticity in sinoatrial node cells (SANCs) largely to G protein-coupled receptor (GPCR) modulation of sarcolemmal ion currents. More recent experimental evidence, however, has indicated that GPCR modulation of SANCs automaticity involves spontaneous, rhythmic, local Ca(2+) releases (LCRs) from the sarcoplasmic reticulum (SR). We explored the GPCR rate modulation of SANCs using a unique and novel numerical model of SANCs in which Ca(2+)-release characteristics are graded by variations in the SR Ca(2+) pumping capability, mimicking the modulation by phospholamban regulated by cAMP-mediated, PKA-activated signaling. The model faithfully predicted the entire range of physiological chronotropic modulation of SANCs by the activation of beta-adrenergic receptors or cholinergic receptors only when experimentally documented changes of sarcolemmal ion channels are combined with a simultaneous increase/decrease in SR Ca(2+) pumping capability. The novel numerical mechanism of GPCR rate modulation is based on numerous complex synergistic interactions between sarcolemmal and intracellular processes via membrane voltage and Ca(2+). Major interactions include changes of diastolic Na(+)/Ca(2+) exchanger current that couple earlier/later diastolic Ca(2+) releases (predicting the experimentally defined LCR period shift) of increased/decreased amplitude (predicting changes in LCR signal mass, i.e., the product of LCR spatial size, amplitude, and number per cycle) to the diastolic depolarization and ultimately to the spontaneous action potential firing rate. Concomitantly, larger/smaller and more/less frequent activation of L-type Ca(2+) current shifts the cellular Ca(2+) balance to support the respective Ca(2+) cycling changes. In conclusion, our model simulations corroborate recent experimental results in rabbit SANCs pointing to a new paradigm for GPCR heart rate modulation by a complex system of dynamically coupled sarcolemmal and intracellular proteins.
AJP Heart and Circulatory Physiology 03/2010; 298(6):H2010-23. · 3.71 Impact Factor
