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

2016 ◽  
Vol 311 (1) ◽  
pp. H251-H267 ◽  
Author(s):  
Syevda G. Sirenko ◽  
Victor A. Maltsev ◽  
Yael Yaniv ◽  
Rostislav Bychkov ◽  
Daniel Yaeger ◽  
...  
Keyword(s):  
Cycle Length ◽  
Surface Membrane ◽  
Ion Currents ◽  
Pacemaker Cells ◽  
Small Reduction ◽  
Complex Interplay ◽  
Digitalis Glycoside ◽  
Intracellular Ca ◽  
Potential Cycle ◽  
Diastolic Potential

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

Normal spontaneous firing of cardiac pacemaker cells is regulated by basal pkc delta activation

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
T Vinogradova ◽  
K Tarasov ◽  
D Riordon ◽  
Y Tarasova ◽  
E Lakatta
Keyword(s):  
Protein Kinase ◽  
Cycle Length ◽  
Cardiac Pacemaker ◽  
Funding Source ◽  
Spontaneous Firing ◽  
Pacemaker Cells ◽  
Patch Clamp Technique ◽  
Sa Node ◽  
Pkc Delta ◽  
Pkc Isoforms

Abstract   The spontaneous beating rate of rabbit sinoatrial node cells (SANC) is regulated by local subsarcolemmal calcium releases (LCRs) from sarcoplasmic reticulum (SR). LCRs appear during diastolic depolarization (DD) and activate an inward sodium/calcium exchange current which increases DD rate and thus accelerates spontaneous SANC firing. High basal level of protein kinase A and calcium/calmodulin-dependent protein kinase II phosphorylation are required to sustain basal LCRs and normal spontaneous SANC firing. Recently we discovered that basal PKC activation is also obligatory for cardiac pacemaker function: inhibition of PKC activity by broad spectrum PKC inhibitors Bis I or calphostin C markedly suppressed SR calcium cycling and decreased or abolished spontaneous beating of freshly isolated rabbit SANC. Here we studied which PKC isoforms mediate PKC-dependent effects on cardiac pacemaker cell automaticity. The PKC superfamily consists of 3 major subgroups: conventional, novel and atypical. All PKC isoforms were detected at the RNA level (RT-qPCR) in the rabbit SA node and ventricle, and expression levels were comparable in both tissues. Expression of PKCβ, however, was markedly higher in the rabbit SA node, compared to other PKC isoenzymes in either tissue. We verified expression of conventional PKC (α, β) and novel PKC-delta at the protein level in SANC and ventricular myocytes (VM). Western blot confirmed RNA results, showing a 6-fold higher PKCβ protein abundance in SANC compared to VM. Expression of PKCα protein was similar in both cell types, while PKC-delta protein was more abundant in VM. To study whether PKCβ regulates spontaneous beating of SANC we employed selective inhibitor of conventional (α, β, gamma) PKC isoforms Go6976 (10 μmol/L), which had no effects on either LCR characteristics (confocal microscopy, calcium indicator Fluo-3AM) or spontaneous beating of freshly isolated rabbit SANC (perforated patch-clamp technique). Because selective PKC-delta inhibitors are not available, we explored effects of PKC-delta inhibition comparing effects of Go6976 (the inhibitor of conventional PKCs) and Go6983, which inhibits conventional PKCs and PKC-delta. In contrast to Go6976, Go6983 (5 μmol/L) markedly decreased the LCR size (from 7.1±0.4 to 4.5±0.3 μm) and number per each spontaneous cycle (from 1.3±0.1 to 0.8±0.1). It also markedly increased the LCR period (time from the prior AP-induced calcium transient to the subsequent LCR) which was paralleled by an increase in the spontaneous SANC cycle length. Rottlerin, another PKC-delta inhibitor, produced similar effects on LCR characteristics, and markedly and time-dependently decreased DD rate, leading to an increase in the spontaneous cycle length, and finally abrogated the spontaneous SANC firing. Thus, our data indicate that basal activity of PKC-delta, but not that of PKCβ, is essential for generation of LCRs and normal spontaneous firing of cardiac pacemaker cells. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Intramural Research Program, National Institute on Aging, National Institute of Health, USA

scholarly journals Cross-talk between Native Plasmalemmal Na+/Ca2+Exchanger and Inositol 1,4,5-Trisphosphate-sensitive Ca2+Internal Store inXenopusOocytes

2004 ◽  
Vol 279 (50) ◽  
pp. 52414-52424 ◽  
Author(s):  
Luisa M. Solís-Garrido ◽  
Antonio J. Pintado ◽  
Eva Andrés-Mateos ◽  
María Figueroa ◽  
Carlos Matute ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Surface Membrane ◽  
Rabbit Serum ◽  
Caged Compounds ◽  
Reverse Transcription Pcr ◽  
Inositol 1,4,5 Trisphosphate ◽  
Intracellular Ca ◽  
Functional Consequences ◽  
Extracellular Sodium ◽  
Cortical Endoplasmic Reticulum

Because the presence of a native plasmalemmal Na+/Ca2+exchange (NCX) activity inXenopus laevisoocytes remains controversial, its possible functional role in these cells is poorly understood. Here, in experiments on control oocytes and oocytes overexpressing a cloned NCX1 cardiac protein, confocal microscopy combined with electrophysiological techniques reveal that these cells express an endogenous NCX protein forming a functional microdomain with inositol 1,4,5-trisphosphate receptors (InsP3R) that controls intracellular Ca2+in a restricted subplasmalemmal space. The following data obtained in control denuded oocytes are consistent with this view: (i) reverse transcription-PCR revealed that the oocyte expresses two transcripts for the NCX1 and NCX3 isoforms; (ii) immunofluorescence experiments showed that native NCX1 and InsP3Rs are largely codistributed in discrete areas of the plasma membrane in close apposition to the cortical endoplasmic reticulum shell; (iii) when stimulated by rabbit serum, which elevates intracellular Ca2+mediated by InsP3, voltage-clamped oocytes display a large and transient inward Ca2+-activated chloride current, ICl(Ca), as a result of the Ca2+rise at the inner surface membrane; (iv) this current is significantly enhanced by KB-R7943 and by an extracellular sodium-depleted medium, two maneuvers that prevent “Ca2+extrusion” via NCX; and (v) blocking NCX enhanced the ICl(Ca)elicited by InsP3but not by Ca2+photolysis in oocytes injected with the respective caged compounds. Moreover, overexpression of cardiac NCX1, confirmed by confocal microscopy, has functional consequences for the “Ca2+influx” but not for the serum-elicited “Ca2+efflux” mode of basal exchange activity and does not alter the number of endogenous NCX/InsP3Rs colocalization sites. Our results suggest that native NCX, because of its strategic position, may regulate InsP3-mediated Ca2+signaling during the early phases of oocyte maturation and/or fertilization, and furthermore foreign cardiac protein is excluded from the Ca2+microdomains surrounding the native NCX/InsP3Rs complex in the oocyte.

scholarly journals Intramural optical mapping of Vm and Cai2+ during long-duration ventricular fibrillation in canine hearts

2012 ◽  
Vol 302 (6) ◽  
pp. H1294-H1305 ◽  
Author(s):  
Wei Kong ◽  
Raymond E. Ideker ◽  
Vladimir G. Fast
Keyword(s):  
Ventricular Fibrillation ◽  
Action Potential ◽  
Short Duration ◽  
Action Potential Duration ◽  
Cycle Length ◽  
The Other ◽  
Membrane Voltage ◽  
Long Duration ◽  
Intracellular Ca ◽  
Rapid Pacing

Intramural gradients of intracellular Ca2+ (Cai2+) Cai2+ handling, Cai2+ oscillations, and Cai2+ transient (CaT) alternans may be important in long-duration ventricular fibrillation (LDVF). However, previous studies of Cai2+ handling have been limited to recordings from the heart surface during short-duration ventricular fibrillation. To examine whether abnormalities of intramural Cai2+ handling contribute to LDVF, we measured membrane voltage ( Vm) and Cai2+ during pacing and LDVF in six perfused canine hearts using five eight-fiber optrodes. Measurements were grouped into epicardial, midwall, and endocardial layers. We found that during pacing at 350-ms cycle length, CaT duration was slightly longer (by ≃10%) in endocardial layers than in epicardial layers, whereas action potential duration (APD) exhibited no difference. Rapid pacing at 150-ms cycle length caused alternans in both APD (APD-ALT) and CaT amplitude (CaA-ALT) without significant transmural differences. For 93% of optrode recordings, CaA-ALT was transmurally concordant, whereas APD-ALT was either concordant (36%) or discordant (54%), suggesting that APD-ALT was not caused by CaA-ALT. During LDVF, Vm and Cai2+ progressively desynchronized when not every action potential was followed by a CaT. Such desynchronization developed faster in the epicardium than in the other layers. In addition, CaT duration strongly increased (by ∼240% at 5 min of LDVF), whereas APD shortened (by ∼17%). CaT rises always followed Vm upstrokes during pacing and LDVF. In conclusion, the fact that Vm upstrokes always preceded CaTs indicates that spontaneous Cai2+ oscillations in the working myocardium were not likely the reason for LDVF maintenance. Strong Vm-Cai2+ desynchronization and the occurrence of long CaTs during LDVF indicate severely impaired Cai2+ handling and may potentially contribute to LDVF maintenance.

scholarly journals Cardiac Pacemaker Cells Generate Cardiomyocytes from Fibroblasts in Long-Term Cultures

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Shigeki Kiuchi ◽  
Akino Usami ◽  
Tae Shimoyama ◽  
Fuminori Otsuka ◽  
Sachiko Yamaguchi ◽  
...  
Keyword(s):  
Guinea Pig ◽  
Cardiac Fibroblasts ◽  
Cluster Formation ◽  
Cardiac Pacemaker ◽  
Pacemaker Cells ◽  
Intracellular Ca ◽  
Specific Proteins ◽  
Cell Densities

Abstract Because cardiomyocyte generation is limited, the turnover of cardiomyocytes in adult heart tissues is much debated. We report here that cardiac pacemaker cells can generate cardiomyocytes from fibroblasts in vitro. Sinoatrial node cells (SANCs) were isolated from adult guinea pig hearts and were cultured at relatively low cell densities. Within a week, a number of fibroblast-like cells were observed to gather around SANCs, and these formed spontaneously beating clusters with cardiomyocyte structures. The clusters expressed genes and proteins that are characteristic of atrial cardiomyocytes. Pharmacological blocking of pacemaker currents inhibited generation of action potentials, and the spontaneous beating were ceased by physically destroying a few central cells. Inhibition of beating during culture also hampered the cluster formation. Moreover, purified guinea pig cardiac fibroblasts (GCFs) expressed cardiac-specific proteins in co-culture with SANCs or in SANC-preconditioned culture medium under electrical stimulation. These results indicate that SANCs can generate cardiomyocytes from cardiac fibroblasts through the influence of humoral factor(s) and electrophysiological activities followed by intracellular Ca2+ oscillations. This potential of SANCs to generate cardiomyocytes indicates a novel mechanism by which cardiomyocytes turns over in the vicinity of pacemaker cells and could be exploited in the development of strategies for cardiac regenerative therapy in adult hearts.

Intracellular Ca2+ Release Sparks Atrial Pacemaker Activity

Physiology ◽  
2001 ◽  
Vol 16 (3) ◽  
pp. 101-106 ◽  
Author(s):  
Stephen L. Lipsius ◽  
Jörg Hüser ◽  
Lothar A. Blatter
Keyword(s):  
Plasma Membrane ◽  
Sarcoplasmic Reticulum ◽  
Right Atrium ◽  
Pacemaker Activity ◽  
Transport Mechanisms ◽  
Electrical Excitation ◽  
Pacemaker Cells ◽  
Pacemaker Function ◽  
Intracellular Ca ◽  
The Right

Electrical excitation of the mammalian heart originates from specialized pacemaker cells in the right atrium. Pacemaker activity depends on multiple ion channels and transport mechanisms that reside primarily within the plasma membrane. However, recent evidence indicates that intracellular Ca2+ release from the sarcoplasmic reticulum also contributes importantly to atrial pacemaker function.

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

2016 ◽  
Vol 310 (9) ◽  
pp. H1259-H1266 ◽  
Author(s):  
Joachim Behar ◽  
Yael Yaniv
Keyword(s):  
Cycle Length ◽  
Cell Function ◽  
Membrane Surface ◽  
Cardiac Pacemaker ◽  
Neonatal Rat ◽  
Hek293 Cells ◽  
Pacemaker Cell ◽  
Pacemaker Cells ◽  
Clock System ◽  
Dominant Component

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., Ca2+ 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 Ca2+-activated AC-cAMP-PKA signaling are involved in the development of tachycardia in R524Q-mutant pacemaker cells.

scholarly journals Molecular Substrates Mediating Lanthanide-Evoked Neurotransmitter Release in Central Synapses

2008 ◽  
Vol 100 (4) ◽  
pp. 2089-2100 ◽  
Author(s):  
ChiHye Chung ◽  
Ferenc Deák ◽  
Ege T. Kavalali
Keyword(s):  
Neurotransmitter Release ◽  
Surface Membrane ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Protein Receptor ◽  
Rapid Action ◽  
Intracellular Ca ◽  
Synaptotagmin 1 ◽  
Molecular Substrates ◽  
Central Synapses

Noncanonical secretagogues such as hypertonicity or α-latrotoxin have been extremely informative in studying neurotransmission. Lanthanum and lanthanides can also trigger neurotransmitter release through an unknown mechanism. Here, we studied the effect of lanthanides on neurotransmission in hippocampal cultures. Application of 2 mM La3+ caused rapid and robust neurotransmitter release within seconds. In addition, transient application of La3+ uncovered a sustained facilitation of miniature neurotransmission. The response to La3+ was detectable at 2 μM and increased in a concentration-dependent manner ≤2 mM. Rapid effect of La3+ was independent of extracellular and intracellular Ca2+ and did not require La3+ entry into cells or activation of phospholipaseCβ. Synapses deficient in synaptobrevin-2, the major synaptic vesicle soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein in the brain, did not display any rapid release in response to La3+, whereas the slow facilitation of release detected after La3+ removal remained intact. In contrast, preincubation with intracellular Ca2+ chelators selectively attenuated the delayed release triggered by La3+. Moreover, synapses deficient in synaptotagmin-1 maintained a rapid response to La3+, suggesting that La3+-triggered neurotransmitter release does not require synaptotagmin-1 as a sensor. Therefore La3+ has two separate effects on synaptic transmission. For its rapid action, La3+ interacts with a target on the surface membrane, and unlike other forms of release, it triggers strictly synaptobrevin-2–dependent fusion, implying that in central synapses synaptobrevin-2 function is secretagogue specific. For the delayed action, La3+ may act intracellularly after its entry or through intracellular Ca2+ via a mechanism that does not require synaptobrevin-2.

scholarly journals Electrophysiological heterogeneity of pacemaker cells in the rabbit intercaval region, including the SA node: insights from recording multiple ion currents in each cell

2018 ◽  
Vol 314 (3) ◽  
pp. H403-H414 ◽  
Author(s):  
Oliver Monfredi ◽  
Kenta Tsutsui ◽  
Bruce Ziman ◽  
Michael D. Stern ◽  
Edward G. Lakatta ◽  
...  
Keyword(s):  
Ion Currents ◽  
Pacemaker Cells ◽  
Sa Node

Early effects of metabolic inhibition on intracellular Ca2+ in toad pacemaker cells: involvement of Ca2+ stores

2003 ◽  
Vol 284 (4) ◽  
pp. H1087-H1094 ◽  
Author(s):  
Yue-Kun Ju ◽  
David G. Allen
Keyword(s):  
Firing Rate ◽  
Substantial Reduction ◽  
Metabolic Inhibition ◽  
Pacemaker Cells ◽  
Cane Toad ◽  
Main Effect ◽  
Intracellular Ca ◽  
Early Effects ◽  
Cytochrome P 450 ◽  
Spontaneous Firing Rate

The early effects of metabolic inhibition on intracellular Ca2+ concentration ([Ca2+]i), Ca2+ current, and sarcoplasmic reticulum (SR) Ca2+ content were studied in single pacemaker cells from the sinus venosus of the cane toad. The amplitude of the spontaneous elevations of systolic [Ca2+]i (Ca2+ transients) was reduced after 5-min exposure to 2 mM NaCN from 338 ± 30 to 189 ± 37 nM ( P < 0.005, n = 9), and the spontaneous firing rate was reduced from 27 ± 2 to 12 ± 4 beats/min ( P < 0.002, n = 9). It has been proposed that CN− acts by inhibition of cytochrome P-450, resulting in a reduction of cAMP and Ca2+ current. To test this proposal, we used clotrimazole, a cytochrome P-450 inhibitor, which also decreased the Ca2+ transients and firing rate. CN− caused an insignificant fall of Ca2+ current (23 ± 11%) but a substantial reduction of SR Ca2+ content (by 65 ± 5%), whereas clotrimazole produced a larger reduction of Ca2+ current and did not affect the SR Ca2+content. Thus the main effect of CN− does not seem to be through inhibition of cytochrome P-450. In conclusion, CN− appears to reduce Ca2+ release from the SR mainly by reducing SR Ca2+ content. A likely cause of the decreased SR content is reduced Ca2+ uptake by the SR pump.

scholarly journals Effect of twitch interval duration on the contractile function of subsequent twitches in isolated rat, rabbit, and dog myocardium under physiological conditions

2011 ◽  
Vol 111 (4) ◽  
pp. 1159-1167 ◽  
Author(s):  
Ying Xu ◽  
Michelle M. Monasky ◽  
Nitisha Hiranandani ◽  
Kaylan M. Haizlip ◽  
George E. Billman ◽  
...  
Keyword(s):  
Cycle Length ◽  
Contractile Function ◽  
Stimulation Frequency ◽  
Mediated Effects ◽  
Relative Contribution ◽  
Intracellular Ca ◽  
State Frequency ◽  
Cycle Lengths ◽  
Primary Cycle ◽  
Contractility Of The Myocardium

Many studies have shown that a change in stimulation frequency leads to altered contractility of the myocardium. However, it remains unclear what changes occur directly after a change in frequency and which ones are a result of the slow processes that lead to the altered homeostasis, which develops after a change in stimulation frequency. To distinguish the immediate from the slow responses, we assessed contractile function in two species that have distinctively different calcium (Ca2+)-handling properties using a recently developed, randomized pacing protocol. In isolated dog and rat right ventricular trabeculae, twitch contractions at five different cycle lengths within the physiologic range of each species were randomized around a steady-state frequency. We found, in both species, that the duration of the cycle length just prior to the analyzed twitch (primary) positively correlated with the increased force of the analyzed twitch. In sharp contrast, the cycle lengths, one and two more removed from the analyzed twitch (“secondary” and “tertiary”), displayed a negative correlation with force of the analyzed twitch. In additional experiments, assessment of intracellular Ca2+ transients in rabbit trabeculae revealed that diastolic Ca2+ levels were closely correlated to contractile function outcome. The relative contribution of the primary cycle length was different between dog (51%) and rat (71%), whereas in neither species was a significant effect on relaxation time observed. With the use of randomized cycle lengths, we have distinguished the intrinsic response from the signaling-mediated effects of frequency-dependent activation on myofilament properties and Ca2+ handling.

Sign in / Sign up

Export Citation Format

Share Document