P1593VEGFR1-PLC signaling is implicated in normal spontaneous firing of cardiac pacemaker cells

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
T Vinogradova ◽  
K Tarasov ◽  
Y Tarasova ◽  
E Lakatta
Keyword(s):  
Cycle Length ◽  
Sinoatrial Node ◽  
Calcium Influx ◽  
Cardiac Pacemaker ◽  
Dependent Manner ◽  
Spontaneous Firing ◽  
Calcium Cycling ◽  
Pacemaker Cells ◽  
Patch Clamp Technique ◽  
Beating Rate

Abstract Spontaneous firing of sinoatrial node cells (SANC) is regulated by sarcoplasmic reticulum (SR) generated local subsarcolemmal calcium releases (LCRs). LCRs appear during diastolic depolarization (DD) and activate an inward sodium-calcium exchange current to accelerate the DD rate and thus spontaneous SANC firing. Vascular endothelial growth factor (VEGF) receptors VEGFR1 and VEGFR2 activate PLC, and suppression of VEGFR-PLC signaling decreases calcium transients and contractility in ventricular myocytes. We tested the idea that VEGFR-PLC signaling may contribute to normal spontaneous beating of SANC. We observed that expression of VEGFR1 (assessed by RT-qPCR) in rabbit sinoatrial node was comparable to that of β1-adrenergic receptors, but less than that in ventricle. The pan VEGFR1/2/3 inhibitor PTK787/ZK222584 (10 μmol/L) in a time-dependent manner: (1) suppressed spontaneous SANC beating rate by ∼50% (perforated patch-clamp technique); (2) markedly decreased the LCR size and number per each spontaneous cycle (confocal microscopy, calcium indicator Fluo-3) and prolonged the LCR period (the interval between action potential-induced calcium transient and occurrence of subsequent LCR). The PTK787/ZK222584-induced increase in the LCR period (from 348.2±30.3 to 619.9±103.4 msec; P<0.05) predicted the concomitant increase in the spontaneous cycle length (from 405.6±31.1 to 702.1±105.1 msec; P<0.05), suggesting that calcium cycling could be a major target of VEGFR-dependent regulation of SANC firing. All effects of PTK787/ZK222584 were reversible upon washout. To elucidate whether signaling of VEGFR1 or VEGFR2 regulated spontaneous SANC firing, we employed a selective VEGFR2 inhibitor ZM-323881 (5 μmol/L), which suppressed spontaneous beating rate in only 2 of 10 SANC. These results indicate that VEGFR1, but not VEGFR2, is likely a key receptor that modulates automaticity in majority of SANC. To clarify downstream targets from VEGFR1 we employed PLC inhibitor U-73122, which decreased the LCR size, number and prolonged the LCR period. The inactive analog U-73343 was without effect. Because LCRs are critically dependent upon the SR calcium load, supplied by L-type calcium current (ICa,L), we examined effects of U-73122 on ICa,L. U-73122, but not U-73343, markedly suppressed ICa,L amplitude by ∼50%, leading to a decrease in the calcium influx and, as a result, to decrease in the LCR parameters, prolongation of the LCR period and spontaneous SANC cycle length. Thus, basal VEGFR1 signaling activates PLC, which modulates intracellular SR calcium cycling and LCR characteristics in SANC. We conclude that VEGFR1-PLC is a novel mechanism involved in the regulation of normal automaticity of cardiac pacemaker cells. Acknowledgement/Funding Intramural Research Program, National Institute on Aging, NIH

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 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.

ANP has no postsynaptic effect on autonomic regulation of cardiac pacemaker rate in the rat

1993 ◽  
Vol 265 (6) ◽  
pp. H1983-H1987 ◽  
Author(s):  
D. J. Atchison ◽  
P. S. Pennefather ◽  
U. Ackermann
Keyword(s):  
Salt Solution ◽  
Action Potentials ◽  
Sinoatrial Node ◽  
Cardiac Pacemaker ◽  
Pacemaker Activity ◽  
Physiological Salt Solution ◽  
Sprague Dawley ◽  
Pacemaker Cells ◽  
Frequency Of Action Potentials ◽  
The Right

We studied whether atrial natriuretic peptide (ANP) influences sinoatrial node pacemaker activity or whether it modifies the response to activation of postsynaptic autonomic receptors. Male Sprague-Dawley rats were anesthetized with pentobarbital sodium (45 mg/kg). Their hearts were removed quickly and placed in physiological salt solution. The atria were isolated; the right intra-atrial chamber was exposed to allow intracellular recording from sinoatrial node pacemaker cells. The tissue was placed in a temperature-regulated recording chamber and superfused with warmed oxygenated physiological salt solution. With use of standard microelectrode recording techniques, action potentials were recorded from spontaneously depolarizing cells in the presence of muscarine (62.5–500 nM) or norepinephrine (0.1 and 1.0 microM). Muscarine reduced the frequency of action potentials dose dependently, whereas norepinephrine increased their frequency. The addition of ANP (0.1–100 nM) to the superfusion had no effect on the frequency of action potentials during the superfusion of physiological salt solution or in the presence of either muscarine or norepinephrine. We conclude that ANP does not act on cardiac pacemaker cells to modulate the effect of neurotransmitters.

Removal of sialic acid alters both T- and L-type calcium currents in cardiac myocytes

1991 ◽  
Vol 260 (3) ◽  
pp. H735-H743 ◽  
Author(s):  
B. Fermini ◽  
R. D. Nathan
Keyword(s):  
Sialic Acid ◽  
Sinoatrial Node ◽  
Calcium Currents ◽  
Pacemaker Cells ◽  
Patch Clamp Technique ◽  
Inactivation Curve ◽  
Node Removal ◽  
Rabbit Sinoatrial Node ◽  
Ca2 Channels ◽  
In Cells

The whole cell configuration of the patch-clamp technique was used to test the hypothesis that the presence of sialic acid residues influences both T- and L-type Ca2+ currents (ICa,T and ICa,L) in cultured pacemaker cells isolated from the rabbit sinoatrial node. Removal of these anionic sugar moieties by neuraminidase (1.0 U/ml for 5-20 min) increased ICa,T in five of nine cells (by a factor of 2.2-5.1) and ICa,L in three of six cells (by a factor of 1.2-1.6). In cells that did not exhibit such an increase, the enzyme reduced ICa,T but had no significant effect on ICa,L. In cells that exhibited an increase in ICa,T, exposure to neuraminidase also shifted the activation curve to more negative potentials and increased the slope of the inactivation curve. The enzyme did not influence the gating of ICa,L or the rates of inactivation of either ICa,T or ICa,L. The enhancement of ICa,T and ICa,L could not be mimicked by including neuraminidase in the patch pipette or by adding a contaminant of the enzyme preparation, phospholipase C, to the bath. When external Ca2+ was replaced by Ba2+, neither ICa,T nor ICa,L was increased significantly by neuraminidase. It is proposed that by removing sialic acid residues neuraminidase might directly alter the gating of T-type Ca2+ channels. On the other hand, the increased amplitudes of ICa,T and ICa,L might be due to a rise in intracellular Ca2+.

scholarly journals Constitutive Phosphodiesterase Activity Restricts Spontaneous Beating Rate of Cardiac Pacemaker Cells by Suppressing Local Ca2+Releases

2008 ◽  
Vol 102 (7) ◽  
pp. 761-769 ◽  
Author(s):  
Tatiana M. Vinogradova ◽  
Syevda Sirenko ◽  
Alexey E. Lyashkov ◽  
Antoine Younes ◽  
Yue Li ◽  
...  
Keyword(s):  
Cardiac Pacemaker ◽  
Phosphodiesterase Activity ◽  
Pacemaker Cells ◽  
Beating Rate

scholarly journals RNA Sequencing of Mouse Sinoatrial Node Reveals an Upstream Regulatory Role for Islet-1 in Cardiac Pacemaker Cells

2015 ◽  
Vol 116 (5) ◽  
pp. 797-803 ◽  
Author(s):  
Vasanth Vedantham ◽  
Giselle Galang ◽  
Melissa Evangelista ◽  
Rahul C. Deo ◽  
Deepak Srivastava
Keyword(s):  
Rna Sequencing ◽  
Sinoatrial Node ◽  
Cardiac Pacemaker ◽  
Regulatory Role ◽  
Pacemaker Cells ◽  
Islet 1

scholarly journals Bi-directional flow of the funny current (If) during the pacemaking cycle in murine sinoatrial node myocytes

2021 ◽  
Author(s):  
Colin H. Peters ◽  
Pin W. Liu ◽  
Stefano Morotti ◽  
Stephanie C. Gantz ◽  
Eleonora Grandi ◽  
...  
Keyword(s):  
Heart Rate ◽  
Conceptual Framework ◽  
Sinoatrial Node ◽  
Cardiac Pacemaker ◽  
Charge Movement ◽  
Firing Cycle ◽  
Pacemaker Cells ◽  
Net Charge ◽  
Precise Role

AbstractSinoatrial node myocytes (SAMs) act as cardiac pacemaker cells by firing spontaneous action potentials (APs) that initiate each heartbeat. The funny current, If, is critical for the generation of these spontaneous APs; however, its precise role during the pacemaking cycle remains unresolved. We used the AP-clamp technique to quantify If during the cardiac cycle in mouse SAMs. We found that If is persistently active throughout the sinoatrial AP, with surprisingly little voltage-dependent gating. As a consequence, it carries both inward and outward current around its reversal potential of -30 mV. Despite operating at only 2-5% of its maximal conductance, If carries a substantial fraction of both depolarizing and repolarizing net charge movement during the firing cycle. We also show that β-adrenergic receptor stimulation increases the percentage of net depolarizing charge moved by If, consistent with a contribution of If to the fight-or-flight increase in heart rate. These properties were confirmed by heterologously-expressed HCN4 channels and by mathematical models of If. Modelling further suggested that the slow activation and deactivation of the HCN4 isoform underlie the persistent activity of If during the sinoatrial AP. These results establish a new conceptual framework for the role of If in pacemaking, in which it operates at a very small fraction of maximal activation but nevertheless drives membrane potential oscillations in SAMs by providing substantial driving force in both inward and outward directions.Significance StatementCardiac pacemaker cells trigger each heartbeat by virtue of spontaneous oscillations in their membrane voltage. Although the funny current (If) is critical for these oscillations and for setting heart rate, its precise role remains an enigma because it activates mostly outside of the physiological voltage range and quite slowly relative to the pacemaker cycle. Here we show that If is persistently active in pacemaker cells; once opened, the small fraction of ion channels that conduct If do not re-close. Consequently, If flows both inward and outward to help propel the voltage oscillations and it paradoxically conducts a large fraction of the net charge movement. These results establish a new conceptual framework for the role of If in driving cardiac pacemaking.

Activation of a calcium-permeable cation channel by insulin-like growth factor II in BALB/c 3T3 cells

1988 ◽  
Vol 255 (4) ◽  
pp. C442-C446 ◽  
Author(s):  
H. Matsunaga ◽  
I. Nishimoto ◽  
I. Kojima ◽  
N. Yamashita ◽  
K. Kurokawa ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Calcium Influx ◽  
Cation Channel ◽  
Dependent Manner ◽  
Insulin Like Growth Factor ◽  
3T3 Cells ◽  
Patch Clamp Technique ◽  
Factor Ii ◽  
Epidermal Growth

Insulin-like growth factor II (IGF II) is a member of somatomedin family and is a potent mitogen in various types of mammalian cells. We have recently reported that IGF II stimulates calcium influx in competent BALB/c 3T3 cells primed with epidermal growth factor (J. Biol. Chem. 262: 12120-12126, 1987). Using patch-clamp technique, we show here an IGF II-sensitive cation channel in BALB/c 3T3 cell plasma membrane. Calcium is permeable to this cation channel and its opening behavior is independent of membrane potential. IGF II increases the opening probability of the identical channel in cells pretreated sequentially with platelet-derived growth factor and epidermal growth factor, whereas IGF II does not affect the opening of the channel in G0-arrested cells. This cation channel activity is observed only when IGF II is included in the patch pipette, indicating direct regulation of the channel by IGF II. We suggest that IGF II stimulates calcium influx by opening this cation channel and that IGF II activates the channel in a unique cell cycle-dependent manner.

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