Beat-to-beat Ca(2+)-dependent regulation of sinoatrial nodal pacemaker cell rate and rhythm

Whether intracellular Ca²⁺ 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 Ca²⁺ regulation of the rate and rhythm of SANC we loaded single isolated SANC with a caged Ca²⁺ buffer, NP-EGTA, and simultaneously recorded membrane potential and intracellular Ca²⁺. Prior to introduction of the caged Ca²⁺ buffer, spontaneous local Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ was acutely released from the caged compound by flash photolysis, intracellular Ca²⁺ 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 Ca²⁺ dynamics by the caged buffer was reestablished. Our results directly support the hypothesis that intracellular Ca²⁺ regulates normal SANC automaticity on a beat-to-beat basis.     

Sarcoplasmic reticulum Ca2+ release is not a dominating factor in sinoatrial node pacemaker activity

Recent work on isolated sinoatrial node cells from rabbit has suggested that sarcoplasmic reticulum Ca2+ release plays a dominant role in the pacemaker potential, and ryanodine at a high concentration (30 micromol/L blocks sarcoplasmic reticulum Ca2+ release) abolishes pacemaking and at a lower concentration abolishes the chronotropic effect of beta-adrenergic stimulation. The aim of the present study was to test this hypothesis in the intact sinoatrial node of the rabbit. Spontaneous activity and the pattern of activation were recorded using a grid of 120 pairs of extracellular electrodes. Ryanodine 30 micromol/L did not abolish spontaneous activity or shift the position of the leading pacemaker site, although it slowed the spontaneous rate by 18.9+/-2.5% (n=6). After ryanodine treatment, beta-adrenergic stimulation still resulted in a substantial chronotropic effect (0.3 micromol/L isoproterenol increased spontaneous rate by 52.6+/-10.5%, n=5). In isolated sinoatrial node cells from rabbit, 30 micromol/L ryanodine slowed spontaneous rate by 21.5+/-2.6% (n=13). It is concluded that sarcoplasmic reticulum Ca2+ release does not play a dominating role in pacemaking in the sinoatrial node. The full text of this article is available at http://www.circresaha.org.     

Functional role of CLC-2 chloride inward rectifier channels in cardiac sinoatrial nodal pacemaker cells

A novel Cl⁻ inward rectifier channel (Cl,ir) encoded by ClC-2, a member of the ClC voltage-gated Cl⁻ channel gene superfamily, has been recently discovered in cardiac myocytes of several species. However, the physiological role of Cl,ir channels in the heart remains unknown. In this study we tested the hypothesis that Cl,ir channels may play an important role in cardiac pacemaker activity. In isolated guinea-pig sinoatrial node (SAN) cells, Cl,ir current was activated by hyperpolarization and hypotonic cell swelling. RT-PCR and immunohistological analyses confirmed the molecular expression of ClC-2 in guinea-pig SAN cells. Hypotonic stress increased the diastolic depolarization slope and decreased the maximum diastolic potential, action potential amplitude, APD₅₀, APD₉₀, and the cycle-length of the SAN cells. These effects were largely reversed by intracellular dialysis of anti-ClC-2 antibody, which significantly inhibited Cl,ir current but not other pacemaker currents, including the hyperpolarization-activated non-selective cationic “funny” current (I_f), the L-type Ca²⁺ currents (I_Ca,L), the slowly-activating delayed rectifier I_Ks and the volume-regulated outwardly-rectifying Cl⁻ current (I_Cl,vol). Telemetry electrocardiograph studies in conscious ClC-2 knockout (Clcn2^−/−) mice revealed a decreased chronotropic response to acute exercise stress when compared to their age-matched Clcn2^+/+ and Clcn2^+/− littermates. Targeted inactivation of ClC-2 does not alter intrinsic heart rate but prevented the positive chronotropic effect of acute exercise stress through a sympathetic regulation of ClC-2 channels. These results provide compelling evidence that ClC-2-encoded endogenous Cl,ir channels may play an important role in the regulation of cardiac pacemaker activity, which may become more prominent under stressed or pathological conditions.     

Differences in atrial septal activation with an intrasinoatrial nodal pacemaker and epicardial sinoatrial nodal pacing.

Changes in Intra-SA nodal pacemaker localization were produced through stimulation of the decentralized cervical vagi and stellate ganglia in the anesthetized dog. Shifts in pacemaker to the rostral, middle, or caudal regions of the SA node produced a change in the timing as well as a change in the sequence of activation of recording sites overlying the AV node. Epicardial pacing with a plaque electrode from either the rostral, middle, or caudal regions of the SA node produced the same activation sequence of the AV nodal electrodes irrespective of the epicardial SA nodal pacing site. The inability of epicardial SA nodal pacing to precisely reproduce the activation pattern of the atrial septum overlying the AV node observed with a natural SA nodal pacemaker can be explained by the geographic relationship of the pacemaker cells within the node to the preferential internodal pathways and the area of atrial tissue stimulated by pacing. Pacing activates a large mass of tissue, whereas an intrinsic pacemaker probably acts as a more localized focus. The inability of pacing to reproduce the activation pattern seen with spontaneous rhythm may be a determinant in the varied P wave morphology seen with coronary sinus or AV nodal junctional rhythms, as compared with more consistent morphology seen with pacing.     

Rhythmic ryanodine receptor Ca2+ releases during diastolic depolarization of sinoatrial pacemaker cells do not require membrane depolarization

Localized, subsarcolemmal Ca2+ release (LCR) via ryanodine receptors (RyRs) during diastolic depolarization of sinoatrial nodal cells augments the terminal depolarization rate. We determined whether LCRs in rabbit sinoatrial nodal cells require the concurrent membrane depolarization, or are intrinsically rhythmic, and whether rhythmicity is linked to the spontaneous cycle length. Confocal linescan images revealed persistent LCRs both in saponin-permeabilized cells and in spontaneously beating cells acutely voltage-clamped at the maximum diastolic potential. During the initial stage of voltage clamp, the LCR spatiotemporal characteristics did not differ from those in spontaneously beating cells, or in permeabilized cells bathed in 150 nmol/L Ca2+. The period of persistent rhythmic LCRs during voltage clamp was slightly less than the spontaneous cycle length before voltage clamp. In spontaneously beating cells, in both transient and steady states, LCR period was highly correlated with the spontaneous cycle length; and regardless of the cycle length, LCRs occurred predominantly at a constant time, ie, 80% to 90% of the cycle length. Numerical model simulations incorporating LCRs reproduce the experimental results. We conclude that diastolic LCRs reflect rhythmic intracellular Ca2+ cycling that does not require the concomitant membrane depolarization, and that LCR periodicity is closely linked to the spontaneous cycle length. Thus, the biological clock of sinoatrial nodal pacemaker cells, like that of many other rhythmic functions occurring throughout nature, involves an intracellular Ca2+ rhythm.     

Effects of muscarinic receptor stimulation on Ca2+ transient, cAMP production and pacemaker frequency of rabbit sinoatrial node cells

We investigated the contribution of the intracellular calcium (Ca _i ²⁺ ) transient to acetylcholine (ACh)-mediated reduction of pacemaker frequency and cAMP content in rabbit sinoatrial nodal (SAN) cells. Action potentials (whole cell perforated patch clamp) and Ca _i ²⁺ transients (Indo-1 fluorescence) were recorded from single isolated rabbit SAN cells, whereas intracellular cAMP content was measured in SAN cell suspensions using a cAMP assay (LANCE^®). Our data show that the Ca _i ²⁺ transient, like the hyperpolarization-activated “funny current” (I _f) and the ACh-sensitive potassium current (I _K,ACh), is an important determinant of ACh-mediated pacemaker slowing. When I _f and I _K,ACh were both inhibited, by cesium (2 mM) and tertiapin (100 nM), respectively, 1 μM ACh was still able to reduce pacemaker frequency by 72%. In these I _f and I _K,ACh-inhibited SAN cells, good correlations were found between the ACh-mediated change in interbeat interval and the ACh-mediated change in Ca _i ²⁺ transient decay (r ² = 0.98) and slow diastolic Ca _i ²⁺ rise (r ² = 0.73). Inhibition of the Ca _i ²⁺ transient by ryanodine (3 μM) or BAPTA-AM (5 μM) facilitated ACh-mediated pacemaker slowing. Furthermore, ACh depressed the Ca _i ²⁺ transient and reduced the sarcoplasmic reticulum (SR) Ca²⁺ content, all in a concentration-dependent fashion. At 1 μM ACh, the spontaneous activity and Ca _i ²⁺ transient were abolished, but completely recovered when cAMP production was stimulated by forskolin (10 μM) and I _K,ACh was inhibited by tertiapin (100 nM). Also, inhibition of the Ca _i ²⁺ transient by ryanodine (3 μM) or BAPTA-AM (25 μM) exaggerated the ACh-mediated inhibition of cAMP content, indicating that Ca _i ²⁺ affects cAMP production in SAN cells. In conclusion, muscarinic receptor stimulation inhibits the Ca _i ²⁺ transient via a cAMP-dependent signaling pathway. Inhibition of the Ca _i ²⁺ transient contributes to pacemaker slowing and inhibits Ca _i ²⁺ -stimulated cAMP production. Thus, we provide functional evidence for the contribution of the Ca _i ²⁺ transient to ACh-induced inhibition of pacemaker activity and cAMP content in rabbit SAN cells.     

T-type Ca2+ channels and cardiac pacemaker activity

It is well known that T-type Ca(2+) channels differ from L-type Ca(2+) channels on the basis of their low-voltage activation range and rapid inactivation, and therefore can contribute to the pacemaker activity of sinoatrial node cells in the heart. However, proper elucidation of their function on the pacemaker activity has been hampered by the lack of selective pharmacology as well as cell-to-cell difference in the amplitude of T-type Ca(2+) current. In the present study, therefore, we investigated the effects of mibefradil, a selective T-type Ca(2+) channel blocker, on the spontaneous action potential of rabbit sinoatrial node cells. Mibefradil strongly inhibited the spontaneous action potential. In particular, suppression of the slow diastolic depolarization was more marked than that had been expected from a sole inhibition of T-type Ca(2+) channels. T-type Ca(2+) channels may be an important contributor to automaticity in heart cells. Alternatively, mibefradil might have blocked other current system (s) which serves as the main pacemaker current, and thereby inhibited the pacemaker activity.     

The role of Ca2+ release from sarcoplasmic reticulum in the regulation of sinoatrial node automaticity

Summary The role of Ca²⁺ release channels in the sarcoplasmic reticulum in modulating physiological automaticity of the sinoatrial (SA) node was studied by recording transmembrane action potentials and membrane ionic currents in small preparations of the rabbit SA node. Ryanodine, which modifies the conductance and gating behavior of the Ca²⁺ release channels, was used to block Ca²⁺ release from the sarcoplasmic reticulum. Superfusion of 1-mM ryanodine decreased the spontaneous firing frequency as well as the maximal rate of depolarization of the SA, and these reductions reached a steady state within approximately 5min. The action potential recordings revealed that the latter part of diastolic depolarization was depressed and that the take-off potential became less negative. This suggested that the negative chronotropic effect of ryanodine resulted from the blockade of physiological Ca²⁺ release from the sarcoplasmic reticulum. In voltage clamp experiments, using double-microelectrode techniques, ryanodine did not markedly reduce the Ca²⁺ current (I_Ca) but decreased the delayed rectifying K+ current (I_K), the steady-state inward current (I_ss), and the hyperpolarization-activated inward current (I_h). These observations suggest that, even when the function of Ca²⁺ channels in the cell membrane is normally maintained, depression of Ca²⁺ release channels in the sarcoplasmic reticulum would prevent sufficient elevation of the Ca²⁺ concentration in SA node cells for the activation of various ionic currents, and, thus adversely affect the physiological automaticity of this primary cardiac pacemaker.     

Ca2+-dependent rapid Ca2+ sensitization of contraction in arterial smooth muscle

Ca(2+) ion is a universal intracellular messenger that regulates numerous biological functions. In smooth muscle, Ca(2+) with calmodulin activates myosin light chain (MLC) kinase to initiate a rapid MLC phosphorylation and contraction. To test the hypothesis that regulation of MLC phosphatase is involved in the rapid development of MLC phosphorylation and contraction during Ca(2+) transient, we compared Ca(2+) signal, MLC phosphorylation, and 2 modes of inhibition of MLC phosphatase, phosphorylation of CPI-17 Thr38 and MYPT1 Thr853, during alpha(1) agonist-induced contraction with/without various inhibitors in intact rabbit femoral artery. Phenylephrine rapidly induced CPI-17 phosphorylation from a negligible amount to a peak value of 0.38+/-0.04 mol of Pi/mol within 7 seconds following stimulation, similar to the rapid time course of Ca(2+) rise and MLC phosphorylation. This rapid CPI-17 phosphorylation was dramatically inhibited by either blocking Ca(2+) release from the sarcoplasmic reticulum or by pretreatment with protein kinase C inhibitors, suggesting an involvement of Ca(2+)-dependent protein kinase C. This was followed by a slow Ca(2+)-independent and Rho-kinase/protein kinase C-dependent phosphorylation of CPI-17. In contrast, MYPT1 phosphorylation had only a slow component that increased from 0.29+/-0.09 at rest to the peak of 0.68+/-0.14 mol of Pi/mol at 1 minute, similar to the time course of contraction. Thus, there are 2 components of the Ca(2+) sensitization through inhibition of MLC phosphatase. Our results support the hypothesis that the initial rapid Ca(2+) rise induces a rapid inhibition of MLC phosphatase coincident with the Ca(2+)-induced MLC kinase activation to synergistically initiate a rapid MLC phosphorylation and contraction in arteries with abundant CPI-17 content.     

Bergmann glia modulate cerebellar Purkinje cell bistability via Ca2+-dependent K+ uptake

Recent studies have shown that cerebellar Bergmann glia display coordinated Ca(2+) transients in live mice. However, the functional significance of Bergmann glial Ca(2+) signaling remains poorly understood. Using transgenic mice that allow selective stimulation of glial cells, we report here that cytosolic Ca(2+) regulates uptake of K(+) by Bergmann glia, thus providing a powerful mechanism for control of Purkinje cell-membrane potential. The decline in extracellular K(+) evoked by agonist-induced Ca(2+) in Bergmann glia transiently increased spike activity of Purkinje cells in cerebellar slices as well as in live anesthetized mice. Thus, Bergmann glia play a previously unappreciated role in controlling the membrane potential and thereby the activity of adjacent Purkinje cells.     

Ca2+-dependent histaminergic regulation of proenkephalin mRNA levels in cultured adrenal chromaffin cells

The effect of histamine on messenger ribonucleic acid levels encoding proenkephalin A (mRNA(enk)) was studied in serum-free cultures of bovine adrenal chromaffin cells. Histamine (10(-7)-10(-4) M) stimulated mRNA(enk), with a maximum response (5-fold) at 10(-5) M, an effect which could be abolished by the H1 receptor antagonist clemastine (10(-7) M) but not by the H2 receptor antagonist cimetidine (10(-7)-10(-5) M). The histamine stimulation was partially reduced by the Ca++ channel blockers D600 (10(-5) M) and nifedipine (10(-7) M). On the other hand, muscarinic receptor stimulation, which similarly to histamine is known to stimulate phosphoinositide hydrolysis in chromaffin cells, did not alter mRNA(enk) in these cells. These data show that mRNA(enk) levels may be modulated by activation of the H1 receptor and that this effect is partially dependent on the activation of voltage-dependent Ca2+ channels.     

Sinoatrial nodal cell ryanodine receptor and Na(+)-Ca(2+) exchanger: molecular partners in pacemaker regulation

The rate of spontaneous diastolic depolarization (DD) of sinoatrial nodal cells (SANCs) that triggers recurrent action potentials (APs) is a fundamental aspect of the heart's pacemaker. Here, in experiments on isolated SANCs, using confocal microscopy combined with a patch clamp technique, we show that ryanodine receptor Ca(2+) release during the DD produces a localized subsarcolemmal Ca(2+) increase that spreads in a wavelike manner by Ca(2+)-induced Ca(2+) release and produces an inward current via the Na(+)-Ca(2+) exchanger (NCX). Ryanodine, a blocker of the sarcoplasmic reticulum Ca(2+) release channel, in a dose-dependent manner reduces the SANC beating rate with an IC(50) of 2.6 micromol/L and abolishes the local Ca(2+) transients that precede the AP upstroke. In voltage-clamped cells in which the DD was simulated by voltage ramp, 3 micromol/L ryanodine decreased an inward current during the voltage ramp by 1.6+/-0.3 pA/pF (SEM, n=4) leaving the peak of L-type Ca(2+) current unchanged. Likewise, acute blockade of the NCX (via rapid substitution of bath Na(+) by Li(+)) abolished SANC beating and reduced the inward current to a similar extent (1.7+/-0.4 pA/pF, n=4), as did ryanodine. Thus, in addition to activation/inactivation of multiple ion channels, Ca(2+) activation of the NCX, because of localized sarcoplasmic reticulum Ca(2+) release, is a critical element in a chain of molecular interactions that permits the heartbeat to occur and determines its beating rate.     

Local Ca2+ entry through L-type Ca2+ channels activates Ca2+-dependent K+ channels in rabbit coronary myocytes.

Large-conductance Ca2+-dependent K+ channels (KCa), which are abundant on the sarcolemma of vascular myocytes, provide negative feedback via membrane hyperpolarization that limits Ca2+ entry through L-type Ca2+ channels (ICaL). We hypothesize that local accumulation of subsarcolemmal Ca2+ during ICaL openings amplifies this feedback. Our goal was to demonstrate that Ca2+ entry through voltage-gated ICaL channels can stimulate adjacent KCa channels by a localized interaction in enzymatically isolated rabbit coronary arterial myocytes voltage clamped in whole-cell or in cell-attached patch clamp mode. During slow-voltage-ramp protocols, we identified an outward KCa current that is activated by a subsarcolemmal Ca2+ pool dissociated from bulk cytosolic Ca2+ pool (measured with indo 1) and is dependent on L-type Ca2+ channel activity. Transient activation of unitary KCa channels in cell-attached patches could be detected during long step depolarizations to +40 mV (holding potential, -40 mV; 219 pS in near-symmetrical K+). This local interaction between the channels required the presence of Ca2+ in the pipette solution, was enhanced by the ICaL agonist Bay K 8644, and persisted after impairment of the sarcoplasmic reticulum by incubation with 10 micromol/L ryanodine and 30 micromol/L cyclopiazonic acid for at least 60 minutes. Furthermore, we provide the first direct evidence of simultaneous openings of single KCa (67 pS) and ICaL (3.9 pS) channels in near-physiological conditions, near resting membrane potential. Our data imply a novel sensitive mechanism for regulating resting membrane potential and tone in vascular smooth muscle.     

Ca2+-dependent structural rearrangements within Na+-Ca2+ exchanger dimers

Cytoplasmic Ca(2+) is known to regulate Na(+)-Ca(2+) exchanger (NCX) activity by binding to two adjacent Ca(2+)-binding domains (CBD1 and CBD2) located in the large intracellular loop between transmembrane segments 5 and 6. We investigated Ca(2+)-dependent movements as changes in FRET between exchanger proteins tagged with CFP or YFP at position 266 within the large cytoplasmic loop. Data indicate that the exchanger assembles as a dimer in the plasma membrane. Addition of Ca(2+) decreases the distance between the cytoplasmic loops of NCX pairs. The Ca(2+)-dependent movements detected between paired NCXs were abolished by mutating the Ca(2+) coordination sites in CBD1 (D421A, E451A, and D500V), whereas disruption of the primary Ca(2+) coordination site in CBD2 (E516L) had no effect. Thus, the Ca(2+)-induced conformational changes of NCX dimers arise from the movement of CBD1. FRET studies of CBD1, CBD2, and CBD1-CBD2 peptides displayed Ca(2+)-dependent movements with different apparent affinities. CBD1-CBD2 showed a Ca(2+)-dependent phenotype mirroring full-length NCX but distinct from both CBD1 and CBD2.     

Apolipoprotein CIII promotes Ca2+-dependent beta cell death in type 1 diabetes

In type 1 diabetes (T1D), there is a specific destruction of the insulin secreting pancreatic beta cell. Although the exact molecular mechanisms underlying beta cell destruction are not known, sera from T1D patients have been shown to promote Ca(2+)-induced apoptosis. We now demonstrate that apolipoprotein CIII (apoCIII) is increased in serum from T1D patients and that this serum factor both induces increased cytoplasmic free intracellular Ca(2+) concentration ([Ca(2+)](i)) and beta cell death. The apoCIII-induced increase in [Ca(2+)](i) reflects an activation of the voltage-gated L-type Ca(2+) channel. Both the effects of T1D sera and apoCIII on the beta cell are abolished in the presence of antibody against apoCIII. Increased serum levels of apoCIII can thus account for the increase in beta cell [Ca(2+)](i) and thereby beta cell apoptosis associated with T1D.     

Ca2+-dependent regulation of synaptic SNARE complex assembly via a calmodulin- and phospholipid-binding domain of synaptobrevin

Synaptic core complex formation is an essential step in exocytosis, and assembly into a superhelical structure may drive synaptic vesicle fusion. To ascertain how Ca(2+) could regulate this process, we examined calmodulin binding to recombinant core complex components. Surface plasmon resonance and pull-down assays revealed Ca(2+)-dependent calmodulin binding (K(d) = 500 nM) to glutathione S-transferase fusion proteins containing synaptobrevin (VAMP 2) domains but not to syntaxin 1 or synaptosomal-associated protein of 25 kDa (SNAP-25). Deletion mutations, tetanus toxin cleavage, and peptide synthesis localized the calmodulin-binding domain to VAMP(77-94), immediately C-terminal to the tetanus toxin cleavage site (Q(76)-F(77)). In isolated synaptic vesicles, Ca(2+)/calmodulin protected native membrane-inserted VAMP from proteolysis by tetanus toxin. Assembly of a (35)S-SNAP-25, syntaxin 1 GST-VAMP(1-96) complex was inhibited by Ca(2+)/calmodulin, but assembly did not mask subsequent accessibility of the calmodulin-binding domain. The same domain contains a predicted phospholipid interaction site. SPR revealed calcium-independent interactions between VAMP(77-94) and liposomes containing phosphatidylserine, which blocked calmodulin binding. Circular dichroism spectroscopy demonstrated that the calmodulin/phospholipid-binding peptide displayed a significant increase in alphahelical content in a hydrophobic environment. These data provide insight into the mechanisms by which Ca(2+) may regulate synaptic core complex assembly and protein interactions with membrane bilayers during exocytosis.