Spontaneous Action Potential Firing Rate Research Articles

Adenosine reduces sinoatrial node cell action potential firing rate by uncoupling its membrane and calcium clocks.

The spontaneous action potential (AP) firing rate of sinoatrial nodal cells (SANC) is regulated by a system of intracellular Ca2+ and membrane ion current clocks driven by Ca2+-calmodulin-activated adenylyl cyclase-protein kinase-A signaling. The mean AP-cycle length (APCL) and APCL variability inform on the effectiveness of clock coupling. Endogenous ATP metabolite adenosine binds to adenosine receptors (A1, A3) that couple to Gi protein-coupled receptors, reducing spontaneous AP firing rate via Gβγ signaling that activates IKAch,Ado. Adenosine also inhibits adenylyl cyclase activity via Gαi signaling, impacting cAMP-mediated protein kinase-A-dependent protein phosphorylation. We hypothesize that in addition to IKAch,Ado activation, adenosine impacts also Ca2+ via Gαi signaling and that both effects reduce AP firing rate by reducing the effectiveness of the Ca2+ and membrane clock coupling. To this end, we measured Ca2+ and membrane potential characteristics in enzymatically isolated single rabbit SANC. 10 µM adenosine substantially increased both the mean APCL (on average by 43%, n = 10) and AP beat-to-beat variability from 5.1 ± 1.7% to 7.2 ± 2.0% (n = 10) measured via membrane potential and 5.0 ± 2.2% to 10.6 ± 5.9% (n = 40) measured via Ca2+ (assessed as the coefficient of variability = SD/mean). These effects were mediated by hyperpolarization of the maximum diastolic membrane potential (membrane clock effect) and suppression of diastolic local Ca2+releases (LCRs) (Ca2+-clock effect): as LCR size distributions shifted to smaller values, the time of LCR occurrence during diastolic depolarization (LCR period) became prolonged, and the ensemble LCR signal became reduced. The tight linear relationship of coupling between LCR period to the APCL in the presence of adenosine "drifted" upward and leftward, i.e. for a given LCR period, APCL was prolonged, becoming non-linear indicating clock uncoupling. An extreme case of uncoupling occurred at higher adenosine concentrations (>100µM): small stochastic LCRs failed to self-organize and synchronize to the membrane clock, thus creating a failed attempt to generate an AP resulting in arrhythmia and cessation of AP firing. Thus, the effects of adenosine to activate Gβγ and IKACh,Ado and to activate Gαi, suppressing adenylyl cyclase activity, both contribute to the adenosine-induced increase in the mean APCL and APCL variability by reducing the fidelity of clock coupling and AP firing rate.

Phosphoprotein Phosphatase 1 but Not 2A Activity Modulates Coupled-Clock Mechanisms to Impact on Intrinsic Automaticity of Sinoatrial Nodal Pacemaker Cells.

Spontaneous AP (action potential) firing of sinoatrial nodal cells (SANC) is critically dependent on protein kinase A (PKA) and Ca2+/calmodulin-dependent protein kinase II (CaMKII)-dependent protein phosphorylation, which are required for the generation of spontaneous, diastolic local Ca2+ releases (LCRs). Although phosphoprotein phosphatases (PP) regulate protein phosphorylation, the expression level of PPs and phosphatase inhibitors in SANC and the impact of phosphatase inhibition on the spontaneous LCRs and other players of the oscillatory coupled-clock system is unknown. Here, we show that rabbit SANC express both PP1, PP2A, and endogenous PP inhibitors I-1 (PPI-1), dopamine and cyclic adenosine 3′,5′-monophosphate (cAMP)-regulated phosphoprotein (DARPP-32), kinase C-enhanced PP1 inhibitor (KEPI). Application of Calyculin A, (CyA), a PPs inhibitor, to intact, freshly isolated single SANC: (1) significantly increased phospholamban (PLB) phosphorylation (by 2–3-fold) at both CaMKII-dependent Thr17 and PKA-dependent Ser16 sites, in a time and concentration dependent manner; (2) increased ryanodine receptor (RyR) phosphorylation at the Ser2809 site; (3) substantially increased sarcoplasmic reticulum (SR) Ca2+ load; (4) augmented L-type Ca2+ current amplitude; (5) augmented LCR’s characteristics and decreased LCR period in intact and permeabilized SANC, and (6) increased the spontaneous basal AP firing rate. In contrast, the selective PP2A inhibitor okadaic acid (100 nmol/L) had no significant effect on spontaneous AP firing, LCR parameters, or PLB phosphorylation. Application of purified PP1 to permeabilized SANC suppressed LCR, whereas purified PP2A had no effect on LCR characteristics. Our numerical model simulations demonstrated that PP inhibition increases AP firing rate via a coupled-clock mechanism, including respective increases in the SR Ca2+ pumping rate, L-type Ca2+ current, and Na+/Ca2+-exchanger current. Thus, PP1 and its endogenous inhibitors modulate the basal spontaneous firing rate of cardiac pacemaker cells by suppressing SR Ca2+ cycling protein phosphorylation, the SR Ca2+ load and LCRs, and L-type Ca2+ current.

β-Adrenergic Stimulation Synchronizes a Broad Spectrum of Action Potential Firing Rates of Cardiac Pacemaker Cells toward a Higher Population Average.

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 (R2 = 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.

Cardiac Overexpression of Adenylyl Cyclase Type VIII Augments Function of the Coupled Oscillatory System and Action Potential Firing Rate of Sinoatrial Nodal Cells

We have shown recently that the intrinsic heart rate (IHR) in vivo (measured during double autonomic blockade) in mice with cardiac-specific overexpression of adenylyl cyclase type VIII(AC VIII) is 30% higher than in wild type littermates (WT). We studied single sinoatrial nodal cell (SANC)to elucidate how AC VIII overexpression affects a coupled-clock system that drives spontaneous action potential (AP) firing in these cells. Spontaneous local Ca2+ releases (LCRs, confocal line-scan microscopy), generated via spontaneous local activation of ryanodine receptors (RyR) of the intracellular Ca2+ oscillator-sarcoplasmic reticulum (SR), occurred earlier in diastole in TgAC8 SANC, accelerating the diastolic AP ignition process that governs the rate of spontaneous diastolic depolarization. This led to an increased rate of AP generation by the ensemble of cell surface membrane ionic current oscillators. In permeabilized (saponin) SANC, Ca2+ signal of individual LCR's, and that of the LCR ensemble were increased in TgAC8 vs WT. Double immunostaining of phosphorylated and total RyR revealed increased phosphorylation of RyR at Ser2808 and Ser2030, and of PLB at Ser16 sites in TgAC8 vs WT SANC. RNA-seq analysis showed an upregulation of several PDEs (including PDE1a, PDE3a, PDE4b, PDE4c, PDE4d, PDE7b and PDE8a), ion channels (Cacna1c, Cacnb2, Kcnb1, Kcnd3, Kcnn1, Kcnq1, Scn1b, Scn4a; Kcnj3 however was downregulated), ATPase Na+/K+ transporters (ATP1a1 and ATP2b1) and calcium cycling proteins (STIM1 and RYR2) were upregulated in TgAC8 vs WT (notably, SIN and PLM were downregulated in TgAC8 vs WT). Thus, cardiac-specific overexpression of AC VIII in SANC increases IHR in vivo via impacting upon both transcriptional and posttranslational proteins modification of the Ca2+-cAMP-PKA-dependent components of the coupled-oscillator system intrinsic to SANC that controls their spontaneous AP firing rate.

Eliminating contraction during culture maintains global and local Ca2+ dynamics in cultured rabbit pacemaker cells

Pacemaker cells residing in the sinoatrial node generate the regular heartbeat. Ca2+ signaling controls the heartbeat rate—directly, through the effect on membrane molecules (NCX exchange, K+ channel), and indirectly, through activation of calmodulin-AC-cAMP-PKA signaling. Thus, the physiological role of signaling in pacemaker cells can only be assessed if the Ca2+ dynamics are in the physiological range. Cultured cells that can be genetically manipulated and/or virally infected with probes are required for this purpose. Because rabbit pacemaker cells in culture experience a decrease in their spontaneous action potential (AP) firing rate below the physiological range, Ca2+ dynamics are expected to be affected. However, Ca2+ dynamics in cultured pacemaker cells have not been reported before. We aim to a develop a modified culture method that sustains the global and local Ca2+ kinetics along with the AP firing rate of rabbit pacemaker cells.We used experimental and computational tools to test the viability of rabbit pacemaker cells in culture under various conditions. We tested the effect of culture dish coating, pH, phosphorylation, and energy balance on cultured rabbit pacemaker cells function. The cells were maintained in culture for 48 h in two types of culture media: one without the addition of a contraction uncoupler and one enriched with either 10 mM BDM (2,3-Butanedione 2-monoxime) or 25 μM blebbistatin. The uncoupler was washed out from the medium prior to the experiments. Cells were successfully infected with a GFP adenovirus cultured with either BDM or blebbistatin. Using either uncoupler during culture led to the cell surface area being maintained at the same level as fresh cells. Moreover, the phospholamban and ryanodine receptor densities and their phosphorylation level remained intact in culture when either blebbistatin or BDM were present. Spontaneous AP firing rate, spontaneous Ca2+ kinetics, and spontaneous local Ca2+ release parameters were similar in the cultured cells with blebbistatin as in fresh cells. However, BDM affects these parameters. Using experimental and a computational model, we showed that by eliminating contraction, phosphorylation activity is preserved and energy is reduced. However, the side-effects of BDM render it less effective than blebbistatin.

Functional Neuroplasticity in the Nucleus Tractus Solitarius and Increased Risk of Sudden Death in Mice with Acquired Temporal Lobe Epilepsy.

Sudden unexpected death in epilepsy (SUDEP) is the leading cause of death in individuals with refractory acquired epilepsy. Cardiorespiratory failure is the most likely cause in most cases, and central autonomic dysfunction has been implicated as a contributing factor to SUDEP. Neurons of the nucleus tractus solitarius (NTS) in the brainstem vagal complex receive and integrate vagally mediated information regarding cardiorespiratory and other autonomic functions, and GABAergic inhibitory NTS neurons play an essential role in modulating autonomic output. We assessed the activity of GABAergic NTS neurons as a function of epilepsy development in the pilocarpine-induced status epilepticus (SE) model of temporal lobe epilepsy (TLE). Compared with age-matched controls, mice that survived SE had significantly lower survival rates by 150 d post-SE. GABAergic NTS neurons from mice that survived SE displayed a glutamate-dependent increase in spontaneous action potential firing rate by 12 wks post-SE. Increased spontaneous EPSC frequency was also detected, but vagal afferent synaptic release properties were unaltered, suggesting that an increase in glutamate release from central neurons developed in the NTS after SE. Our results indicate that long-term changes in glutamate release and activity of GABAergic neurons emerge in the NTS in association with epileptogenesis. These changes might contribute to increased risk of cardiorespiratory dysfunction and sudden death in this model of TLE.

The Autonomic Nervous System Regulates the Heart Rate through cAMP-PKA Dependent and Independent Coupled-Clock Pacemaker Cell Mechanisms.

Sinoatrial nodal cells (SANCs) generate spontaneous action potentials (APs) that control the cardiac rate. The brain modulates SANC automaticity, via the autonomic nervous system, by stimulating membrane receptors that activate (adrenergic) or inactivate (cholinergic) adenylyl cyclase (AC). However, these opposing afferents are not simply additive. We showed that activation of adrenergic signaling increases AC-cAMP/PKA signaling, which mediates the increase in the SANC AP firing rate (i.e., positive chronotropic modulation). However, there is a limited understanding of the underlying internal pacemaker mechanisms involved in the crosstalk between cholinergic receptors and the decrease in the SANC AP firing rate (i.e., negative chronotropic modulation). We hypothesize that changes in AC-cAMP/PKA activity are crucial for mediating either decrease or increase in the AP firing rate and that the change in rate is due to both internal and membrane mechanisms. In cultured adult rabbit pacemaker cells infected with an adenovirus expressing the FRET sensor AKAR3, PKA activity and AP firing rate were tightly linked in response to either adrenergic receptor stimulation (by isoproterenol, ISO) or cholinergic stimulation (by carbachol, CCh). To identify the main molecular targets that mediate between PKA signaling and pacemaker function, we developed a mechanistic computational model. The model includes a description of autonomic-nervous receptors, post- translation signaling cascades, membrane molecules, and internal pacemaker mechanisms. Yielding results similar to those of the experiments, the model simulations faithfully reproduce the changes in AP firing rate in response to CCh or ISO or a combination of both (i.e., accentuated antagonism). Eliminating AC-cAMP-PKA signaling abolished the core effect of autonomic receptor stimulation on the AP firing rate. Specifically, disabling the phospholamban modulation of the SERCA activity resulted in a significantly reduced effect of CCh and a failure to increase the AP firing rate under ISO stimulation. Directly activating internal pacemaker mechanisms led to a similar extent of changes in the AP firing rate with respect to brain receptor stimulation. Thus, Ca2+ and cAMP/PKA-dependent phosphorylation limits the rate and magnitude of chronotropic changes in the spontaneous AP firing rate.

Real-time relationship between PKA biochemical signal network dynamics and increased action potential firing rate in heart pacemaker cells: Kinetics of PKA activation in heart pacemaker cells

cAMP-PKA protein kinase is a key nodal signaling pathway that regulates a wide range of heart pacemaker cell functions. These functions are predicted to be involved in regulation of spontaneous action potential (AP) generation of these cells. Here we investigate if the kinetics and stoichiometry of increase in PKA activity match the increase in AP firing rate in response to β-adrenergic receptor (β-AR) stimulation or phosphodiesterase (PDE) inhibition, that alters the AP firing rate of heart sinoatrial pacemaker cells. In cultured adult rabbit pacemaker cells infected with an adenovirus expressing the FRET sensor AKAR3, the EC50 in response to graded increases in the intensity of β-AR stimulation (by Isoproterenol) the magnitude of the increases in PKA activity and the spontaneous AP firing rate were similar (0.4±0.1nM vs. 0.6±0.15nM, respectively). Moreover, the kinetics (t1/2) of the increases in PKA activity and spontaneous AP firing rate in response to β-AR stimulation or PDE inhibition were tightly linked. We characterized the system rate-limiting biochemical reactions by integrating these experimentally derived data into a mechanistic–computational model. Model simulations predicted that phospholamban phosphorylation is a potent target of the increase in PKA activity that links to increase in spontaneous AP firing rate. In summary, the kinetics and stoichiometry of increases in PKA activity in response to a physiological (β-AR stimulation) or pharmacological (PDE inhibitor) stimuli match those of changes in the AP firing rate. Thus Ca2+-cAMP/PKA-dependent phosphorylation limits the rate and magnitude of increase in spontaneous AP firing rate.

Hydroxychloroquine reduces heart rate by modulating the hyperpolarization-activated current If: Novel electrophysiological insights and therapeutic potential

BackgroundBradycardic agents are of interest for the treatment of ischemic heart disease and heart failure, as heart rate is an important determinant of myocardial oxygen consumption.ObjectivesThe purpose of this study was to investigate the propensity of hydroxychloroquine (HCQ) to cause bradycardia.MethodsWe assessed the effects of HCQ on (1) cardiac beating rate in vitro (mice); (2) the “funny” current (If) in isolated guinea pig sinoatrial node (SAN) myocytes (1, 3, 10 µM); (3) heart rate and blood pressure in vivo by acute bolus injection (rat, dose range 1–30 mg/kg), (4) blood pressure and ventricular function during feeding (mouse, 100 mg/kg/d for 2 wk, tail cuff plethysmography, anesthetized echocardiography).ResultsIn mouse atria, spontaneous beating rate was significantly (P < .05) reduced (by 9% ± 3% and 15% ± 2% at 3 and 10 µM HCQ, n = 7). In guinea pig isolated SAN cells, HCQ conferred a significant reduction in spontaneous action potential firing rate (17% ± 6%, 1 μM dose) and a dose-dependent reduction in If (13% ± 3% at 1 µM; 19% ± 2% at 3 µM). Effects were also observed on L-type calcium ion current (ICaL) (12% ± 4% reduction) and rapid delayed rectifier potassium current (IKr) (35% ± 4%) at 3 µM. Intravenous HCQ decreased heart rate in anesthetized rats (14.3% ± 1.1% at 15mg/kg; n = 6) without significantly reducing mean arterial blood pressure. In vivo feeding studies in mice showed no significant change in systolic blood pressure nor left ventricular function.ConclusionsWe have shown that HCQ acts as a bradycardic agent in SAN cells, in atrial preparations, and in vivo. HCQ slows the rate of spontaneous action potential firing in the SAN through multichannel inhibition, including that of If.

Culture and adenoviral infection of sinoatrial node myocytes from adult mice.

Pacemaker myocytes in the sinoatrial node of the heart initiate each heartbeat by firing spontaneous action potentials. However, the molecular processes that underlie pacemaking are incompletely understood, in part because of our limited ability to manipulate protein expression within the native cellular context of sinoatrial node myocytes (SAMs). Here we describe a new method for the culture of fully differentiated SAMs from adult mice, and we demonstrate that robust expression of introduced proteins can be achieved within 24-48 h in vitro via adenoviral gene transfer. Comparison of morphological and electrophysiological characteristics of 48 h-cultured versus acutely isolated SAMs revealed only minor changes in vitro. Specifically, we found that cells tended to flatten in culture but retained an overall normal morphology, with no significant changes in cellular dimensions or membrane capacitance. Cultured cells beat spontaneously and, in patch-clamp recordings, the spontaneous action potential firing rate did not differ between cultured and acutely isolated cells, despite modest changes in a subset of action potential waveform parameters. The biophysical properties of two membrane currents that are critical for pacemaker activity in SAMs, the "funny current" (If) and voltage-gated Ca(2+) currents (ICa), were also indistinguishable between cultured and acutely isolated cells. This new method for culture and adenoviral infection of fully-differentiated SAMs from the adult mouse heart expands the range of experimental techniques that can be applied to study the molecular physiology of cardiac pacemaking because it will enable studies in which protein expression levels can be modified or genetically encoded reporter molecules expressed within SAMs.

Reduced Heart Rate in Mice Harboring an SR Luminal Ca2+ Sensor Mutation (E4872Q) is Linked to Abnormal Ca2+ Release and Pacemaker Function in Isolated Cardiocytes Derived from the Mutant RyR Clone

Rationale: The coupled-clock theory of cardiac pacemaker normal automaticity integrates numerous facets of pacemaker cell Ca2+ cycling and electrophysiology. Details of intrinsic RyR molecular mechanisms that regulate spontaneous RyR activation to generate local Ca2+ releases (LCRs) of the “Ca2+ clock”, that drive normal automaticity, however, have not been elucidated. Objective: We hypothesized that spontaneous RyR activation to generate LCRs of the “Ca2+ clock” will be attenuated in cells harboring mutant SR luminal Ca2+ sensor (E4872Q). Methods and Results: We measured the spontaneous beating rate, action potential (AP) triggered RyR Ca2+ releases, characteristics of spontaneous LCRs in single ES derived cardiocytes from wild type (WT) and the RyR mutant (E4872Q) mouse embryonic stem cells (ES) with intact sarcolemma and in permeabilized cells in the absence of AP's, and the expression of SR Ca2+ proteins in SAN lysates. We also measured the heart rate in vivo, which was 15% lower in mutant mice vs WT mice. Compared to WT cardiocytes, cells harboring the RyR mutation had a reduced spontaneous AP firing rate and reduced spontaneous RyR Ca2+ release. Expression of RyR protein was reduced, and calsequestrin and calreticulin were increased in mutant vs that of WT cells. Conclusions: Numerous luminal SR Ca2+ sensing mechanisms linked to regulation of spontaneous RyR activation, regulator of the spontaneous AP firing rate of pacemaker cells. Alterations in spontaneous RyR activation in RyR mutant cells were a mechanism for a reduction of their AP firing rate and for reduced heart rate in mutant mice in vivo.

Diabetes Slows Heart Rate via Electrical Remodeling of K+ Currents in Sinoatrial Node Myocytes

Diabetes mellitus is associated with sinoatrial node dysfunction, as evidenced by an increased risk of atrial fibrillation, pacemaker implantation due to bradycardia and cardiac death in diabetic patients. While sinoatrial node myocytes (SAMs) generate the spontaneous action potentials (APs) that initiate each heartbeat, little is known about how diabetes affects SAMs directly. In this study, we used streptozoticin (STZ) -treated mice as a model of diabetic hyperglycemia. Four weeks after STZ injections, we found that both intrinsic heart rate (measured during autonomic blockade) and maximum heart rate (measured during restraint stress) were reduced in diabetic animals compared to pre-treatment values. Current-clamp recordings from acutely isolated SAMs from diabetic animals revealed corresponding reductions in spontaneous AP firing rates. AP waveform analysis showed that the reduced firing rates in diabetic cells resulted from a prolongation of the AP duration and a slowing of the rate of repolarization. Accordingly, we observed significant decreases in steady state and transient outward K+ current densities in whole-cell voltage-clamp recordings from SAMs from diabetic animals. Diabetes caused little or no change in voltage-gated calcium currents in diabetic SAMs. The effects of diabetes on the AP waveform and firing rate were mimicked by application of 10 μM 4-aminopyridine in current clamp experiments and when the transient outward K+ current was reduced in a mathematical model of the sinoatrial AP. These results suggest that diabetic sinoatrial node dysfunction results in part from electrical remodeling of K+ currents in sinoatrial node myocytes, which paradoxically slows the spontaneous AP firing rate and thus heart rate.

Glycoside-Induced Collapse of the Sodium and Calcium Gradients Leads to a Biphasic Effect on Cardiac Cell Pacemaker Function

A major postulate of a modern coupled-clock pacemaker-cell theory is based upon electrochemical driving force for sodium and calcium (ENa and ECa). However, [sodium] gradients and their changes induced by glycosides (inhibiting sodium/potassium-ATPase) have never been measured. We measured time-dependent changes in intracellular [sodium](Nai), [calcium](Cai), and action-potentials (AP) [using Sodium-Binding-Benzofuran-Isophthalate (SBFI), Indo-1, and perforated patch-clamp, respectively] induced by cardiac glycosides (digoxegenin, 10μM) in single, isolated rabbit sinoatrial node cells. During 5 minutes of drug application spontaneous AP firing rate underwent biphasic changes, i.e. an increase followed by a decrease. During this time Nai monotonically increased from 8.5±0.6 mM to 13.1±1.1 mM (n=7 cells). The diastolic Cai also monotonically increased 124±6 nM to 149±11 nM (n=5). This Cai increase and biphasic drug effect on AP firing rate were closely reproduced by a coupled-clock Maltsev-Lakatta model simulations when the experimentally measured Nai time course was included in the model as an independent parameter. Model simulations also reproduced the initial AP rate increase via a moderate Cai increase (predicted by the model and validated by our Cai measurements). Specifically, when more cell calcium is available for pumping and release, it boosts the calcium clock, driving membrane clocks and the entire coupled-clock system. According to model predictions, subsequent marked increases in Nai and Cai collapse ENa and ECa and lead to AP rate decrease and dysrhythmic beating as sodium/calcium-exchanger current decreases, resulting in clock uncoupling. Thus, our results not only explain the complex glycoside effects on pacemaker cell AP-firing rate, but also provide new evidence supporting the idea that the normal automaticity of SANC emerges from coupled-clock functions, i.e. Cai and membrane ionic currents, that are controlled by sodium and calcium gradients to regulate sodium/calcium-exchanger current.

Role of connexin channels in the retinal light response of a diurnal rodent.

Several studies have shown that connexin channels play an important role in retinal neural coding in nocturnal rodents. However, the contribution of these channels to signal processing in the retina of diurnal rodents remains unclear. To gain insight into this problem, we studied connexin expression and the contribution of connexin channels to the retinal light response in the diurnal rodent Octodon degus (degu) compared to rat, using in vivo ERG recording under scotopic and photopic light adaptation. Analysis of the degu genome showed that the common retinal connexins present a high degree of homology to orthologs expressed in other mammals, and expression of Cx36 and Cx43 was confirmed in degu retina. Cx36 localized mainly to the outer and inner plexiform layers (IPLs), while Cx43 was expressed mostly in cells of the retinal pigment epithelium. Under scotopic conditions, the b-wave response amplitude was strongly reduced by 18-β-glycyrrhetinic acid (β-GA) (−45.1% in degu, compared to −52.2% in rat), suggesting that connexins are modulating this response. Remarkably, under photopic adaptation, β-GA increased the ERG b-wave amplitude in degu (+107.2%) while reducing it in rat (−62.3%). Moreover, β-GA diminished the spontaneous action potential firing rate in ganglion cells (GCs) and increased the response latency of ON and OFF GCs. Our results support the notion that connexins exert a fine-tuning control of the retinal light response and have an important role in retinal neural coding.

Mechanisms that match ATP supply to demand in cardiac pacemaker cells during high ATP demand

The spontaneous action potential (AP) firing rate of sinoatrial node cells (SANCs) involves high-throughput signaling via Ca(2+)-calmodulin activated adenylyl cyclases (AC), cAMP-mediated protein kinase A (PKA), and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII)-dependent phosphorylation of SR Ca(2+) cycling and surface membrane ion channel proteins. When the throughput of this signaling increases, e.g., in response to β-adrenergic receptor activation, the resultant increase in spontaneous AP firing rate increases the demand for ATP. We hypothesized that an increase of ATP production to match the increased ATP demand is achieved via a direct effect of increased mitochondrial Ca(2+) (Ca(2+)m) and an indirect effect via enhanced Ca(2+)-cAMP/PKA-CaMKII signaling to mitochondria. To increase ATP demand, single isolated rabbit SANCs were superfused by physiological saline at 35 ± 0.5°C with isoproterenol, or by phosphodiesterase or protein phosphatase inhibition. We measured cytosolic and mitochondrial Ca(2+) and flavoprotein fluorescence in single SANC, and we measured cAMP, ATP, and O₂ consumption in SANC suspensions. Although the increase in spontaneous AP firing rate was accompanied by an increase in O₂ consumption, the ATP level and flavoprotein fluorescence remained constant, indicating that ATP production had increased. Both Ca(2+)m and cAMP increased concurrently with the increase in AP firing rate. When Ca(2+)m was reduced by Ru360, the increase in spontaneous AP firing rate in response to isoproterenol was reduced by 25%. Thus, both an increase in Ca(2+)m and an increase in Ca(2+) activated cAMP-PKA-CaMKII signaling regulate the increase in ATP supply to meet ATP demand above the basal level.

Ca2+/Calmodulin-Dependent Protein Kinase II (CaMKII) Activity and Sinoatrial Nodal Pacemaker Cell Energetics

Ca2+-activated basal adenylate cyclase (AC) in rabbit sinoatrial node cells (SANC) guarantees, via basal cAMP/PKA-calmodulin/CaMKII-dependent protein phosphorylation, the occurrence of rhythmic, sarcoplasmic-reticulum generated, sub-membrane Ca2+ releases that prompt rhythmic, spontaneous action potentials (APs). This high-throughput signaling consumes ATP.AimsWe have previously demonstrated that basal AC-cAMP/PKA signaling directly, and Ca2+ indirectly, regulate mitochondrial ATP production. While, clearly, Ca2+-calmodulin-CaMKII activity regulates ATP consumption, whether it has a role in the control of ATP production is unknown.Methods and ResultsWe superfused single, isolated rabbit SANC at 37°C with physiological saline containing CaMKII inhibitors, (KN-93 or autocamtide-2 Related Inhibitory Peptide (AIP)), or a calmodulin inhibitor (W-7) and measured cytosolic Ca2+, flavoprotein fluorescence and spontaneous AP firing rate. We measured cAMP, ATP and O2 consumption in cell suspensions. Graded reductions in basal CaMKII activity by KN-93 (0.5–3 µmol/L) or AIP (2–10 µmol/L) markedly slow the kinetics of intracellular Ca2+ cycling, decrease the spontaneous AP firing rate, decrease cAMP, and reduce O2 consumption and flavoprotein fluorescence. In this context of graded reductions in ATP demand, however, ATP also becomes depleted, indicating reduced ATP production.ConclusionsCaMKII signaling, a crucial element of normal automaticity in rabbit SANC, is also involved in SANC bioenergetics.

Reduced Excitability and Ca2+ Current Density in Sinoatrial Myocytes from Aged Mice

Cardiac pacemaking results from the generation of spontaneous action potentials (APs) by specialized myocytes in the sinoatrial node (sinoatrial myocytes, SAMs). These APs are produced by the coordinated activity of a number of different ion channels, including hyperpolarization-activated cyclic nucleotide-sensitive (HCN) channels and voltage-gated Ca2+ channels. Across all mammalian species, the maximum heart rate that can be elicited by stress or exercise decreases with age. However, little is known about age-related changes in the intrinsic excitability of SAMs. In this study, we examined differences in heart rate in young (<3 mo) versus old (>32 mo) mice, and the corresponding differences in spontaneous AP firing rate and ionic currents in isolated SAMs from young versus old animals. ECG measurements in vivo confirmed previous observations of age-related reductions in maximum heart rate and intrinsic heart rate (i.e., in the absence of autonomic input). Current clamp recordings from acutely isolated SAMs revealed age-related deficits in spontaneous AP firing rate that mirrored the age-related changes in heart rate; the basal AP firing rate was significantly slower in SAMs from elderly animals, and maximal stimulation with 1 μM isoproterenol (Iso, a β adrenergic agonist) resulted in a slower average maximum firing rate in SAMs from old mice. To explore the mechanistic bases of these age-related deficiencies in sinoatrial AP generation, we have recently begun voltage-clamp recordings of hyperpolarization-activated currents and voltage-gated calcium currents from isolated SAMs from young and old mice. To date, we have found significant reductions in Ca2+ current density in cells from older animals. These studies may provide mechanistic information about fundamental processes that limit the physical capability of the elderly.

Basal cAMP/Pka/Ca2+ Signaling is Linked to Action Potential (AP) Rhythmicity of Sinoatrial Nodal Cells (SANC) as well as to their Firing Rate

Compared to freshly isolated SANC (f-SANC), the spontaneous AP firing rate at 34±0.5oC of cultured adult rabbit SANC (c-SANC) is reduced by 50% (from 2.79Hz to 1.35Hz), due to Gi protein suppression of basal cAMP/PKA/Ca2+-dependent signaling. Here we demonstrate that altered PKA-dependent modulation of basal intracellular Ca2+ cycling also reduces AP rhythmicity of c-SANC.The AP rhythmicity index (RI, fig.1A), i.e. the offset of the 3rd peak from the autocorrelation function of AP records, or from power spectrum analysis is reduced in c- vs. f-SANC, and is associated with prolongation of spontaneous Local Ca2+ Releases (LCR) period during diastolic depolarization and an increase in its coefficient of variation (0.199 ± 0.014 (n=41) for c-SANC vs. 0.122 ± 0.009 (n=32) for f-SANC, p<0.001). Acute β-adrenergic receptor stimulation by isoproterenol (ISO), phosphodiesterase inhibition by 3-isobutyl-1-methylxanthine (IBMX), or prolonged Gi suppression by pertussis toxin (PTX), which rescues impaired cAMP/PKA signaling in c-SANC, not only rescues the reduced AP firing rate, but also restores normal variability of LCR period and restores the rhythmicity of AP firing to the f-SANC level (fig.1B).View Large Image | View Hi-Res Image | Download PowerPoint Slide

RGS2 overexpression or Gi inhibition rescues the impaired PKA signaling and slow AP firing of cultured adult rabbit pacemaker cells

Freshly isolated adult rabbit sinoatrial node cells (f-SANC) are an excellent model for studies of autonomic signaling, but are not amenable to genetic manipulation. We have developed and characterized a stable cultured rabbit SANC (c-SANC) model that is suitable for genetic manipulation to probe mechanisms of spontaneous action potential (AP) firing.After 48h in culture, c-SANC generate stable, rhythmic APs at 34±0.5°C, at a rate that is 50% less than f-SANC. In c- vs. f-SANC: AP duration is prolonged; phosphorylation of phospholamban at Ser16 and type2 ryanodine receptor (RyR2) at Ser2809 are reduced; and the level of type2 regulator of G-protein signaling (RGS2), that facilitates adenylyl cyclases/cAMP/protein kinase A (PKA) via Gi inhibition, is substantially reduced. Consistent with the interpretation that cAMP/PKA signaling becomes impaired in c-SANC, acute β-adrenergic receptor stimulation increases phospholamban and RyR2 phosphorylation, enhances RGS2-labeling density, and accelerates the AP firing rate to the similar maximum in c- and f-SANC. Specific PKA inhibition completely inhibits all β-adrenergic receptor effects. Adv-RGS2 infection, or pertussis toxin treatment to disable Gi-signaling, each partially rescues the c-SANC spontaneous AP firing rate.Thus, a Gi-dependent reduction in PKA-dependent protein phosphorylation, including that of Ca2+ cycling proteins, reduces the spontaneous AP firing rate of c-SANC, and can be reversed by genetic or pharmacologic manipulation of PKA signaling.

The “Funny” Current (If) Inhibition by Ivabradine at Membrane Potentials Encompassing Spontaneous Depolarization in Pacemaker Cells

Recent clinical trials have shown that ivabradine (IVA), a drug that inhibits the funny current (If) in isolated sinoatrial nodal cells (SANC), decreases heart rate and reduces morbidity and mortality in patients with cardiovascular diseases. While IVA inhibits If, 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 If 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 If was 18 ± 3 pA/pF (at −120 mV) and the maximal If reduction by IVA was 60 ± 8% observed at −92 ± 4 mV. At the maximal diastolic depolarization (~−60 mV) If amplitude was only −2.9 ± 0.4 pA/pF, and was reduced by only 41 ± 6% by IVA. Thus, If 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.