Membrane Clock Research Articles

Mitochondrial dysfunction is a key link involved in the pathogenesis of sick sinus syndrome: a review.

Sick sinus syndrome (SSS) is a grave medical condition that can precipitate sudden death. The pathogenesis of SSS remains incompletely understood. Existing research postulates that the fundamental mechanism involves increased fibrosis of the sinoatrial node and its surrounding tissues, as well as disturbances in the coupled-clock system, comprising the membrane clock and the Ca2+ clock. Mitochondrial dysfunction exacerbates regional tissue fibrosis and disrupts the functioning of both the membrane and calcium clocks. This plays a crucial role in the underlying pathophysiology of SSS, including mitochondrial energy metabolism disorders, mitochondrial oxidative stress damage, calcium overload, and mitochondrial quality control disorders. Elucidating the mitochondrial mechanisms involved in the pathophysiology of SSS and further investigating the disease's mechanisms is of great significance.

Voltage-gated ion channel’s gene expression in the myocardium of embryo and adult chickens

The functioning of the cardiovascular system is critical for embryo survival. Cardiac contractions depend on the sequential activation of different classes of voltage-gated ion channels. Understanding the fundamental features of these interactions is important for identifying the mechanisms of pathologies development in the myocardium. However, at present there is no consensus on which ion channels are involved in the formation of automaticity in the early embryonic stages. The aim of this study was to elucidate the expression of genes encoding various types of ion channels that are involved in the generation of electrical activity chicken heart at different stages of ontogenesis. We analyzed the expression of 14 genes from different families of ion channels. It was revealed that the expression profiles of ion channel genes change depending on the stages of ontogenesis. The HCN4, CACNA1D, SCN1A, SCN5A, KCNA1 genes have maximum expression at the tubular heart stage. In adult, a switch occurs to the higher expression of CACNA1C, KCNH6, RYR and SLC8A1 genes. This data correlated with the results obtained by the microelectrode method. It can be assumed that the automaticity of the tubular heart is mainly due to the mechanism of the «membrane–clock» (hyperpolarization-activated current (If), Ca2+–current L–type (ICaL), Na+–current (INa) and the slow component of the delayed rectifier K+–current (IKs)). Whereas in adult birds, the mechanism for generating electrical impulses is determined by both « membrane– clock» and «Ca2+–clock».

Computational insight into energy control balance by Ca2+ and cAMP-PKA signaling in pacemaker cells

Cyclic adenosine monophosphate (cAMP)−protein kinase A (PKA) signaling controls sinoatrial node cell (SANC) function by affecting the degree of coupling between Ca2+ and membrane clocks. PKA is known to phosphorylate ionic channels, Ca2+ pump and release from the sarcoplasmic reticulum, and enzymes controlling ATP production in the mitochondria. While the PKA cytosolic targets in SANC have been extensively explored, its mitochondrial targets and its ability to maintain SANC energetic balance remain to be elucidated.To investigate the role of PKA in SANC energetics, we tested three hypotheses: (i) PKA is an important regulator of the ATP supply-to-demand balance, (ii) Ca2+ regulation of energetics is important for maintenance of NADH level and (iii) abrupt reduction in ATP demand first reduces the AP firing rate and, after dropping below a certain threshold, leads to a reduction in ATP. To gain mechanistic insights into the ATP supply-to-demand matching regulators, a modified model of mitochondrial energy metabolism was integrated into our coupled-clock model that describes ATP demand.Experimentally, increased ATP demand was accompanied by maintained ATP and NADH levels. Ca2+ regulation of energetics was found by the model to be important in the maintenance of NADH and PKA regulation was found to be important in the maintenance of intracellular ATP and the increase in oxygen consumption. PKA inhibition led to a biphasic reduction in AP firing rate, with the first phase being rapid and ATP-independent, while the second phase was slow and ATP-dependent. Thus, SANC energy balance is maintained by both Ca2+ and PKA signaling.

The role of P21-activated kinase (Pak1) in sinus node function

Sinoatrial node (SAN) dysfunction (SND) and atrial arrhythmia frequently occur simultaneously with a hazard ratio of 4.2 for new onset atrial fibrillation (AF) in SND patients. In the atrial muscle attenuated activity of p21-activated kinase 1 (Pak1) increases the risk for AF by enhancing NADPH oxidase 2 dependent production of reactive oxygen species (ROS). However, the role of Pak1 dependent ROS regulation in SAN function has not yet been determined. We hypothesize that Pak1 activity maintains SAN activity by regulating the expression of the hyperpolarization activated cyclic nucleotide gated cation channel (HCN).To determine Pak1 dependent changes in heart rate (HR) regulation we quantified the intrinsic sinus rhythm in wild type (WT) and Pak1 deficient (Pak1−/−) mice of both sexes in vivo and in isolated Langendorff perfused hearts. Pak1−/− hearts displayed an attenuated HR in vivo after autonomic blockage and in isolated hearts. The contribution of the Ca2+ clock to pacemaker activity remained unchanged, but Ivabradine (3 μM), a blocker of HCN channels that are a membrane clock component, eliminated the differences in SAN activity between WT and Pak1−/− hearts. Reduced HCN4 expression was confirmed in Pak1−/− right atria. The reduced HCN activity in Pak1−/− could be rescued by class II HDAC inhibition (LMK235), ROS scavenging (TEMPOL) or attenuation of Extracellular Signal-Regulated Kinase (ERK) 1/2 activity (SCH772984). No sex specific differences in Pak1 dependent SAN regulation were determined.Our results establish Pak1 as a class II HDAC regulator and a potential therapeutic target to attenuate SAN bradycardia and AF susceptibility.

3D spatial modeling of sinoatrial node cells reveals a critical role of subcellular ryanodine receptor distribution in pacemaker automaticity.

Ryanodine receptors (RyRs) represent a central component in sinoatrial node (SAN) pacemaker system, as dysregulated RyRs associated with catecholaminergic polymorphic ventricular tachycardia are linked with SAN dysfunction. Within each SAN cell (SANC), the subcellular distribution pattern of RyRs is region-dependent, varying from central SAN (mostly underneath the surface sarcolemma) to peripheral SAN (striated pattern). A similar distribution pattern is also reported for the PKA- and/or Ca2+/calmodulin-dependent protein kinase II (CaMKII)-dependent phosphorylation of RyRs. The impact and functional implication of their distribution patterns on the SANC pacemaker activity have not been fully elucidated. Here, we employed our recently developed 3D spatial model of mouse SANCs to investigate how the subcellar distribution and phosphorylation of RyRs affect spontaneous firing. Our model that features a high surface-to-center gradient of RyR expression well recapitulates the coupled-clock mechanisms, and the biomarkers of simulated spontaneous action potentials agree well with those from previous experimental studies. When abolishing the gradient without changing the whole-cell expression of RyRs in the SANCs, the model predicts 15.0% slowing of the firing rate. Reversing the surface-to-center gradient of RyR distribution (i.e., higher density in the cell center vs surface sarcolemma) further reduced the firing rate (by 25.3%) while increasing the beat-to-beat variation in cycle length, due to disrupted coupling between local Ca2+ releases and the membrane clock. Likewise, preventing RyR phosphorylation caused a substantial slowing in the firing rate (17.6%) while also augmenting beat-to-beat variability. Our simulations mechanistically uncover the functional significance of subcellar RyR distribution and its phosphorylation in maintaining robust automaticity of SANCs. The modeling framework will be applied to systematically delineate the impact of Ca2+-clock protein phosphorylation, distribution, and their interplay with the membrane-clock proteins on the SANC automaticity.

Synchronization of pacemaker cell firing underlies autonomic modulation of beating intervals and their variability in a numerical model of heterogeneous SA node tissue.

The normal heart beats regularly but not metronomically. It is initiated by pacemaker cells within the sinoatrial node (SAN), an extremely heterogeneous cellular network. The functional role of SAN cell heterogeneity remains unknown. Here we investigated the problem in a SAN tissue model comprised of 2025 single cell models. Each cell model features a coupled operation of the Ca and membrane clocks. We modeled tissue heterogeneity by varying the ICaL conductance (gCaL) and SR Ca pumping (Pup). In our simulations heterogeneous cells interacted in tissue and acquired new properties: while cells in isolation had all rhythmic or no firing, cells firing irregular APs emerged in local cell communities with similar properties. The heart rate is naturally increased via β-adrenergic receptors (βAR) and decreased via cholinergic receptors (ChR) in the SAN. βAR stimulation increased the rate and synchronization of AP firing of individual cell models within the SAN tissue, whereas ChR stimulation decreased the rate and synchronization of AP firing. The number of firing cells increased in βAR stimulation to 99% from 80%, and the average cycle length of the SAN decreased by ∼20%. Meanwhile the standard deviation of the cycle length decreased by a multiple of 25, reflecting a much higher level of synchronization. During ChR stimulation, the number of firing cells remained almost unchanged, but the average AP cycle length increased by almost 20%. The standard deviation also increased by a multiple of 2, demonstrating the higher level of desynchronization. The number of cells with emergent irregular APs firing was substantially greater under ChR stimulation than BAR stimulation, indicating importance of these cells as an intrinsic source of heart rate variability at various conditions.

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.

CAMP-PKA signaling modulates the automaticity of human iPSC-derived cardiomyocytes.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been used to screen and characterize drugs and to reveal mechanisms underlying cardiac diseases. However, before hiPSC-CMs can be used as a reliable experimental model, the physiological mechanisms underlying their normal function should be further explored. Accordingly, a major feature of hiPSC-CMs is automaticity, which is regulated by both Ca2+ and membrane clocks. To investigate the mechanisms coupling these clocks, we tested three hypotheses: (1) normal automaticity of spontaneously beating hiPSC-CMs is regulated by local Ca2+ releases (LCRs) and cAMP/PKA-dependent coupling of Ca2+ clock to M clock; (2) the LCR period indicates the level of crosstalk within the coupled-clock system; and (3) perturbing the activity of even one clock can lead to hiPSC-CM-altered automaticity due to diminished crosstalk within the coupled-clock system. By measuring the local and global Ca2+ transients, we found that the LCRs properties are correlated with the spontaneous beat interval. Changes in cAMP-dependent coupling of the Ca2+ and M clocks, caused by a pharmacological intervention that either activates the β-adrenergic or cholinergic receptor or upregulates/downregulates PKA signaling, affected LCR properties, which in turn altered hiPSC-CMs automaticity. Clocks' uncoupling by attenuating the pacemaker current If or the sarcoplasmic reticulum Ca2+ kinetics, decreased hiPSC-CMs beating rate, and prolonged the LCR period. Finally, LCR characteristics of spontaneously beating (at comparable rates) hiPSC-CMs and rabbit SAN are similar. In conclusion, hiPSC-CM automaticity is controlled by the coupled-clock system whose function is mediated by Ca2+-cAMP-PKA signaling.

PO-690-06 A COUPLED-CLOCK SYSTEM DRIVES THE AUTOMATICITY OF SPONTANEOUSLY BEATING HUMAN INDUCED PLURIPOTENT STEM CELL-DERIVED CARDIOMYOCYTES

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been used to screen and characterize drugs and to reveal mechanisms underlying cardiac diseases. However, before hiPSC-CMs can be used as a reliable experimental model, the physiological mechanisms underlying their normal function should be further explored. Accordingly, a major feature of hiPSC-CMs is automaticity which is regulated by both Ca2+ and membrane (M) clocks.

Saltatory Ca2+-induced-Ca2+ release propagation among RyR2 clusters in sinoatrial node cells isolated from GFP-targeted RYR2 mice

Rhythmic heart excitations are generated by sinoatrial node cells (SANC) operating as a coupled-clock system: an ion channel membrane clock couples to sarcoplasmic reticulum Ca2+clock that generates diastolic local Ca2+releases (LCRs) via release channels RyR2. In contrast to stochastic Ca2+sparks in ventricular myocytes, LCRs are roughly periodic at physiological [Ca2+] due to partial phosphorylation (PKA/CaMKII) of Ca2+ cycling proteins in the basal state, i.e. in the absence of beta-adrenergic-receptor stimulation.

Mechanisms of Sinoatrial Node Dysfunction in Heart Failure With Preserved Ejection Fraction.

The ability to increase heart rate during exercise and other stressors is a key homeostatic feature of the sinoatrial node (SAN). When the physiological heart rate response is blunted, chronotropic incompetence limits exercise capacity, a common problem in patients with heart failure with preserved ejection fraction (HFpEF). Despite its clinical relevance, the mechanisms of chronotropic incompetence remain unknown. Dahl salt-sensitive rats fed a high-salt diet and C57Bl6 mice fed a high-fat diet and an inhibitor of constitutive nitric oxide synthase (Nω-nitro-L-arginine methyl ester [L-NAME]; 2-hit) were used as models of HFpEF. Myocardial infarction was created to induce HF with reduced ejection fraction. Rats and mice fed with a normal diet or those that had a sham surgery served as respective controls. A comprehensive characterization of SAN function and chronotropic response was conducted by in vivo, ex vivo, and single-cell electrophysiologic studies. RNA sequencing of SAN was performed to identify transcriptomic changes. Computational modeling of biophysically-detailed human HFpEF SAN was created. Rats with phenotypically-verified HFpEF exhibited limited chronotropic response associated with intrinsic SAN dysfunction, including impaired β-adrenergic responsiveness and an alternating leading pacemaker within the SAN. Prolonged SAN recovery time and reduced SAN sensitivity to isoproterenol were confirmed in the 2-hit mouse model. Adenosine challenge unmasked conduction blocks within the SAN, which were associated with structural remodeling. Chronotropic incompetence and SAN dysfunction were also found in rats with HF with reduced ejection fraction. Single-cell studies and transcriptomic profiling revealed HFpEF-related alterations in both the "membrane clock" (ion channels) and the "Ca2+ clock" (spontaneous Ca2+ release events). The physiologic impairments were reproduced in silico by empirically-constrained quantitative modeling of human SAN function. Chronotropic incompetence and SAN dysfunction were seen in both models of HF. We identified that intrinsic abnormalities of SAN structure and function underlie the chronotropic response in HFpEF.

TRPC7 regulates the electrophysiological functions of embryonic stem cell-derived cardiomyocytes

BackgroundBiological pacemakers consisting of pluripotent stem cell-derived cardiomyocytes are potentially useful for treating bradycardia. However, tachyarrhythmia caused by derived cardiomyocytes themselves is one of main barriers hampering their clinical translation. An in-depth understanding of the mechanisms underlying the spontaneous action potential (a.k.a. automaticity) might provide potential approaches to solve this problem. The aim of this project is to study the role of canonical transient receptor potential isoform 7 (TRPC7) channels in regulating the automaticity of embryonic stem cell-derived cardiomyocytes (ESC-CMs).Methods and resultsBy Western blotting, the expression of TRPC7 was found to be increased during the differentiation of mouse ESC-CMs (mESC-CMs). Adenovirus-mediated TRPC7 knockdown decreased while overexpression increased the frequency of Ca2+ transients (CaTs), local Ca2+ releases (LCRs), and action potentials (APs) as detected by confocal microscopy and whole-cell patch-clamping. TRPC7 was found to be positively associated with the activity of ryanodine receptor 2 (RyR2), sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), and sodium-calcium exchanger (NCX) but not hyperpolarization-activated, cyclic nucleotide-gated channel (HCN), and inositol trisphosphate receptor (IP3R). Knockdown or overexpression of TRPC7 did not alter the expression of HCN4, Cav1.3, Cav3.1, Cav3.2, IP3R1, RyR2, and SERCA but positively regulated the phosphorylation of RyR2 at S2814 and phospholamban (PLN) at T17. Moreover, the positive regulation of APs by TRPC7 was Ca2+-dependent, as overexpression of N-terminus of TRPC7 (dominant negative of TRPC7) which diminished the Ca2+ permeability of TRPC7 decreased the AP frequency.ConclusionsTRPC7 regulates the automaticity of mESC-CMs through two mechanisms. On the one hand, TRPC7 positively regulates the intracellular Ca2+ clock through the regulation of activities of both RyR2 and SERCA; on the other hand, TRPC7 also positively regulates the membrane clock via its influence on NCX activity. Altogether, our study reveals that TRPC7 is a potential drug target to manipulate the action potential firing rate of pluripotent stem cell-derived cardiomyocyte-based biological pacemakers to prevent tachyarrhythmia, a condition that might be encountered after transplantation.

L-type Cav1.3 calcium channels are key actors in beta-adrenergic triggered automaticity in dormant mouse sino-atrial myocytes

Cardiac pacemaking relies on the spontaneous electrical activity of sino-atrial myocytes (SANCs) in the right atrium arising from a robust interplay of membrane ion channels activity (membrane clock) and intracellular calcium dynamics (calcium clock). Strikingly, most of isolated SANCs exhibit a dormant state (no apparent spontaneous activity) whereas only a small fraction shows regular firing. It has recently been shown that these non-firing SANCs are also present in intact SAN and can switch into a firing mode under beta-adrenergic stimulation. However, their functional relevance in vivo is unknown. To study the role of L-type Ca v 1.3 calcium channels in dormant SANCs using a knock-in mouse strain in which the sensitivity of Ca v 1.2 α1 subunits to dihydropyridines (DHP) was inactivated (Ca v 1.2 DHP−/− ). We performed voltage and current-clamp recordings and confocal live imaging on isolated SANCs under isoprenaline (ISO, 100 nM) in the absence or presence of the DHP blocker Nifedipine (Nife, 3 μM). Preliminary results seem to show less L-type calcium current I Ca,L in dormant SANCs (D) compared to rhythmic SANCs (R) (D: −6.3 ± 1.2, R: −15.6 pA/pF). Dormant SANCs also have less diastolic calcium (D: 96.9 ± 12, R: 218 ± 51.2 a.u, P = 0.14) but significant higher sarcoplasmic reticulum calcium load (D: 6.3 ± 0.6, R: 3.1 ± 0.3 a.u, P = 0.004). Interestingly, ISO perfusion could only switch half of dormant SANCs into a firing mode in both action potential (AP) and calcium transients (CT) recordings whereas the others remained dormant. Further Nife perfusion completely stopped this ISO-induced firing switching the cells back into a dormant state for the vast majority of them (75% in AP-recorded SANCs and 100% in CT-recorded SANCs). Taken together, these results indicate that Cav1.3 channels play a key role in sustaining the coupling of membrane and calcium clock in dormant SANCs under beta-adrenergic stimulation.

CAMP-Dependent Signaling Restores AP Firing in Dormant SA Node Cells via Enhancement of Surface Membrane Currents and Calcium Coupling.

Action potential (AP) firing rate and rhythm of sinoatrial nodal cells (SANC) are controlled by synergy between intracellular rhythmic local Ca2+ releases (LCRs) (“Ca2+ clock”) and sarcolemmal electrogenic mechanisms (“membrane clock”). However, some SANC do not fire APs (dormant SANC). Prior studies have shown that β-adrenoceptor stimulation can restore AP firing in these cells. Here we tested whether this relates to improvement of synchronization of clock coupling. We characterized membrane potential, ion currents, Ca2+ dynamics, and phospholamban (PLB) phosphorylation, regulating Ca2+ pump in enzymatically isolated single guinea pig SANC prior to, during, and following β-adrenoceptor stimulation (isoproterenol) or application of cell-permeant cAMP (CPT-cAMP). Phosphorylation of PLB (Serine 16) was quantified in the same cells following Ca2+ measurement. In dormant SANC LCRs were small and disorganized at baseline, membrane potential was depolarized (−38 ± 1 mV, n = 46), and ICaL, If, and IK densities were smaller vs SANC firing APs. β-adrenoceptor stimulation or application of CPT-cAMP led to de novo spontaneous AP generation in 44 and 46% of dormant SANC, respectively. The initial response was an increase in size, rhythmicity and synchronization of LCRs, paralleled with membrane hyperpolarization and small amplitude APs (rate ∼1 Hz). During the transition to steady-state AP firing, LCR size further increased, while LCR period shortened. LCRs became more synchronized resulting in the growth of an ensemble LCR signal peaked in late diastole, culminating in AP ignition; the rate of diastolic depolarization, AP amplitude, and AP firing rate increased. ICaL, IK, and If amplitudes in dormant SANC increased in response to β-adrenoceptor stimulation. During washout, all changes reversed in order. Total PLB was higher, but the ratio of phosphorylated PLB (Serine 16) to total PLB was lower in dormant SANC. β-adrenoceptor stimulation increased this ratio in AP-firing cells. Thus, transition of dormant SANC to AP firing is linked to the increased functional coupling of membrane and Ca2+ clock proteins. The transition occurs via (i) an increase in cAMP-mediated phosphorylation of PLB accelerating Ca2+ pumping, (ii) increased spatiotemporal LCR synchronization, yielding a larger diastolic LCR ensemble signal resulting in an earlier increase in diastolic INCX; and (iii) increased current densities of If, ICaL, and IK.

Insights into sinus arrhythmia of the dog: Acetylcholine perfusion of canine right atrium results in beat-to-beat patterns that mimic sinus arrhythmia supporting exit block in the sinoatrial conduction pathways

Sinus arrhythmia of the dog is unique because of the pronounced alternating beat-to-beat intervals. The clustering of these short (faster rates) and long (slower rates) intervals is not just influenced by autonomic input from breathing; sinus arrhythmia can persist in the panting or apneic dog. The multiplicity of central and peripheral influences on the sinus node complicates the unraveling of the mechanisms of sinus arrhythmia. Studies of the sinus node suggest that acetylcholine can slow cellular depolarization and block sinoatrial conduction. Electrocardiographic monitoring of the dog supports this notion in that abrupt bifurcation into short and long intervals develop at lower heart rates. We sought to determine whether this phenomenon could be recapitulated in canine atrial preparations perfused with acetylcholine and whether selective pharmacologic blockade of the voltage and calcium clocks could provide insight into its mechanism. Spontaneous beat to beat (A–A) intervals were obtained from monophasic action potential recordings of perfused canine right atrial preparations before and during perfusion with acetylcholine (2−5 μM). The calcium clock was blocked with ryanodine (2−3 μM). The membrane clock was blocked with diltiazem hydrochloride (ICa,L blocker; 0.25 μM) and ZD7288 (If blocker; 3 μM). Hyperpolarization was hindered by blockade of IK,Ado/IK,Ach with tertiapin Q (100 nM) before and during acetylcholine perfusion.Acetylcholine resulted in beat clusters similar to those seen in sinus arrhythmia of the dog. Beat clusters were consistent with intermittent 2:1 and 3:1 sinoatrial conduction block. Tertiapin Q abolished this patterning suggesting a role of IK,Ado/IK,ACh in the mechanism of these acetylcholine-induced beat-to-beat patterns.

Differential Roles of Calcium-Activated and Calcium-Inhibited Adenylyl Cyclase Isoforms in the Regulation of SAN Automaticity

Sinoatrial node (SAN) is the primary pacemaker of the heart and is tightly regulated by β-adrenergic receptor (β-AR) signaling. Adenylyl cyclase (AC) is a key enzyme in the β-AR pathway that catalyzes the production of cAMP from ATP, leading to the activation of a cascade of downstream effectors. There are 10 known isoforms of AC, but their relative expression and distribution vary considerably among different regions of the heart. Additionally, a knowledge gap exists regarding their contribution to SAN automaticity. We hypothesize that Ca2+-activated AC isoforms play important regulatory roles in SAN automaticity. To determine regional distribution of AC isoforms in the heart, single-cell RT-PCR was performed. Global knockout (KO) of AC isoforms and WT C57Bl/6 mice were utilized to assess the role of AC in the SAN at basal and after β-AR stimulation with isoproterenol (ISO). Cardiac structure and function were assessed by echocardiography as well as hemodynamic measurements. Ambulatory electrocardiography was used to determine in vivo SAN function during basal condition, after β-AR stimulation, and after autonomic nervous system blockade. Pacemaker and Ca2+ currents, as well as local Ca2+ release, were performed in isolated SAN cells using conventional patch-clamp techniques and confocal microscopy. cAMP biosensors were used to directly evaluate compartmentalization of the different isoforms. Indeed, we found distinct regulation of SAN automaticity by different isoforms of ACs. Differential compartmentalization of AC isoforms with the Ca2+vs. membrane clocks further validated these findings. Localization and contribution to SAN automaticity vary with different isoforms of ACs due to their distinct compartmentalization with the Ca2+vs. membrane clocks. The findings have broad therapeutic ramifications. Embedded in these findings are novel insights that can be exploited in developing successful therapeutics for SAN dysfunction.

Abstract 17006: Reversing Membrane Clock Remodeling (MCR) Associated With Heart Failure With Preserved Ejection Fraction (HFpEF) in the Sinoatrial Node (SAN) Eliminates Chronotropic Incompetence (CI)

Introduction: While CI, or the inability of heart rate (HR) to adequately respond to exercise, is a strong indicator of mortality in HFpEF, treatment options remain limited as its mechanisms remain underexplored. Ionic remodeling of the SAN, both of the membrane (MC) and calcium clocks (CC), is associated with HFpEF and CI. Aim: Using an in silico approach, we investigated whether SAN ionic remodeling can contribute to 3 different CI presentations (Fig A) and explored the effectiveness of targeting the remodeling of the MC or CC (MCR and CCR, resp.) in eliminating CI. Methods: A human SAN model (control) was used, in which ionic remodeling was integrated using experimental data from HFpEF rat model with CI (Fig B top). A family of HFpEF SAN models (n = 147) was generated because the uniqueness of Ca 2+ handling parameters constrained to experimentally-based Ca 2+ transients could not be guaranteed (Fig B middle). To assess the level of CI, max beat rate (BR), BR response (τ on ), and BR recovery (τ off ) were quantified for each model in response to a transient 40s pulse of 1 μM isoproterenol (ISO) (Fig B bottom). Lastly, the effect of reversing either CCR or MCR on CI was determined. Results: Without ISO, BR of HFpEF SAN models was similar to that of control; when ISO was applied (Fig C), HFpEF models exhibited all aspects of CI: submaximal BR (Fig D), and elevated τ on and τ off (Fig E). In CCR reversed models, BR without ISO was similar to that of control; with ISO, they exhibited submaximal BR (Fig F). In MCR reversed models, BR without ISO was slightly lower compared to control; with ISO, while they exhibited submaximal BR, the increase in BR with ISO was similar to that of control (Fig F). τ on and τ off were elevated in CCR reversed models, but they were not in MCR reversed models (Fig G). In summary, CCR reversed models exhibited all aspects of CI, but MCR reversed models did not. Conclusion: Ionic remodeling of the SAN contributes to CI in HFpEF, and targeting MCR could be a promising strategy for eliminating CI.

The effect of forskolin on membrane clock and calcium clock in the hypoxic/reoxygenation of sinoatrial node cells and its mechanism.

In this study, we investigated the effect of forskolin (FSK, a selective adenylate cyclase agonist) on the automatic diastolic depolarization of sinus node cells (SNC) with hypoxia/reoxygenation (H/R) injury. The SNC of the newborn rat was randomly assigned into the control group, the H/R (H/R injury) group, or the H/R + FSK (H/R injury + FSK treatment) group. Patch-clamp was performed to record the action potential and electrophysiological changes. The cellular distribution of intracellular calcium concentration was analyzed by fluorescence staining. Compared with the control cells, spontaneous pulsation frequency (SPF) and diastolic depolarization rate (DDR) of H/R cells were reduced from 244.3 ± 10.6 times/min and 108.7 ± 7.8mV/s to 130.5 ± 7.6 times/min and 53.4 ± 6.5mV/s, respectively. FSK significantly increased SPF and DDR of H/R cells to 208.3 ± 8.3 times/min and 93.2 ± 8.9mV/s (n = 15, both p < 0.01), respectively. H/R reduced the current densities of If, ICa,T and inward INCX, which were significantly increased by 10μM FSK treatment (n = 15, p < 0.01). Furthermore, reduced expression of HCN4 and NCX1.1 channel protein were significantly increased by FSK. Inhibitor studies showed that both SQ22536 (a selective adenylate cyclase inhibitor) and H89 (a selective protein kinases A [PKA] inhibitor) blocked the effects of FSK on SPF and DDR. H/R causes pacemaker dysfunction in newborn rat sinoatrial node cells leading to divergence of the DD and the slow of spontaneous APs, which change can be dramatically reversed by FSK through increasing INCX and If current in H/R injury.

Functional Microdomain of Adenylyl Cyclase Isoform 1 Contributes to Sinoatrial Node Automaticity via β-Adrenergic Receptor Pathway

The sinoatrial node (SAN) is the primary pacemaker of the heart and is tightly regulated by β-adrenergic receptor (β-AR) signaling. Adenylyl cyclase (AC) is a key enzyme in the β-AR pathway that catalyzes the production of cAMP. We hypothesize that compartmentalization of different isoforms of ACs with the membrane and Ca2+ clocks are critical for the function of SAN cells (SANC). Immunofluorescence confocal microscopy, stimulated emission depletion microscopic imaging, and proximity ligation assay (PLA) were used to examine compartmentalization of ACI and caveolin-3 (Cav3), HCN4, or Cav1.2. Spontaneous action potentials, pacemaker and Ca2+ currents, as well as local Ca2+ release were recorded. cAMP-FRET biosensors targeting plasma membrane, sarcoplasmic reticulum, and cytosol were expressed in cultured SANC isolated from ACI-/-and wild type (WT) mice to examine AC functional microdomains in the SAN. We demonstrate that ACI is the most abundant isoform in the SAN. ACI-/-mice exhibited SAN dysfunction after β-AR stimulation. To elucidate the mechanistic underpinnings, we examined subcellular co-localization of ACI to key proteins that contribute to functional integrity of the SAN. We observed robust PLA signals of SANCs co-labeled for ACI with Cav3, HCN4, or Cav1.2, Functionally, there were significant differences in action potential firing frequencies, Ca2+ and pacemaker currents between SANCs isolated from ACI-/-and WT mice. Novel cAMP biosensors showed a blunted response in local cAMP generation during β-AR stimulation in ACI-/-SANC. ACI is the predominant isoform in the SAN and co-localizes with Cav3 and HCN4 channels. Compartmentalization of ACI and HCN4 plays critical roles in the regulation of the “membrane clock” via β-AR pathway. Since ACI is Ca2+-activated, it may provide the molecular link between Ca2+ and membrane clocks of the SAN.

Inhibitory Effects of Cyclopiazonic Acid on the Pacemaker Current in Sinoatrial Nodal Cells

Objective: The spontaneous action potential of isolated sinoatrial node (SAN) cells is regulated by a coupled-clock system of two clocks: the calcium clock and membrane clock. However, it remains unclear whether calcium clock inhibitors have a direct effect on the membrane clock. The purpose of this study was to investigate the direct effect of cyclopiazonic acid (CPA), a selective calcium clock inhibitor, on the function of the membrane clock of SAN cells. Methods: at SAN cells were isolated by trypsinization and identified based on morphology and electrophysiology. If and HCN currents were recorded via patch clamp technique. The expression of the HCN channel protein was determined by Western blotting analysis. Results: The diastolic depolarization rate of spontaneous action potentials and the current densities of If were reduced by exposure to 10 μM CPA. The inhibitory effect of CPA was concentration-dependent with an IC50 value of 16.3 μM and a Hill coefficient of 0.98. The effect of CPA on If current was also time-dependent, and the If current amplitude was partially restored after washout. Furthermore, the steady-state activation curve of the If current was shifted to a negative potential, indicating that channel activation slowed down. Finally, the protein expression of HCN4 in HEK293 cells was markedly downregulated by CPA. Conclusions: These results indicate that the direct inhibition effect of CPA on the If current in SAN cells is both concentration- and time-dependent. The underlying mechanisms may involve slowing down steady-state activation and the downregulation of pacemaker channel protein expression.