Sinoatrial Node Cells Research Articles

Genetic Ablation of G Protein-Gated Inwardly Rectifying K+ Channels Prevents Training-Induced Sinus Bradycardia.

Background: Endurance athletes are prone to bradyarrhythmias, which in the long-term may underscore the increased incidence of pacemaker implantation reported in this population. Our previous work in rodent models has shown training-induced sinus bradycardia to be due to microRNA (miR)-mediated transcriptional remodeling of the HCN4 channel, leading to a reduction of the “funny” (If) current in the sinoatrial node (SAN).Objective: To test if genetic ablation of G-protein-gated inwardly rectifying potassium channel, also known as IKACh channels prevents sinus bradycardia induced by intensive exercise training in mice.Methods: Control wild-type (WT) and mice lacking GIRK4 (Girk4–/–), an integral subunit of IKACh were assigned to trained or sedentary groups. Mice in the trained group underwent 1-h exercise swimming twice a day for 28 days, 7 days per week. We performed electrocardiogram recordings and echocardiography in both groups at baseline, during and after the training period. At training cessation, mice were euthanized and SAN tissues were isolated for patch clamp recordings in isolated SAN cells and molecular profiling by quantitative PCR (qPCR) and western blotting.Results: At swimming cessation trained WT mice presented with a significantly lower resting HR that was reversible by acute IKACh block whereas Girk4–/– mice failed to develop a training-induced sinus bradycardia. In line with HR reduction, action potential rate, density of If, as well as of T- and L-type Ca2+ currents (ICaT and ICaL) were significantly reduced only in SAN cells obtained from WT-trained mice. If reduction in WT mice was concomitant with downregulation of HCN4 transcript and protein, attributable to increased expression of corresponding repressor microRNAs (miRs) whereas reduced ICaL in WT mice was associated with reduced Cav1.3 protein levels. Strikingly, IKACh ablation suppressed all training-induced molecular remodeling observed in WT mice.Conclusion: Genetic ablation of cardiac IKACh in mice prevents exercise-induced sinus bradycardia by suppressing training induced remodeling of inward currents If, ICaT and ICaL due in part to the prevention of miR-mediated transcriptional remodeling of HCN4 and likely post transcriptional remodeling of Cav1.3. Strategies targeting cardiac IKACh may therefore represent an alternative to pacemaker implantation for bradyarrhythmias seen in some veteran athletes.

Deciphering Cellular Signals in Adult Mouse Sinoatrial Node Cells

The flexibility of manipulating the mouse genome makes cells from this species an ideal model to study cellular signals that control organ function. Genetically-modified mice provide opportunities to decipher how pacemaking sinoatrial node (SAN) cells are regulated by several second messengers and signaling molecules. However, detailed information on the spatiotemporal dynamics of these signaling events in adult mouse SAN cells has been limited due to the challenge of visualizing them in live cells. Here, a method is described for culturing and immediate infection of adult mouse SAN cells with genetically-encoded FRET-based biosensors to examine cellular dynamics of signaling events. SAN cells maintained in culture media containing blebbistatin or (S)-nitro-blebbistatin retain their elongated morphology and action potential (AP) waveform for up to 40 hours. The culturing condition does not change β-adrenergic-mediated cAMP signal as determined in freshly dissociated and cultured SAN cells from a cardiac-specific cAMP reporter mouse. SAN cells expressing cAMP sensors in different cellular compartments show distinct β-adrenergic-mediated cAMP pools. Examination of cyclic GMP (cGMP), protein kinase A (PKA), Ca2+/CaM kinase II (CaMKII) and protein kinase D (PKD) activity with specific FRET biosensors also show unique responses to different stimuli in SAN cells. Isolation and culture of SAN cells from heart failure mice reveal a decrease in cAMP and cGMP signaling. Thus, a reliable and efficient method for culturing adult mouse SAN cells has been developed to facilitate studies of signaling network dynamics during physiological and pathological conditions.

Tongyang Huoxue Decoction (TYHX) Ameliorating Hypoxia/Reoxygenation-Induced Disequilibrium of Calcium Homeostasis and Redox Imbalance via Regulating Mitochondrial Quality Control in Sinoatrial Node Cells.

Sick sinus syndrome (SSS) is a disease with bradycardia or arrhythmia. The pathological mechanism of SSS is mainly due to the abnormal conduction function of the sinoatrial node (SAN) caused by interstitial lesions or fibrosis of the SAN or surrounding tissues, SAN pacing dysfunction, and SAN impulse conduction accompanied by SAN fibrosis. Tongyang Huoxue Decoction (TYHX) is widely used in SSS treatment and amelioration of SAN fibrosis. It has a variety of active ingredients to regulate the redox balance and mitochondrial quality control. This study mainly discusses the mechanism of TYHX in ameliorating calcium homeostasis disorder and redox imbalance of sinoatrial node cells (SANCs) and clarifies the protective mechanism of TYHX on the activity of SANCs. The activity of SANCs was determined by CCK-8 and the TUNEL method. The levels of apoptosis, ROS, and calcium release were analyzed by flow cytometry and immunofluorescence. The mRNA and protein levels of calcium channel regulatory molecules and mitochondrial quality control-related molecules were detected by real-time quantitative PCR and Western Blot. The level of calcium release was detected by laser confocal. It was found that after H/R treatment, the viability of SANCs decreased significantly, the levels of apoptosis and ROS increased, and the cells showed calcium overload, redox imbalance, and mitochondrial dysfunction. After treatment with TYHX, the cell survival level was improved, calcium overload and oxidative stress were inhibited, and mitochondrial energy metabolism and mitochondrial function were restored. However, after the SANCs were treated with siRNA (si-β-tubulin), the regulation of TYHX on calcium homeostasis and redox balance was counteracted. These results suggest that β-tubulin interacts with the regulation of mitochondrial function and calcium release. TYHX may regulate mitochondrial quality control, maintain calcium homeostasis and redox balance, and protect SANCs through β-tubulin. The regulation mechanism of TYHX on mitochondrial quality control may also become a new target for SSS treatment.

Modeling the Human Sinoatrial Node Based on Sequential Discharge Hypothesis

The electrical synchronization of human sinoatrial (SA) node cells for propagating the pacemaker activity has always been an important challenge in literatures. Nevertheless, most literatures, that provide hypotheses for the electrical synchronization of SA node cells, only investigated the activity of a very small group of coupled SA node cells. Therefore, this study proposes a novel SA node model based on the sequential discharge hypothesis to estimate the electrical potential around the SA node, which electrodes measure it. This model consists of a three-dimensional regular network of Hodgkin-Huxley (HH) model modified by time constantthat propagates the action potentials of dominant pacemaker through the gap junctions. The results show that the HH model modified in this study not only has adequate capacity for generating oscillations between 40 and 180 beats per minute (BPM), but also their three-dimensional regular network can provide a suitable estimate from the electrical potential around the SA node. Therefore, this model is a good suggestion for the electrical synchronization of large mammalian SA node cells, especially human.

Mechanistic Insights Into the Reduced Pacemaking Rate of the Rabbit Sinoatrial Node During Postnatal Development: A Simulation Study.

Marked age- and development- related differences have been observed in morphology and characteristics of action potentials (AP) of neonatal and adult sinoatrial node (SAN) cells. These may be attributable to a different set of ion channel interactions between the different ages. However, the underlying mechanism(s) have yet to be elucidated. The objective of this study was to determine the mechanisms underlying different spontaneous APs and heart rate between neonatal and adult SAN cells of the rabbit heart by biophysical modeling approaches. A mathematical model of neonatal rabbit SAN cells was developed by modifying the current densities and/or kinetics of ion channels and transporters in an adult cell model based on available experimental data obtained from neonatal SAN cells. The single cell models were then incorporated into a multi-cellular, two-dimensional model of the intact SAN-atrium to investigate the functional impact of altered ion channels during maturation on pacemaking electrical activities and their conduction at the tissue level. Effects of the neurotransmitter acetylcholine on the pacemaking activities in neonatal cells were also investigated and compared to those in the adult. Our results showed: (1) the differences in ion channel properties between neonatal and adult SAN cells are able to account for differences in their APs and the heart rate, providing mechanistic insight into understanding the reduced pacemaking rate of the rabbit sinoatrial node during postnatal development; (2) in the 2D model of the intact SAN-atria, it was shown that cellular changes during postnatal development impaired pacemaking activity through increasing the activation time and reducing the conduction velocity across the SAN; (3) the neonatal SAN model, with its faster beating rates, showed a greater sensitivity to parasympathetic modulation in response to acetylcholine than did the adult model. These results provide novel insights into the understanding of the cellular mechanisms underlying the differences in the cardiac pacemaking activities of the neonatal and adult SAN.

Concomitant genetic ablation of L-type Cav1.3 (\u03b11D) and T-type Cav3.1 (\u03b11G) Ca2+ channels disrupts heart automaticity

Cardiac automaticity is set by pacemaker activity of the sinus node (SAN). In addition to the ubiquitously expressed cardiac voltage-gated L-type Cav1.2 Ca2+ channel isoform, pacemaker cells within the SAN and the atrioventricular node co-express voltage-gated L-type Cav1.3 and T-type Cav3.1 Ca2+ channels (SAN-VGCCs). The role of SAN-VGCCs in automaticity is incompletely understood. We used knockout mice carrying individual genetic ablation of Cav1.3 (Cav1.3−/−) or Cav3.1 (Cav3.1−/−) channels and double mutant Cav1.3−/−/Cav3.1−/− mice expressing only Cav1.2 channels. We show that concomitant loss of SAN-VGCCs prevents physiological SAN automaticity, blocks impulse conduction and compromises ventricular rhythmicity. Coexpression of SAN-VGCCs is necessary for impulse formation in the central SAN. In mice lacking SAN-VGCCs, residual pacemaker activity is predominantly generated in peripheral nodal and extranodal sites by f-channels and TTX-sensitive Na+ channels. In beating SAN cells, ablation of SAN-VGCCs disrupted late diastolic local intracellular Ca2+ release, which demonstrates an important role for these channels in supporting the sarcoplasmic reticulum based “Ca2+clock” mechanism during normal pacemaking. These data implicate an underappreciated role for co-expression of SAN-VGCCs in heart automaticity and define an integral role for these channels in mechanisms that control the heartbeat.

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

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

Hypercalcemia impairs sino-atrial automaticity through excessive Cav1.2-mediated Ca2+ influx

We investigated whether abnormal activity of the L-type Ca2+ channels (LTCCs), Cav1.2 and Cav1.3, under extracellular hypercalcemia, impaired heart pacemaker activity. Membrane currents and intracellular Ca2+ ([Ca2 +]i) dynamics of the sino-atrial node (SAN) generate the heart pacemaker activity. Impairments of SAN activity, also called SAN dysfunction (SND), harms heart automaticity. Recent studies have associated abnormal [Ca2 +]i dynamics with SNDs. Cav1.2 and Cav1.3 carry the main Ca2+ influx of SAN cells to sustain their [Ca2 + ]i dynamics. Modified extracellular Ca2+ ([Ca2 +]o) could alter Ca2+ influx through these channels. For example, cancer and hyperparathyroidism can raise [Ca2 + ]o, causing an extracellular hypercalcemia that could alter [Ca2 + ]i dynamics and impair SAN activity and heart automaticity. To test this hypothesis, we measured contractions, [Ca2 +]i release and L-type Ca2+ current (ICa,L) in spontaneous cells of the murine SAN. Then, we recorded rate and propagation of the spontaneous action potentials (APs) generated by the SAN tissue ex-vivo. In spontaneous SAN cells, we observed that the modification of [Ca2 +]o modulates [Ca2 +]i and cells contraction through changes of ICa,L. In particular, the increase of [Ca2 +]o dysregulated pacemaker activity, likely through excessive Ca2+ influx mediated by Cav1.2. [Ca2 +]o increase to hypercalcemic levels induced arrhythmia also in the intact SAN tissues, activating ectopic leading regions of pacemaking and impairing conduction towards the atria. We recovered these pacemaker dysfunctions by blocking Ca2 + -activated small-conductance K+ (SK) channels. Hypercalcemia causes excessive Cav1.2-mediated Ca2+ influx, which alters [Ca2 +]i and could hyperactivate SK channels, leading to pacemaker impairment. Cav1.2 or SK modulation may reduce pacemaker dysfunctions, preventing SND progression.

Genetic ablation of G protein-gated inwardly rectifying K+ (Girk)4 channels prevents heart rate reduction induced by intensive exercise training

The incidence of brady-arrhythmias is known to be higher in athletes. In rodent models of exercise training, bradycardia results from downregulation of f-(HCN4) channels leading to a reduction of the If current in the sino-atrial node (SAN). To test if genetic ablation of G-protein-gated inwardly rectifying potassium 4 (Girk4) channel prevents sinus bradycardia induced by intensive exercise training in mice. Control (WT) and homozygous Girk4 knock-out (Girk4-/-) mice were assigned to trained or sedentary groups. Mice in the trained group underwent 1-hour exercise swimming twice a day for 28 days, 7 days per week whereas sedentary mice underwent 5-min swimming daily in the same period. We performed telemetric electrocardiogram recordings in both groups at baseline and during the training period. At training cessation, mice were euthanized and SAN tissues were isolated for patch clamp recordings in isolated SAN cells and transcriptional profiling by quantitative PCR (qPCR). At day 17 of the swimming regimen, heart rate (HR) reduction in trained WT mice was significantly different vs. WT sedentary mice, whereas Girk4-/- mice failed to develop sinus bradycardia. Action potential recordings in isolated SAN cells from trained WT mice showed lower rates of pacemaking in comparison to cells from mice of the other groups. In line with HR reduction, the density of If was significantly reduced only in SAN cells obtained from WT-trained mice. Correspondingly, qPCR analysis showed mRNA expression of HCN4 was significantly lower in WT-trained SAN relative to WT sedentary animals, but unchanged in Girk4-/- animals. This finding could be attributed to a significant increase in the expression of miR-423-5p (a transcriptional repressor of HCN4) observed in trained WT, but not trained Girk4-/- animals. Genetic ablation of Girk4 channels prevents sinus bradycardia induced by down regulation of f-HCN4 channels in trained WT mice.

Evidence for a selective blockade of Cav1.2 versus Cav1.3 by the mamba toxin calciseptine in the mouse heart

Selective blockers are important tools for identifying the physiological role of ion channels isoforms. L-type calcium channels are high voltage activated channels sensitive to dihydropyridine (DHP). Two distinct isoforms (a1 C/Cav1.2 and a1D/Cav1.3) mediate calcium entry into cardiac cells to trigger contraction or contribute to heart rate generation. Many classes of drugs have been shown to target L-type calcium channels but poor selectivity has been reported between Cav1.2 and Cav1.3. Our goal is to look for an animal toxin able to discriminate between these two L-type calcium channel isoforms. We focused on calciseptine, a snake toxin purified from mamba venom which decreases, via Cav1.2 inhibition, vascular and cardiac contraction without affecting heart rate (De Weille et al., 2001). We compared the effect of calciseptine and of a classical DHP on the contraction amplitude and heart rate in Langendorf perfused hearts from wild-type mice and mice carrying Cav1.2 channels insensitive to DHP. Calciseptine 100 nM strongly decreased the amplitude of contractions by 82%. Increasing the concentration up to 1 μM further reduced the contraction while no modification of the heart rate was noticed. The frequency of spontaneous AP recorded from sinus node using the patch clamp technique was also unaffected by 100 nM calciseptine. (216 ± 20 bpm ctrl vs. 212 ± 22 bpm calciseptine). On the other hand, 3 μM nifedipine, which blocks both Cav1.2 and Cav1.3 channels decreased contraction and heart rate in isolated mouse heart, induced arrhythmias and strongly reduced AP frequency in isolated sinus node cells (193 ± 10 bpm ctrl vs. 48 ± 9 bpm nifedipine). These preliminary data suggest that calciseptine may act as a specific blocker of Cav1.2 versus Cav1.3, a result to be confirmed by direct exploration of the effect of calciseptine on native ventricular and SAN cells calcium currents or on recombinant Cav1.2 and Cav1.3 currents.

Electrophysiological insights into the relationship between calcium dynamics and cardiomyocyte beating function in chronic hemodialysis treatment.

For patients in which the Ca2+ concentration of dialysis fluid is lower than that in plasma, chronic hemodialysis treatment often leads to cardiac beating dysfunction. By applying these conditions to an electrophysiological mathematical model, we evaluated the impact of body fluid Ca2+ dynamics during treatment on cardiomyocyte beating and, moreover, explored measures that may prevent cardiomyocyte beating dysfunction. First, Ca2+ concentrations in both plasma and interstitial fluid were decreased with treatment time, which induced both a slight decline in beating rhythm on a sinoatrial nodal cell and a wane in contraction force on a ventricular cell. These simulated results were in agreement with clinical observations. Next, a relationship between the intracellular Ca2+ concentration and ion current dynamics of ion transporters were examined to elucidate the mechanism underlying cardiomyocyte beating dysfunction. The inward current of the Na/Ca exchanger (NCX) increased with a decrease in Ca2+ concentration in interstitial fluid and induced a reduction in intracellular Ca2+ concentration during treatment. Furthermore, the decline in intracellular Ca2+ concentration reduced the contraction force. These findings implied that ion transport through the NCX is a dominant factor that induces cardiomyocyte beating dysfunction during hemodialysis. Finally, the replenishment of Ca2+ or application of an NCX inhibitor during treatment suppressed the decrease in intracellular Ca2+ concentration and contributed to the stabilization of cardiomyocyte beating function. In summary, the clinical implementation of hepatically cleared NCX inhibitor may be a suitable approach to improving the quality of life for patients on chronic hemodialysis.

Contribution of estrogen to the pregnancy-induced increase in cardiac automaticity

BackgroundThe heart rate progressively increases throughout pregnancy, reaching a maximum in the third trimester. This elevated heart rate is also present in pregnant mice and is associated with accelerated automaticity, higher density of the pacemaker current If and changes in Ca2+ homeostasis in sinoatrial node (SAN) cells. Strong evidence has also been provided showing that 17β-estradiol (E2) and estrogen receptor α (ERα) regulate heart rate. Accordingly, we sought to determine whether E2 levels found in late pregnancy cause the increased cardiac automaticity associated with pregnancy. Methods and resultsVoltage- and current-clamp experiments were carried out on SAN cells isolated from female mice lacking estrogen receptor alpha (ERKOα) or beta (ERKOβ) receiving chronic E2 treatment mimicking late pregnancy concentrations. E2 treatment significantly increased the action potential rate (284 ± 24 bpm, +E2 354 ± 23 bpm, p = 0.040) and the density of If (+52%) in SAN cells from ERKOβ mice. However, If density remains unchanged in SAN cells from E2-treated ERKOα mice. Additionally, E2 also increased If density (+67%) in nodal-like human-induced pluripotent stem cell-derived cardiomyocytes (N-hiPSC-CM), recapitulating in a human SAN cell model the effect produced in mice. However, the L-type calcium current (ICaL) and Ca2+ transients, examined using N-hiPSC-CM and SAN cells respectively, were not affected by E2, indicating that other mechanisms contribute to changes observed in these parameters during pregnancy. ConclusionThe accelerated SAN automaticity observed in E2-treated ERKOβ mice is explained by an increased If density mediated by ERα, demonstrating that E2 plays a major role in regulating SAN function during pregnancy.

Overexpression of TBX3 in human induced pluripotent stem cells (hiPSCs) increases their differentiation into cardiac pacemaker-like cells

BackgroudThe TBX3(T-box 3)transcription factor is considered as an essential factor in sinoatrial node formation. While the effect of TBX3 in the differentiation of sinoatrial node cells from embryonic stem cells(ESCs) has been recognized, its role in human induced pluripotent stem cell derived cardiomyocytes(hiPSCMs) has not been addressed. Therefore, the purpose of the present study was to investigate whether overexpression of TBX3 in hiPSCs could increase their differentiation into pacemaker-like cells. MethodsThe hiPSCs were transfected with TBX3 gene during differentiation into cardiomyocytes(CMs). The hiPSCMs were analyzed using immunofluorescence, RT-qPCR, flow cytometry, whole-cell patch clamp recording to identify the differentiation effect exerted by TBX3. We discovered that hiPSCs transfected with TBX3 showed more proportions of NKX2.5-cTNT + sinoatrial node cells and faster contracting rates. ResultsThe results showed increment in transcription factor TBX18, SHOX2; hyperpolarization-activated cyclic nucleotide (HCN) channel: HCN1, HCN2, HCN4, connexin 45(CX45), Na + Ca2+ exchanger(NCX) in TBX3 transfected hiPSCMs. Sinoatrial node cell specific If current and action potential were also confirmed by patch clamp in TBX3 transfected hiPSCMs and the pacemaker-like cells were able to pace hiPSCMs ex vivo. ConclusionIn conclusion, the present study demonstrated that overexpression of TBX3 could increase the differentiation of hiPSCs into pacemaker-like cells. Our study provide new strategy to construct a biological pacemaker, however, further study is still needed to identify the efficacy and safety of using the pacemaker-like cells to produce biological pacemaker in vivo.

Synchronized Cardiac Impulses Emerge From Heterogeneous Local Calcium Signals Within and Among Cells of Pacemaker Tissue.

This study sought to identify subcellular Ca2+ signals within and among cells comprising the sinoatrial node (SAN) tissue. The current paradigm of SAN impulse generation: 1) is that full-scale action potentials (APs) of a common frequency are initiated at 1 site and are conducted within the SAN along smooth isochrones; and 2) does not feature fine details of Ca2+ signaling present in isolated SAN cells, in which small subcellular, subthreshold local Ca2+ releases (LCRs) self-organize to generate cell-wide APs. Immunolabeling was combined with a novel technique to detect the occurrence of LCRs and AP-induced Ca2+ transients (APCTs) in individual pixels (chronopix) across the entire mouse SAN images. At high magnification, Ca2+ signals appeared markedly heterogeneous in space, amplitude, frequency, and phase among cells comprising an HCN4+/CX43- cell meshwork. The signaling exhibited several distinguishable patterns of LCR/APCT interactions within and among cells. Rhythmic APCTs that were apparently conducted within the meshwork were transferred to a truly conducting HCN4-/CX43+ network of striated cells via narrow functional interfaces where different cell types intertwine, that is, the SAN anatomic/functional unit. At low magnification, the earliest APCT of each cycle occurred within a small area of the HCN4 meshwork, and subsequent APCT appearance throughout SAN pixels was discontinuous and asynchronous. The study has discovered a novel, microscopic Ca2+ signaling paradigm of SAN operation that has escaped detection using low-resolution, macroscopic tissue isochrones employed in prior studies: synchronized APs emerge from heterogeneous subcellular subthreshold Ca2+ signals, resembling multiscale complex processes of impulse generation within clusters of neurons in neuronal networks.

Signatures of the autonomic nervous system and the heart\u2019s pacemaker cells in canine electrocardiograms and their applications to humans

Heart rate and heart rate variability (HRV) are mainly determined by the autonomic nervous system (ANS), which interacts with receptors on the sinoatrial node (SAN; the heart’s primary pacemaker), and by the “coupled-clock” system within the SAN cells. HRV changes are associated with cardiac diseases. However, the relative contributions of the ANS and SAN to HRV are not clear, impeding effective treatment. To discern the SAN’s contribution, we performed HRV analysis on canine electrocardiograms containing basal and ANS-blockade segments. We also analyzed human electrocardiograms of atrial fibrillation and heart failure patients, as well as healthy aged subjects. Finally, we used a mathematical model to simulate HRV under decreased “coupled-clock” regulation. We found that (a) in canines, the SAN and ANS contribute mainly to long- and short-term HRV, respectively; (b) there is evidence suggesting a similar relative SAN contribution in humans; (c) SAN features can be calculated from beat-intervals obtained in-vivo, without intervention; (d) ANS contribution can be modeled by sines embedded in white noise; (e) HRV changes associated with cardiac diseases and aging can be interpreted as deterioration of both SAN and ANS; and (f) SAN clock-coupling can be estimated from changes in HRV. This may enable future non-invasive diagnostic applications.

GPCR-dependent biasing of GIRK channel signaling dynamics by RGS6 in mouse sinoatrial nodal cells

How G protein-coupled receptors (GPCRs) evoke specific biological outcomes while utilizing a limited array of G proteins and effectors is poorly understood, particularly in native cell systems. Here, we examined signaling evoked by muscarinic (M2R) and adenosine (A1R) receptor activation in the mouse sinoatrial node (SAN), the cardiac pacemaker. M2R and A1R activate a shared pool of cardiac G protein-gated inwardly rectifying K+ (GIRK) channels in SAN cells from adult mice, but A1R-GIRK responses are smaller and slower than M2R-GIRK responses. Recordings from mice lacking Regulator of G protein Signaling 6 (RGS6) revealed that RGS6 exerts a GPCR-dependent influence on GIRK-dependent signaling in SAN cells, suppressing M2R-GIRK coupling efficiency and kinetics and A1R-GIRK signaling amplitude. Fast kinetic bioluminescence resonance energy transfer assays in transfected HEK cells showed that RGS6 prefers Gαo over Gαi as a substrate for its catalytic activity and that M2R signals preferentially via Gαo, while A1R does not discriminate between inhibitory G protein isoforms. The impact of atrial/SAN-selective ablation of Gαo or Gαi2 was consistent with these findings. Gαi2 ablation had minimal impact on M2R-GIRK and A1R-GIRK signaling in SAN cells. In contrast, Gαo ablation decreased the amplitude and slowed the kinetics of M2R-GIRK responses, while enhancing the sensitivity and prolonging the deactivation rate of A1R-GIRK signaling. Collectively, our data show that differences in GPCR-G protein coupling preferences, and the Gαo substrate preference of RGS6, shape A1R- and M2R-GIRK signaling dynamics in mouse SAN cells.

In This Issue

HomeCirculation ResearchVol. 126, No. 12In This Issue Free AccessIn BriefPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessIn BriefPDF/EPUBIn This Issue Ruth Williams Ruth WilliamsRuth Williams Search for more papers by this author Originally published4 Jun 2020https://doi.org/10.1161/RES.0000000000000397Circulation Research. 2020;126:1667is related toAssociation of Inpatient Use of Angiotensin-Converting Enzyme Inhibitors and Angiotensin II Receptor Blockers With Mortality Among Patients With Hypertension Hospitalized With COVID-19ACEI/ARB on COVID-19 in Patients With Hypertension (p 1671)Hypertensive patients taking ACE inhibitors or angiotensin II blockers should continue such treatments if infected with coronavirus, say Zhang et al.Download figureDownload PowerPointPatients with hypertension have an increased risk of death from COVID-19. While the high blood pressure itself is likely to blame, concerns have been raised that medications used to treat hypertension—specifically ACE inhibitors (ACEI) and angiotensin II receptor blockers (ARB)—may worsen coronavirus infection. Research in animals showed these drugs increased expression of ACE2—the protein on lung cells used by the virus to gain entry. Conflicting evidence showed these drugs might reduce lung injury in pneumonia patients, however. To weigh up the benefits and risks of ACEI/ARB use, Zhang and colleagues performed a retrospective analysis of 1128 patients with COVID-19 and hypertension who were treated at nine hospitals in Hubei province, China. Of the patients, 188 took ACEI/ARB during their hospital stay and 940 did not. The ages, sexes, and comorbidities in the two groups were similar. After 28 days of follow up, 99 of the patients had died—7 from the group taking ACEI/ARB (3.7%) and 92 from the group that did not (9.8%). The team concludes that treatment of hypertension patients with ACEI/ARB does not increase risk of COVID-19 mortality and may even reduce the threat.ERR Signaling and Cardiac Maturation (p 1685)ERR is a maturation switch controlling heart cell development, say Sakamoto et al.Download figureDownload PowerPointFrom fetal to postnatal development, the human heart goes through significant changes including an expansion of mitochondrial numbers, a change in fuel utilization, replacement of fetal contractile proteins with adult ones, and increases in ion uptake and release. The transcription factor ERR was known to drive postnatal mitochondria biogenesis. Now Sakamoto and colleagues show it drives these other developmental changes, too. They developed a genetic method to knockdown expression of ERR in early postnatal mice and, when the animals were 5 weeks old, analyzed their transcriptomes. In the mice lacking ERR, there was a reduction in expression of genes involved in ion channeling and handling, fatty acid oxidation (the major metabolic process of the adult heart) as well as adult versions of contractile proteins compared with that seen in control animals. By contrast, expression of genes encoding fetal contractile proteins and factors involved in glycolysis—the metabolic pathway characteristic of fetal hearts—was upregulated. In heart failure, cardiomyocytes can revert to fetal-like cells. The authors, therefore, suggest that boosting ERR might be a way to counteract such pathology as well as a way to induce and study cardiomyocyte maturation in cultured progenitor cells.CIRP Governs the Heart Rate Response to Stress (p 1706)Xie et al discover a factor that keeps heart rate under control during stress.Download figureDownload PowerPointDuring the acute stress response (also known as fight-or-flight), heart rate increases rapidly due to the effect of adrenergic signaling on the cells of the sinoatrial node (SAN)—the heart’s pacemaker. Within SAN cells, levels of the signaling factor cAMP ramp up, which in turn increases the cells’ calcium handling and firing rates. But, a racing heartbeat can be damaging if excessive or prolonged, so what keeps the system in check? Xie and colleagues now show that cold-induced RNA-binding protein (CIRP) puts the brakes on by limiting cAMP levels. In rats that lack CIRP, the team showed, adrenergic signaling (treatment with isoproterenol) caused hearts to beat faster for longer than it did in wild-type animals, while basal rates were comparable. Cardiac tissue from the CIRP-lacking animals showed higher-than-usual levels of cAMP after isoproterenol treatment, and this was due to lower-than-usual levels of phosphodiesterase (PDE), the enzyme that normally degrades cAMP. The team went on to show that CIRP normally binds and stabilizes PDE messenger RNA ensuring a ready supply of the enzyme to restrain cAMP signaling. As well as revealing this crucial control mechanism, the work highlights CIRP as a potential new target for future heart-rate–lowering medications, the authors say. Previous Back to top Next FiguresReferencesRelatedDetailsRelated articlesAssociation of Inpatient Use of Angiotensin-Converting Enzyme Inhibitors and Angiotensin II Receptor Blockers With Mortality Among Patients With Hypertension Hospitalized With COVID-19Peng Zhang, et al. Circulation Research. 2020;126:1671-1681 June 5, 2020Vol 126, Issue 12 Advertisement Article InformationMetrics © 2020 American Heart Association, Inc.https://doi.org/10.1161/RES.0000000000000397 Originally publishedJune 4, 2020 PDF download Advertisement

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.

Ivabradine in Postural Orthostatic Tachycardia Syndrome: A Review of the Literature.

Postural orthostatic tachycardia syndrome (POTS) is an autonomic disorder characterized by symptoms such as palpitations, dyspnea, chest discomfort, and lightheadedness affecting various systems. The pathophysiology of POTS is not completely understood due to a variety of symptoms showing that the disease is multifactorial. There is no approved uniform management strategy for POTS and hence, no drug has been approved by the United States (US) Food and Drug Administration (FDA) for it. Ivabradine is an FDA-approved drug for stable symptomatic heart failure (HF) and patients with an ejection fraction (EF) of ≤35%. Previous studies have depicted improvement in symptoms of POTS with the use of ivabradine. It is a selective inhibitor of funny sodium channels (If) in the sinoatrial (SA) node cells resulting in the prolongation of the slow diastolic depolarization (phase IV) and reduction in the heart rate (HR). Although beta-adrenoceptor blockers are commonly used to lower HR in patients with POTS, they are less ideal due to numerous adverse effects. This review aims to provide a comprehensive and up-to-date picture of all the studies and case reports that utilized ivabradine for the treatment of POTS along with a precise overview of epidemiology, pathophysiology, and types of POTS. To conclude, we recommend further research on the effectiveness of ivabradine in patients who experience symptoms of POTS. Other than stable chronic angina pectoris, its application in this setting has been proven to be effective and safe. Further evaluation by means of randomized control trials is required to encourage use of this HR-lowering agent in common disorders other than HF and stable angina, i.e. POTS.

Distinct mechanisms mediate pacemaker dysfunction associated with catecholaminergic polymorphic ventricular tachycardia mutations: Insights from computational modeling

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a stress-induced ventricular arrhythmia associated with rhythm disturbance and impaired sinoatrial node cell (SANC) automaticity (pauses). Mutations associated with dysfunction of Ca2+-related mechanisms have been shown to be present in CPVT. These dysfunctions include impaired Ca2+ release from the ryanodine receptor (i.e., RyR2R4496C mutation) or binding to calsequestrin 2 (CASQ2). In SANC, Ca2+ signaling directly and indirectly mediates pacemaker function.We address here the following research questions: (i) what coupled-clock mechanisms and pathways mediate pacemaker mutations associated with CPVT in basal and in response to β-adrenergic stimulation? (ii) Can different mechanisms lead to the same CPVT-related pacemaker pauses? (iii) Can the mutation-induced deteriorations in SANC function be reversed by drug intervention or gene manipulation?We used a numerical model of mice SANC that includes membrane and intracellular mechanisms and their interconnected signaling pathways. In the basal state of RyR2R4496C SANC, the model predicted that the Na+-Ca2+ exchanger current (INCX) and T-type Ca2+ current (ICaT) mediate between changes in Ca2+ signaling and SANC dysfunction. Under β-adrenergic stimulation, changes in cAMP-PKA signaling and the sodium currents (INa), in addition to INCX and ICaT, mediate between changes in Ca2+ signaling and SANC automaticity pauses. Under basal conditions in Casq2−/−, the same mechanisms drove changes in Ca2+ signaling and subsequent pacemaker dysfunction. However, SANC automaticity pauses in response to β–AR stimulation were mediated by ICaT and INa. Taken together, distinct mechanisms can lead to CPVT-associated SANC automaticity pauses. In addition, we predict that specifically increasing SANC cAMP-PKA activity by either a pharmacological agent (IBMX, a phosphodiesterase (PDE) inhibitor), gene manipulation (overexpression of adenylyl cyclase 1/8) or direct manipulation of the SERCA phosphorylation target through changes in gene expression, compensate for the impairment in SANC automaticity. These findings suggest new insights for understanding CPVT and its therapeutic approach.