The objective of this study was to determine how neurons within the right atrial ganglionated plexus (RAGP) and posterior atrial ganglionated plexus (PAGP) interact to modulate right atrial chronotropic, dromotropic, and inotropic function, particularly with respect to their extracardiac vagal and sympathetic efferent neuronal inputs. Surgical ablation of the PAGP (PAGPx) attenuated vagally mediated bradycardia by 26%; it reduced heart rate slowing evoked by vagal stimulation superimposed on sympathetically mediated tachycardia by 36%. RAGP ablation (RAGPx) eliminated vagally mediated bradycardia, while retaining the vagally induced suppression of sympathetic-mediated tachycardia (-83%). After combined RAGPx and PAGPx, vagal stimulation still reduced sympathetic-mediated tachycardia (-47%). After RAGPx alone and after PAGPx alone, stimulation of the vagi still produced negative dromotropic effects, although these changes were attenuated compared with the intact state. Negative dromotropic responses to vagal stimulation were further attenuated after combined ablation, but parasympathetic inhibition of atrioventricular nodal conduction was still demonstrable in most animals. Finally, neither RAGPx nor PAGPx altered autonomic regulation of right atrial inotropic function. These data indicate that multiple aggregates of neurons within the intrinsic cardiac nervous system are involved in sinoatrial nodal regulation. Whereas parasympathetic efferent neurons regulating the right atrium, including the sinoatrial node, are primarily located within the RAGP, prejunctional parasympathetic-sympathetic interactions regulating right atrial function also involve neurons within the PAGP.

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Introduction: Beat-to-beat variability in cycle length exists in spontaneously beating cardiac preparations of varying complexities from the level of the isolated whole heart to the single sinoatrial nodal cell (SANC). The nature of this variability is poorly characterised as are its fundamental physiological origins. Methods: Recordings of spontaneous electrical activity were made from hearts in vivo, during Langendorff-perfusion, and from single SANC. Heart rate variability (HRV) was calculated in the time- and frequency-domains at baseline and in response to pharmacological mediators that interfered with critical processes involved in automaticity (catecholamines, carbachol, ivabradine, zatebradine, ryanodine and thapsigargin). In addition, a novel 2D technique for imaging Ca2+ fluorescence in spontaneously beating, fluo4-AM loaded, patched single sinoatrial nodal cells was developed to investigate the biophysical behaviour of Ca2+ during pacemaking to see if variability in this was responsible for SANC HRV. Results: Under baseline, temperature-stable conditions, levels of HRV were greatest in vivo (human > rat). SANC exhibited slightly lower levels of HRV, whereas HRV levels expressed by Langendorff-perfused hearts were the least (rabbit > rat), although still comprised a significant proportion of the variability witnessed in vivo. Anaesthetising in vivo rabbits decreased HRV to levels similar to those seen in the Langendorff-perfused heart. HRV was decreased by catecholamines and by ryanodine/thapsigargin in the Langendorff heart. Conversely, HRV was increased by carbachol, ivabradine, zatebradine and ryanodine in SANC. Heart rate changes had a marked effect on levels of HRV. 2D Ca2+ imaging of SANC showed that diastolic local Ca2+ releases (LCRs) occurred earlier than previously thought, with early LCRs having characteristics that were distinct from later LCRs. Mean time of occurrence of all the LCRs within a given diastole closely predicted the duration of the cycle. The rate of restitution of the whole cell Ca2+ transient (used as a surrogate for the pumping function of SERCA) in turn closely predicted the mean time of occurrence of LCRs. Tight synchronisation of the electrical activity of the cell with the biophysical behaviour of Ca2+ appeared to predict shorter cycle lengths. Isoprenaline increased LCR amplitude, though did not increase LCR number, size or duration. Isoprenaline caused LCRs to occur earlier, and synchronised their occurrence and the rate of pumping of Ca2+ back into the sarcoplasmic reticulum. Finally, LCRs were found to preferentially recur in certain regions of the cell, dubbed hotspots. Isoprenaline favoured hotspot production. Conclusion: Whilst greatest in vivo, significant HRV exists in spontaneously beating cardiac preparations devoid of a functioning autonomic nervous system. Studies in SANC indicate that the origin of this is likely to be variability in release of LCRs from the SR via ryanodine receptors. This in turn is controlled by SR refilling kinetics via SR Ca2+ pumping. The coupled system of membrane- and Ca2+-pacemaker clocks are so heavily intertwined that myriad factors will come to bear on generating such variability, including the amount of Ca2+ available for pumping and the phosphorylation state of key proteins, to the extent that variability in no one process can take the credit for generating such HRV.

The sinoatrial node is the primary cardiac pacemaker, which is responsible for generating spontaneous depolarisation of cellular membranes, leading to pacemaking action potentials that control the initiation and regulation of the rhythms of the heart. Previous studies in experimental electrophysiology have gathered a large amount of experimental data about the mechanisms of cardiac pacemaking activities at the molecular, ionic and cellular levels, however, the precise mechanisms underlying the genesis of spontaneous pacemaking action potentials still remain controversial. Mathematical models of the electrophysiology provide a unique alternative tool complimentary to experimental investigations, enabling us to analyse the fundamental physiological mechanisms of cardiac pacemaking activities in an efficient way that would be more difficult to conduct in experimental approaches. In this thesis, an integrated model, incorporating the detailed cellular ion channel kinetics, multi-compartment intracellular Ca2+ handling system and cell morphology, was developed for simulating the spontaneous pacemaking action potentials as well as the stochastic nature of local Ca2+ dynamics in the murine SA node cells. By using the model, the ionic mechanisms underlying the automaticity of primary cardiac pacemaking cells were investigated, the individual role of the ‘membrane clock’ (the cell membrane events) and ‘Ca2+ clock’ (intracellular Ca2+ activities) on generating the pacemaking action potentials were examined. In addition, the model also considered the regulation of the autonomic nervous systems on cardiac pacemaking action potentials. For the first time, competitive regulation of electrical action potentials of the murine SA node cells by the circadian sympathetic and parasympathetic systems during 24-hours were investigated. Furthermore, the individual role of the neurotransmitters, ACh- and ISO-induced actions on variant ion channel and Ca2+ handling in regulating cardiac pacemaking action potentials were also analysed. At the tissue level, an anatomically detailed 2D model of the intact SA node and atrium was developed to investigate the ionic mechanisms underlying sinus node dysfunctions in variant genetic defect conditions. Effects of these genetic defects in impairing cardiac pacemaker ability in pacing and driving the surrounding atrium as seen in the sinus node dysfunction were investigated.

Le canal calcique de libération du Ca2+, appelé récepteur à la ryanodine (RyR) est localisé dans la membrane du réticulum sarcoplasmique des cardiomyocytes, en incluant ceux du pacemaker, et a un rôle important dans le couplage excitation contraction et la génération du rythme cardiaque. Des mutations dans leur gène sont responsables de la tachycardie catécholergique (CPVT), qui est une maladie létale, manifestée par des syncopes ou mort subite lors de stress émotionnel ou physique. Au repos, ces patients ont un électrocardiogramme normal, mais une tendance plus importante à la bradycardie.Nos collaborateurs ont identifié la mutation RyR2R420Q dans une famille espagnole atteinte de CPVT. Nous avons construit une souris portant cette mutation et étudié l’activité du nœud sinoatrial (NSA, pacemaker principale) afin d’élucider les mécanismes.Nous avons trouvé que les cellules du NSA présentent une activité spontanée plus lente que les souris sauvages (WT). Dans la cellule in situ, on peut étudier l’activité des RyRs par l’analyse des « sparks » Ca2+, qui sont des évenements élémentaires produits par l’activation d’un cluster des RyRs. Nos analyses en microscopie confocale sur des NSA disséquées on montré que la fréquence des sparks Ca2+ était légèrement augmentée. Par contre, la longueur de ces sparks est fortement prolongée dans les cellules KI. Ceci produit une libération plus importante de Ca2+ pendant la diastole dans les cellules KI qui réduit l’automatisme, en réduisant la charge en Ca2+ du réticulum sarcoplasmique et en inactivant le courant calcique type L. Donc les thérapies en étude qi favoriseraient la stabilisation du RyR2 en état fermé pourraient ne pas Être efficaces, et il faudra plutôt essayer des thérapies qui faciliteraient la fermeture du canal, une fois il est ouvert. / The cardiac type-2 ryanodine receptor (RyR2) encodes a Ca2+ release channel on sarcoplasmic reticulum (SR) membrane in cardiomyocytes, including sinoatrial node (SAN) myocytes, and releases Ca2+ required for contraction and SAN spontaneous rhythm. Its genetic defects are related to catecholaminergic polymorphic ventricular tachycardia (CPVT), which is a lethal heritable disease characterized by exercise/stress-induced syncope and/or sudden cardiac death. Interestingly, CPVT patients frequently present SAN dysfunction as bradycardia at rest.In a previous study, a novel CPVT-related RyR2 mutation (RyR2R420Q) in a Spanish family, associated with SAN dysfunction was reported. R420 is located at the N-terminal portion of the channel and seems to be an important site for maintaining a stable A/B/C domain of N-terminus in RyR2. As N-terminal mutation resultant RyR2 behaviour and SAN function are never analyzed before, we created the KI mice model bearing mutation R420Q to understand the underlying mechanism.In this thesis, we found increased Ca2+ release during diastole, indicating a gain-of-function effect of RyR2 N-terminal mutation R420Q. Interestingly, this defect may not be only an enhanced activity, as the Ca2+ sparks frequency was only slightly increased in KI, but also the closing mechanism, producing longer Ca2+ sparks. That is, the number of Ca2+ sparks is increased by the RyR2R420Q mutation, and meanwhile the amount of Ca2+ released in each Ca2+ spark is also dramatically enhanced. This increased Ca2+ release retards SR Ca2+ replenishment, disrupting the Ca2+ clock and the coupled clock, resulting in the slower SAN function. Thus favouring RyR stabilization in the closing state might not be an adequate therapy but accelerating its closure.

Rationale: Cardiac function is under exquisite intrinsic cardiac neural control. Neuroablative techniques to modulate control of cardiac function are currently being studied in patients, albeit with variable and sometimes deleterious results. Objective: Recognizing the major gaps in our understanding of cardiac neural control, we sought to evaluate neural regulation of impulse initiation in the sinoatrial node (SAN) as an initial discovery step. Methods and Results: We report an in-depth, multiscale structural and functional characterization of the innervation of the SAN by the right atrial ganglionated plexus (RAGP) in porcine and human hearts. Combining intersectional strategies, including tissue clearing, immunohistochemical, and ultrastructural techniques, we have delineated a comprehensive neuroanatomic atlas of the RAGP-SAN complex. The RAGP shows significant phenotypic diversity of neurons while maintaining predominant cholinergic innervation. Cellular and tissue-level electrophysiological mapping and ablation studies demonstrate interconnected ganglia with synaptic convergence within the RAGP to modulate SAN automaticity, atrioventricular conduction, and left ventricular contractility. Using this approach, we comprehensively demonstrate that intrinsic cardiac neurons influence the pacemaking site in the heart. Conclusions: This report provides an experimental demonstration of a discrete neuronal population controlling a specific geographic region of the heart (SAN) that can serve as a framework for further exploration of other parts of the intrinsic cardiac nervous system (ICNS) in mammalian hearts and for developing targeted therapies.

Lau Chui Pik. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 104-122). / Abstracts in English and Chinese. / Chapter Chapter 1 --- --- Introduction --- p.1 / Chapter 1.1 --- Importance of sinoatrial node in heart functions --- p.3 / Chapter 1.2 --- The importance of Adenosine 5'-triphosphate (ATP) --- p.5 / "ATP as a neurotransmitter, cotransmitter and neuromodulator" --- p.5 / Role of ATP in the heart --- p.7 / Chapter 1.3 --- Importance of delayed rectifier potassium channels (Ik) in the heart --- p.9 / Delayed rectifier potassium channel --- p.10 / Properties of Ik channels in the sinoatrial nodal (SAN)cells --- p.11 / Importance of Ik on heart function --- p.14 / Chapter 1.4 --- Drug/hormone/neurotransmitter modulation of Ik --- p.15 / Drugs modulations of Ik --- p.15 / Hormones/neurotransmitters modulations of Ik --- p.18 / Chapter 1.5 --- Problems encountered in using extracellular ATP on experiments --- p.23 / Chapter 1.6 --- Classification of P2-purinergic receptors --- p.24 / Major nucleotide receptors --- p.24 / p2X receptors --- p.26 / p2Y receptors --- p.28 / 1.7Objectives of the experiment --- p.30 / Chapter Chapter 2 --- --- Materials & Methods --- p.31 / Chapter 2.1 --- Materials --- p.32 / Chapter 2.1.1 --- Solutions --- p.32 / Chapter 2.1.2 --- Enzymes --- p.34 / Chapter 2.1.3 --- Drugs --- p.34 / Chapter 2.2 --- Methods --- p.35 / Chapter 2.2.1 --- Isolation of guinea pig SAN cells --- p.35 / Chapter 2.2.2 --- Identification of SAN region --- p.36 / Chapter 2.2.3 --- Obtaining of single SAN cells --- p.38 / Chapter 2.2.4 --- Preparation of micro-pipettes --- p.40 / Chapter 2.2.5 --- The Patch Clamp Technique --- p.40 / Recording configurations --- p.41 / Electrical recordings --- p.44 / Formation of gigaseal on cell membrane and the development of whole-cell configuration --- p.45 / The changing of bathing solution and addition of drugs --- p.46 / The voltage clamp protocol --- p.47 / Data acquisition and analysis --- p.48 / Statistics --- p.48 / Chapter Chapter 3 --- --- Results --- p.49 / Chapter 3.1 --- The modulatory effect of different concentrations of [ATP]0 on IKs in guinea pig SAN cells --- p.50 / Chapter 3.1.1 --- Characterization of IKs currents --- p.50 / Chapter 3.1.2 --- Stimulatory effect of extracellular A TP on IKs current --- p.51 / Chapter 3.1.3 --- Current-Voltage relationship of ATP on IKs current --- p.57 / Chapter 3.1.4 --- Percentage increase of IKs current in the presence of different [ATP] o --- p.63 / Chapter 3.2 --- Investigation on whether the enhancement effect on IKs is due to ATP or its metabolite adenosine --- p.71 / Chapter 3.2.1 --- Effect of 100 μMATP-γS and adenosine on IKs --- p.71 / Chapter 3.2.2 --- Percentage increase of IKs in the presence of adenosine and ATP-γS --- p.76 / Chapter 3.3 --- Investigation on whether or not G-protein signalling pathway involved in ATP-mediated response on SAN IKs --- p.80 / Chapter 3.3.1 --- Effects of GTP-γS alone on IKs --- p.80 / Chapter 3.3.2 --- Effect of 100 μM ATP in the presence of GTP-yS on IKs --- p.83 / Chapter Chapter 4 --- --- Discussion --- p.86 / Chapter 4.1 --- The modulatory effect of different concentrations of [ATP]0 on IKs in guinea pig SAN cells --- p.87 / Chapter 4.2 --- Investigation on whether the enhancement effect on IKs is due to ATP or its metabolite adenosine --- p.92 / Chapter 4.3 --- Investigation on whether or not G-protein signalling pathway involved in ATP-mediated response on SAN IKs --- p.97 / Chapter 4.4 --- Limitations of this study --- p.102 / Chapter 4.5 --- Future studies --- p.102 / Chapter Chapter 5 --- --- References --- p.104

Submitted by Natalia Vieira (natalia.vieira@famerp.br) on 2016-05-20T17:54:02Z No. of bitstreams: 1 danielleliliadantastukamoto_dissert.pdf: 6750015 bytes, checksum: a409b5df80783b77a0948c423b3fbcb5 (MD5) / Made available in DSpace on 2016-05-20T17:54:02Z (GMT). No. of bitstreams: 1 danielleliliadantastukamoto_dissert.pdf: 6750015 bytes, checksum: a409b5df80783b77a0948c423b3fbcb5 (MD5) Previous issue date: 2015-06-12 / Introduction: The frequency of events of total anomalous pulmonary venous connection (TAPVC) varies in the different series from 5.9 to 7.1 / 100 000 live births, representing 1 to 5% of cases of congenital heart disease. Since the current care has developed; the need to approach possible late complications, such as arrhythmias, is important. Objective: To evaluate the distribution of arrhythmias and possible related variables in patients in the late postoperative TAPVC. Method: Medical record review and clinical and noninvasive arrhythmia evaluation were used for data analysis of 20 patients who underwent isolated TAPVC. Statistical analysis for the detection of related variables. Results: Clinical and echocardiographic evaluations showed no significant changes. Of the 20 patients, 13 (65%) showed some abnormalities for age according to the 12-led EGC. By Holter, six patients (30%) presented atrial and / or ventricular ectopy with higher frequency than mild. Junctional rhythm and 2: 1 atrioventricular occurred in one patient (5%). Analyzing the criteria for sinus node dysfunction, bradycardia was observed by Holter in 30% of the patients. There were no pauses longer than two seconds. By Ergometric test, 82% of children had chronotropic deficit. Follow-up more than 60 months and abnormal ECG were related to the presence of arrhythmias. Conclusion: The long term follow-up of patients undergoing anomalous pulmonary venous connection showed that regardless of the good clinical and echocardiographic results, the possibility of occurrence of atrial and / or ventricular arrhythmias highlight attention to the need for maintenance of regular clinical evaluations. / Introdução: A frequência de ocorrência da conexão anômala total de veias pulmonares (CATVP) varia nas diversas séries entre 5,9 a 7,1/100 mil nascidos vivos, representando 1 a 5 % dos casos de cardiopatia congênita. A evolução nos cuidados atuais leva à necessidade de abordagem de eventuais complicações tardias, como as arritmias. Objetivo: Avaliar a distribuição das arritmias e possíveis variáveis relacionadas em pacientes em pós operatório tardio de CATVP. Método: Análise dos dados de 20 pacientes operados de CATVP isolada, com revisão de prontuário e avaliação clínica e não invasiva de arritmias. Estudo estatístico para detecção de variáveis relacionadas. Resultados: As avaliações clínica e ecocardiográfica não evidenciaram alterações significativas. O eletrocardiograma (ECG) de 12 derivações apresentou anormalidades para a idade em 13 dos 20 pacientes estudados (65%). Ao Holter, 6 pacientes (30%) apresentaram ectopias atriais e/ou ventriculares de frequência maior que discreta. Ritmo juncional e bloqueio atrioventricular 2:1 aconteceram em um paciente (5%). Analisando-se os critérios para disfunção do nó sinusal, observou-se bradicardia ao holter em 30% dos pacientes. Não houve pausas maiores que 2 segundos. Ao teste ergométrico, 82% das crianças apresentaram déficit cronotrópico. Seguimento maior que 60 meses e ECG anormal relacionaram-se à presença de arritmias. Conclusão: O seguimento em longo prazo de pacientes operados de conexão anômala total de veias pulmonares mostrou que independentemente dos bons resultados clínicos e ecocardiográficos, existe a possibilidade de ocorrência de arritmias atriais e/ou ventriculares, chamando a atenção para a necessidade de manutenção de avaliações clinicas regulares.

Background: The Wnt/β-catenin signalling pathway is activated in arrhythmogenic heart diseases such as myocardial infarction and heart failure, but it is unclear if the pathway regulates cardiac ion channels and thus may play a role in arrhythmogenesis. Previous PCR array screening from our lab showed that the transcript level of the T-type calcium channel gene Cacna1g was reduced in primary culture of neonatal rat ventricular myocytes (NRVMs) after activation of Wnt/β-catenin signalling with Wnt3a protein (100 ng/ml) or a small molecule activator of the pathway, CHIR (3 µM) (n=3, p<0.01). In this study, we examined the effects of Wnt/β-catenin signalling on T-type calcium channels (Caᴠ3.1), which play a key role in the pacemaker function of the sinoatrial node (SAN). Results: RT-qPCR and western blot demonstrated dose-dependent reductions in Cacna1g mRNA (n=7, p<0.01) and Cav3.1 protein (n=4, p<0.01) in NRVMs after treatment with CHIR (3 μM). There was also a decrease in Cacna1g mRNA in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) after treatment with CHIR (5 μM) (n=4; p<0.001). Patch-clamp recording demonstrated reduced T-type calcium current (ICa,T) in NRVMs after Wnt3a treatment (3 μg/ml) (n=5, p<0.05). In isolated mouse SAN tissue, perfusion with an ICa,T blocker, ML-218 (30 µM), led to dose-dependent reductions in spontaneous beating rate (n=4, p<0.0001) indicating a critical role of ICa,T in SAN pacemaking. In adult rats, activation of Wnt/β-catenin signalling through the application of CHIR in a poloxamer gel to the SAN region did not alter the in vivo heart rate in electrocardiogram (ECG) (n=8, p=0.12). However, ex vivo culture of SAN tissue from the in vivo experiments revealed a reduction intrinsic beating rate in the CHIR treated group (n=7) compared to the control (DMSO) (n=8) (p<0.05). Summary: Wnt/β-catenin signalling inhibits T-type Ca²⁺ current in cardiomyocytes by, at least partly, reduced Cacna1g mRNA and Cav3.1 protein. Activation of Wnt/β-catenin signalling reduces the intrinsic heart rate likely by inhibition of T-type Ca²⁺ current in SAN pacemaker cells.

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