Background: Sick sinus syndrome (SSS) is common and may require electronic pacemaker implantation, gene therapy (biopacemaking) may be an alternative. For SSS, repair may be better than the generation of a de novo biopacemaker, due to the complex nature of the sinoatrial node (SAN). We hypothesised that an ex vivo model of SSS could be created by the identification of a subsidiary pacemaker in the SAN region, and that expression of a pacemaker channel (HCN4 or HCN212) in this region would accelerate the pacing rate. Methods: A bradycardic SSS model was generated by the removal of the upper two thirds of a rat SAN and a system to record the intrinsic activity during tissue culture was developed. The leading pacemaker site of the SSS preparations were identified by activation mapping then characterised by If blockade, β-adrenergic stimulation, histology and immunohistochemistry. Further SSS preparations were injected in this region with recombinant adenovirus (RAd) expressing no functional ion channel (Ad5-GFP or Ad5-GFP-HCN4Δ); or RAd expressing a functional If channel (Ad5-HCN212 or Ad5-PREK-HCN4). During tissue culture electrical activity was monitored using bipolar electrodes. Results: Tissue culture and monitoring of the rat SAN is feasible and does not induce significant changes in HCN4 or connexin-43 expression. The uninjected SSS preparations displayed a slower rate than the control SAN (p<0.001). In 5/6 cases the subsidiary pacemaker was HCN4 -ve and Connexin-43 +ve (in contrast to the SAN) and was close to the superior aspect of the inferior vena cava. The cell size of the subsidiary pacemaker was comparable to that of the SAN and smaller than working myocardium (p<0.001). Pacing was responsive to β-adrenergic stimulation and was partially dependent on If current. The pacing rate of the AdHCN212-injected SSS preparations was significantly faster than the uninjected SSS preparations (p<0.001). The remaining RAd did not significantly affect the pacing rate of the SSS model. Conclusions: There is subsidiary pacemaker tissue close to the superior aspect of the IVC that shares some characteristics of the SAN. These results suggest that adenovirus-mediated expression of HCN channels in subsidiary pacemaker tissue of the right atrium may be a useful strategy in biopacemaking for SSS.

The sinus node (SN) is the natural pacemaker of the heart. In the human, the SN is surrounded by the paranodal area (PNA), the function of which is currently unknown. The PNA may act as subsidiary atrial pacemakers (SAP) and become the dominant pacemaker during sinus node dysfunction (SND). Creation of an animal model of SND allows characterisation of SAP, which can be a target for novel treatment strategies other than the currently available electronic pacemakers. I developed a large animal model of SND by ablating the SN in the goat and validated it by mapping the location of the newly emergent SAP. Functional characterisation of the SAP revealed reduced atrioventricular (AV) conduction time consistent with a location of the SAP close to the AV junction. SAP recovery time showed an initially significant prolongation compared to the SN recovery time, followed by a gradual decrease over 4 weeks. SAP pauses, and temporary reliance on electronic pacemaker activity have also been demonstrated then disappeared over time, suggesting possible modulation, maturation of the SAP. Structural characterisation of the SN revealed an extensive pacemaking complex within the right atrium (RA); the SN was surrounded by the PNA, extending down to the inferior vena cava (IVC) and into the interatrial groove. The PNA had a histological appearance that is intermediate to the SN and the RA. 3D reconstruction demonstrated, for the first time in a large animal model, an extensive and almost complete circle of pacemaking tissue at the junction of the embryologically different sinus venosus and the muscular right atrium. The SAP emerged in a location close to the IVC along the crista terminalis. Expression of key ion channel proteins in the SAP showed abundance of the pacemaker channel (HCN4) and the sodium/calcium exchanger (NCX1) compared to RA, similar to the expression pattern of the SN. The expression of the main high conductance connexin (Cx43) was not significantly different between SAP and RA, and both expressed Cx43 more abundantly than the SN.Conclusion: Destruction of the sinus node in this experimental model resulted in the generation of chronic SAP activity in the majority of the animals. The SAP displayed maturation over time and located in the inferior part of the RA, in the same area where the PNA was found in controls, suggesting the role of PNA as the dominant pacemaker in sinus node dysfunction. The SAP in the goat constitutes a promising stable target for electrophysiological modification to construct a fully functioning biological pacemaker.

Background: Biopacemaking is the attempt to replicate sinoatrial node (SAN)-like pacemaker activity in other areas of the heart by manipulating genes involved in pacemaking. Application of this could emulate the electronic pacemaker without the need for implantation of permanent hardware, or directly repair dysfunctional SAN tissue in human disease. We upregulated the transcription factors Tbx18, Tbx3 and the membrane ion exchanger NCX1 in bradycardic subsidiary atrial pacemaker (SAP) tissue which we used as a model of SAN dysfunction. We aimed to show that one or more of these gene targets could improve pacemaker function and alter the molecular character of SAP tissue and thus could potentially be used for the repair of dysfunctional SAN tissue. Methods: SAP tissue was isolated from the right atria of rats and kept beating in culture at 37°C for 48 hours. Recombinant adenoviruses were injected into SAP preparations to upregulate Tbx18, Tbx3 and NCX1 individually. Beating rate, overdrive suppression and pharmacological response to If blockade and β-adrenergic stimulation were measured along with molecular changes in pacemaker and atrial genes and proteins using RT-qPCR and immunohistochemistry. Results: Tbx18 upregulation significantly increased SAP beating rate after 48 hours of culture (a final rate of 141 ± 9 bpm in uninfected SAP tissue versus 215 ± 16 bpm in Ad-Tbx18 infected SAP tissue, p<0.01). It induced upregulation of HCN2 (p<0.01) and RYR2 (p<0.05), downregulation of HCN4 (p<0.05) and no change HCN1, Tbx3, Kv1.5, Kir2.1, Nav1.5, NCX1, Cx43, Cx45, Cav1.2 or Cav3.1. There was also no change in overdrive suppression and no change in response to pharmacology. No increase in beating rate was seen with either Tbx3 or NCX1 upregulation. Tbx3 preparations induced downregulation of the atrial genes Kir2.1 (p<0.01) and Nav1.5 (p<0.05), along with HCN1 (p<0.05), HCN4 (p<0.01), Tbx18 (p<0.05) and NCX1 (p<0.01), upregulated Cx43 (p<0.05) and showed no change in Cx45, RYR2, Kv1.5. NCX1 preparations demonstrated reduced overdrive suppression (p<0.05). Conclusion: Tbx18 showed the most potential for biopacemaking in SAP tissue, however both Tbx3 and NCX1 could be applied as secondary targets to fine tune biopacemaker function. Future work would focus on applying these targets to dysfunctional SAN tissue in larger animals.

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.

X-Linked Sinus Node Dysfunction and Atrial Fibrillation. Introduction: Atrial fibrillation (AF) is a heritable disorder with male predilection, suggesting a sex chromosome defect in certain patients. Loss-of-function truncation mutations in EMD, encoding the nuclear membrane protein emerin, cause X-linked Emery-Dreifuss muscular dystrophy (EDMD) characterized by localized contractures and skeletal myopathy in adolescence, sinus node dysfunction (SND) in early adulthood, and atrial fibrillation as a variably associated trait. This study sought to identify the genetic basis for male-restricted, nonsyndromic sinus node dysfunction and AF in a multigenerational family. Methods and Results: Genealogical and medical records, and DNA samples, were obtained. Progressive SND and AF occurred in four males related through maternal lineages, consistent with X-linked inheritance. Skeletal myopathy was absent, even at advanced ages. Targeted X chromosome genotyping mapped the disease locus to Xq28, implicating EMD as a positional candidate gene. DNA sequencing revealed hemizygosity for an in-frame 3-bp deletion in EMD (Lys37del) in affected males, disrupting a residue within the LEM binding domain critical for nuclear assembly but leaving the remainder of the protein intact. Buccal epithelial cell staining with emerin antibody demonstrated near-total functional loss of emerin. Female relatives underwent prospective electrocardiographic and genetic testing. Those heterozygous for Lys37del had ∼50-70% emerin-positive nuclei and variable degrees of paroxysmal supraventricular arrhythmia. Conclusions: Mutation of EMD can underlie X-linked familial AF. Lys37del is associated with epithelial cell emerin deficiency, as in EDMD, yet it causes electrical atriomyopathy in the absence of skeletal muscle disease. Targeted genetic testing of EMD should be considered in patients with SND-associated AF and/or family history suggesting X-linked inheritance.

Bradyarrhythmias are common clinical findings consisting of physiologic and pathologic conditions (sinus node dysfunction and atrioventricular [AV] conduction disturbances). Bradyarrhythmias can be benign, requiring no treatment; however, acute unstable bradycardia can lead to cardiac arrest. In patients with confirmed or suspected bradycardia, a thorough history and physical examination should include possible causes of sinoatrial node dysfunction or AV block. Management of bradycardia is based on the severity of symptoms, the underlying causes, presence of potentially reversible causes, presence of adverse signs, and risk of progression to asystole. Pharmacologic therapy and/or pacing are used to manage unstable or symptomatic bradyarrhythmias.

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Atrial fibrillation and atrial flutter are the most common sustained arrhythmias, however their underlying mechanisms are yet to be fully characterised. This thesis evaluates the electrophysiological and electroanatomical substrate of the atria in patients with these arrhythmias. Experimental studies of atrial fibrillation have demonstrated effective refractory period shortening and conduction slowing as a result of atrial fibrillation giving rise to the concept that "atrial fibrillation begets atrial fibrillation". However, cardioversion to prevent electrical remodelling does not prevent progression of disease, suggesting a "second factor" drives this process. Chapters 2 and 3 evaluate the atrial substrate in patients with "lone" atrial fibrillation. These studies demonstrate such patients, remote from an arrhythmic event, have prolongation of atrial refractoriness, conduction slowing, impairment of sinus node function, site-specific conduction delay, lower voltage and a greater proportion of complex electrograms compared to reference patients. These abnormalities constitute the "second factor" critical to the development and progression of atrial fibrillation. Atrial flutter has a close inter-relationship with atrial fibrillation and these rhythms frequently co-exist. Atrial fibrillation often occurs in patients with heart disease known to demonstrate abnormal atrial substrate; whether similar substrate exists in patients with atrial flutter to account for the co-existence of both arrhythmias is unknown. Chapters 4 and 5 evaluate the atrial substrate in patients with atrial flutter, remote from arrhythmia, demonstrating structural abnormalities characterised by loss of myocardial voltage, conduction slowing and impaired sinus node function, without reduction in atrial refractoriness. These findings implicate a common substrate as the cause of the close inter-relationship between these arrhythmias. There is a frequent association between atrial arrhythmia and sinus node disease for which several mechanisms have been postulated. In addition, there is a size discrepancy between the anatomical sinus node and the much larger functional sinus node complex. little is known about normal sinus node function or the effects of remodelling due to arrhythmia. Chapter 6 characterises sinus node activation to determine the nature and extent of the functional sinus node complex in patients with and without chronic atrial flutter. The functional sinus node complex demonstrates dynamic shifts in activation with preferential pathways of conduction to atrial myocardium. Patients with atrial flutter demonstrate lesser voltage, longer conduction times along preferential pathways and a smaller functional sinus node complex. These findings provide insights into the function of the human sinus node in health and disease. Sites of complex fractionated atrial electrograms and highest dominant frequency are implicated in maintaining atrial fibrillation. Chapter 7 determines the minimum recording duration that accurately characterises electrogram complexity and activation frequency. An electrogram duration of 5 seconds is required to accurately identify these sites. Chapter 8 evaluates the relationship between sites of fractionation and high frequency activation during atrial fibrillation. Greater fractionation and higher dominant frequency are seen in persistent atrial fibrillation and left atria. Preferential areas of high dominant frequency are observed in paroxysmal but not persistent atrial fibrillation. Areas of complex fractionated atrial electrograms are found adjacent to sites of high dominant frequency. / Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2009

Symptomatic sinus bradycardia, due to either sick sinus syndrome or vagotonia, can be familial, affecting several members of a family. We report an 18-year-old male patient with palpitations and limited exercise capacity who was noted to have severe sinus bradycardia. His resting heart rate was 40/min, with normal PR and corrected QT intervals, and sinus pauses up to 6 seconds during sleep. Exercise treadmill test and pharmacologic autonomic blockade during electrophysiologic studies abolished the bradycardia, suggestive of vagotonia rather than intrinsic sinus node dysfunction. This patient's father and a female cousin had a similar clinical history but associated with syncope and severe sinus bradycardia. The mode of transmission appeared to be autosomal dominant. All three have permanent demand pacemakers implanted and are asymptomatic.

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