Intrinsic Electrical Remodeling Underlies Atrioventricular Block in Athletes.

[Figure: see text].

MicroRNAs: Midfielders of Cardiac Health, Disease and Treatment.

MicroRNAs (miRNAs) are a class of small non-coding RNA molecules that play a role in post-transcriptional gene regulation. It is generally accepted that their main mechanism of action is the negative regulation of gene expression, through binding to specific regions in messenger RNA (mRNA) and repressing protein translation. By interrupting protein synthesis, miRNAs can effectively turn genes off and influence many basic processes in the body, such as developmental and apoptotic behaviours of cells and cardiac organogenesis. Their importance is highlighted by inhibiting or overexpressing certain miRNAs, which will be discussed in the context of coronary artery disease, atrial fibrillation, bradycardia, and heart failure. Dysregulated levels of miRNAs in the body can exacerbate or alleviate existing disease, and their omnipresence in the body makes them reliable as quantifiable markers of disease. This review aims to provide a summary of miRNAs as biomarkers and their interactions with targets that affect cardiac health, and intersperse it with current therapeutic knowledge. It intends to succinctly inform on these topics and guide readers toward more comprehensive works if they wish to explore further through a wide-ranging citation list.

Repolarizing potassium currents in working myocardium of Japanese quail: a novel translational model for cardiac electrophysiology.

Birds developed endothermy and four-chambered high-performance heart independently from mammals. Though avian embryos are extensively studied and widely used as various models for heart research, little is known about cardiac physiology of adult birds. Meanwhile, cardiac electrophysiology is in search for easily accessible and relevant model objects which resemble human myocardium in the pattern of repolarizing currents (IKr, IKs, IKur and Ito). This study focuses on the configuration of electrical activity and electrophysiological phenotype of working myocardium in adult Japanese quails (Coturnix japonica). The resting membrane potential and action potential (AP) waveform in quail atrial myocardium were similar to that in working myocardium of rodents. Using whole-cell patch clamp and sharp glass microelectrodes, we demonstrated that the repolarization of quail atrial and ventricular myocardium is determined by voltage-dependent potassium currents IKr, IKs and Ito - the latter was previously considered as an exclusive evolutionary feature of mammals. The specific blockers of these currents, dofetilide (3 µmol l-1), HMR 1556 (30 µmol l-1) and 4-aminopyridine (3 mmol l-1), prolonged AP in both ventricular and atrial myocardial preparations. The expression of the corresponding channels responsible for these currents in quail myocardium was investigated with quantitative RT-PCR and western blotting. In conclusion, the described pattern of repolarizing ionic currents and channels in quail myocardium makes this species a novel and suitable experimental model for translational cardiac research and reveals new information related to the evolution of cardiac electrophysiology in vertebrates.

Supraventricular Arrhythmias in Athletes: Basic Mechanisms and New Directions.

Athletes are prone to supraventricular rhythm disturbances including sinus bradycardia, heart block, and atrial fibrillation. Mechanistically, this is attributed to high vagal tone and cardiac electrical and structural remodeling. Here, we consider the supporting evidence for these three pro-arrhythmic mechanisms in athletic human cohorts and animal models, featuring current controversies, emerging data, and future directions of relevance to the translational research agenda.

Targeting miR-423-5p Reverses Exercise Training-Induced HCN4 Channel Remodeling and Sinus Bradycardia.

RATIONALE: Downregulation of the pacemaking ion channel, HCN4 (hyperpolarization-activated cyclic nucleotide gated channel 4), and the corresponding ionic current, If, underlies exercise training-induced sinus bradycardia in rodents. If this occurs in humans, it could explain the increased incidence of bradyarrhythmias in veteran athletes, and it will be important to understand the underlying processes. OBJECTIVE: To test the role of HCN4 in the training-induced bradycardia in human athletes and investigate the role of microRNAs (miRs) in the repression of HCN4. METHODS AND RESULTS: As in rodents, the intrinsic heart rate was significantly lower in human athletes than in nonathletes, and in all subjects, the rate-lowering effect of the HCN selective blocker, ivabradine, was significantly correlated with the intrinsic heart rate, consistent with HCN repression in athletes. Next-generation sequencing and quantitative real-time reverse transcription polymerase chain reaction showed remodeling of miRs in the sinus node of swim-trained mice. Computational predictions highlighted a prominent role for miR-423-5p. Interaction between miR-423-5p and HCN4 was confirmed by a dose-dependent reduction in HCN4 3'-untranslated region luciferase reporter activity on cotransfection with precursor miR-423-5p (abolished by mutation of predicted recognition elements). Knockdown of miR-423-5p with anti-miR-423-5p reversed training-induced bradycardia via rescue of HCN4 and If. Further experiments showed that in the sinus node of swim-trained mice, upregulation of miR-423-5p (intronic miR) and its host gene, NSRP1, is driven by an upregulation of the transcription factor Nkx2.5. CONCLUSIONS: HCN remodeling likely occurs in human athletes, as well as in rodent models. miR-423-5p contributes to training-induced bradycardia by targeting HCN4. This work presents the first evidence of miR control of HCN4 and heart rate. miR-423-5p could be a therapeutic target for pathological sinus node dysfunction in veteran athletes.

Mechanistic insights from targeted molecular profiling of repolarization alternans in the intact human heart.

AIMS: Action potential duration (APD) alternans is an established precursor or arrhythmia and sudden cardiac death. Important differences in fundamental electrophysiological properties relevant to arrhythmia exist between experimental models and the diseased in vivo human heart. To investigate mechanisms of APD alternans using a novel approach combining intact heart and cellular cardiac electrophysiology in human in vivo. METHODS AND RESULTS: We developed a novel approach combining intact heart electrophysiological mapping during cardiac surgery with rapid on-site data analysis to guide myocardial biopsies for laboratory analysis, thereby linking repolarization dynamics observed at the organ level with underlying ion channel expression. Alternans-susceptible and alternans-resistant regions were identified by an incremental pacing protocol. Biopsies from these sites (n = 13) demonstrated greater RNA expression in Calsequestrin (CSQN) and Ryanodine (RyR) and ion channels underlying IK1 and Ito at alternans-susceptible sites. Electrical restitution properties (n = 7) showed no difference between alternans-susceptible and resistant sites, whereas spatial gradients of repolarization were greater in alternans-susceptible than in alternans-resistant sites (P = 0.001). The degree of histological fibrosis between alternans-susceptible and resistant sites was equivalent. Mathematical modelling of these changes indicated that both CSQN and RyR up-regulation are key determinants of APD alternans. CONCLUSION: Combined intact heart and cellular electrophysiology show that regions of myocardium in the in vivo human heart exhibiting APD alternans are associated with greater expression of CSQN and RyR and show no difference in restitution properties compared to non-alternans regions. In silico modelling identifies up-regulation and interaction of CSQN with RyR as a major mechanism underlying APD alternans.

Mechanism underlying impaired cardiac pacemaking rhythm during ischemia: A simulation study.

Ischemia in the heart impairs function of the cardiac pacemaker, the sinoatrial node (SAN). However, the ionic mechanisms underlying the ischemia-induced dysfunction of the SAN remain elusive. In order to investigate the ionic mechanisms by which ischemia causes SAN dysfunction, action potential models of rabbit SAN and atrial cells were modified to incorporate extant experimental data of ischemia-induced changes to membrane ion channels and intracellular ion homeostasis. The cell models were incorporated into an anatomically detailed 2D model of the intact SAN-atrium. Using the multi-scale models, the functional impact of ischemia-induced electrical alterations on cardiac pacemaking action potentials (APs) and their conduction was investigated. The effects of vagal tone activity on the regulation of cardiac pacemaker activity in control and ischemic conditions were also investigated. The simulation results showed that at the cellular level ischemia slowed the SAN pacemaking rate, which was mainly attributable to the altered Na+-Ca2+ exchange current and the ATP-sensitive potassium current. In the 2D SAN-atrium tissue model, ischemia slowed down both the pacemaking rate and the conduction velocity of APs into the surrounding atrial tissue. Simulated vagal nerve activity, including the actions of acetylcholine in the model, amplified the effects of ischemia, leading to possible SAN arrest and/or conduction exit block, which are major features of the sick sinus syndrome. In conclusion, this study provides novel insights into understanding the mechanisms by which ischemia alters SAN function, identifying specific conductances as contributors to bradycardia and conduction block.

Insights from echocardiography, magnetic resonance imaging, and microcomputed tomography relative to the mid-myocardial left ventricular echogenic zone.

BACKGROUND: The anatomical substrate for the mid-mural ventricular hyperechogenic zone remains uncertain, but it may represent no more than ultrasound reflected from cardiomyocytes orientated orthogonally to the ultrasonic beam. We sought to ascertain the relationship between the echogenic zone and the orientation of the cardiomyocytes. METHODS: We used 3D echocardiography, diffusion tensor imaging, and microcomputed tomography to analyze the location and orientation of cardiomyocytes within the echogenic zone. RESULTS: We demonstrated that visualization of the echogenic zone is dependent on the position of the transducer and is most clearly seen from the apical window. Diffusion tensor imaging and microcomputed tomography show that the echogenic zone seen from the apical window corresponds to the position of the circumferentially orientated cardiomyocytes. An oblique band seen in the parasternal view relates to cardiomyocytes orientated orthogonally to the ultrasonic beam. CONCLUSIONS: The mid-mural ventricular hyperechogenic zone represents reflected ultrasound from cardiomyocytes aligned orthogonal to the ultrasonic beam. The echogenic zone does not represent a space, a connective tissue sheet, a boundary between ascending and descending limbs of a hypothetical helical ventricular myocardial band, nor an abrupt change in cardiomyocyte orientation.

Ivabradine protects against ventricular arrhythmias in acute myocardial infarction in the rat.

Ventricular arrhythmias are an important cause of mortality in the acute myocardial infarction (MI). To elucidate effect of ivabradine, pure heart rate (HR) reducing drug, on ventricular arrhythmias within 24 h after non-reperfused MI in the rat. ECG was recorded for 24 h after MI in untreated and ivabradine treated rats and episodes of ventricular tachycardia/fibrillation (VT/VF) were identified. Forty-five minutes and twenty-four hours after MI epicardial monophasic action potentials (MAPs) were recorded, cardiomyocyte Ca(2+) handling was assessed and expression and function of ion channels were studied. Ivabradine reduced average HR by 17%. Combined VT/VF incidence and arrhythmic mortality were higher in MI versus MI + Ivabradine rats. MI resulted in (1) increase of Ca(2+) sensitivity of ryanodine receptors 24 h after MI; (2) increase of HCN4 expression in the left ventricle (LV) and funny current (IF) in LV cardiomyocytes 24 h after MI, and (3) dispersion of MAP duration both 45 min and 24 h after MI. Ivabradine partially prevented all these three potential proarrhythmic effects of MI. Ivabradine is antiarrhythmic in the acute MI in the rat. Potential mechanisms include prevention of: diastolic Ca(2+)-leak from sarcoplasmic reticulum, upregulation of IF current in LV and dispersion of cardiac repolarization. Ivabradine could be an attractive antiarrhythmic agent in the setting of acute MI.

Chronic effects of mild hyperglycaemia on left ventricle transcriptional profile and structural remodelling in the spontaneously type 2 diabetic Goto-Kakizaki rat.

Heart failure in chronic type 2 diabetes mellitus is partly attributable to adverse structural remodelling of the left ventricle (LV), but the contribution of hyperglycaemia (HG) per se in remodelling processes is debated. In this study, we examined the molecular signature of LV remodelling in 18-month-old spontaneously diabetic male Goto-Kakizaki (GK) rats that represent a long-term mildly diabetic phenotype, using histological, immunoblotting and quantitative gene expression approaches. Relative to age-matched Wistar controls, mildly diabetic GK rats presented with LV hypertrophy, increased expression of natriuretic peptides and phosphorylation of pro-hypertrophic Akt. Fibrosis proliferation in the GK LV paralleled increased transcriptional and biologically active pro-fibrogenic transforming growth factor-ß1 (TGFß1) in the LV with upregulated mRNA abundance for key extracellular matrix (ECM) components such as fibronectin, collagen type(s) 1 and 3α and regulators including matrix metalloproteinases 2 and 9, and their tissue inhibitor (TIMP) 4, connexin 43 and α5-integrin. GK rats also presented with altered mRNA expression for cardiac sarcoplasmic reticulum Ca(2+)ATPase, Na(+)/Ca(2+) exchanger and the L-type Ca(2+) channels which may contribute to the altered Ca(2+) transient kinetics previously observed in this model at 18 months of age (t test, p < 0.05 vs. age-matched Wistar control for all parameters). The results indicate that chronic mild HG can produce the molecular and structural correlates of a hypertrophic myopathy. Diffuse ECM proliferation in this model is possibly a product of HG-induced TGFß1 upregulation and altered transcriptional profile of the ECM.

Epistatic rescue of Nkx2.5 adult cardiac conduction disease phenotypes by prospero-related homeobox protein 1 and HDAC3.

RATIONALE: Nkx2.5 is one of the most widely studied cardiac-specific transcription factors, conserved from flies to man, with multiple essential roles in both the developing and adult heart. Specific dominant mutations in NKX2.5 have been identified in adult congenital heart disease patients presenting with conduction system anomalies and recent genome-wide association studies implicate the NKX2.5 locus, as causative for lethal arrhythmias ("sudden cardiac death") that occur at a frequency in the population of 1 in 1000 per annum worldwide. Haploinsufficiency for Nkx2.5 in the mouse phenocopies human conduction disease pathology yet the phenotypes, described in both mouse and man, are highly pleiotropic, implicit of unknown modifiers and/or factors acting in epistasis with Nkx2.5/NKX2.5. OBJECTIVE: To identify bone fide upstream genetic modifier(s) of Nkx2.5/NKX2.5 function and to determine epistatic effects relevant to the manifestation of NKX2.5-dependent adult congenital heart disease. METHODS AND RESULTS: A study of cardiac function in prospero-related homeobox protein 1 (Prox1) heterozygous mice, using pressure-volume loop and micromannometry, revealed rescue of hemodynamic parameters in Nkx2.5(Cre/+); Prox1(loxP/+) animals versus Nkx2.5(Cre/+) controls. Anatomic studies, on a Cx40(EGFP) background, revealed Cre-mediated knock-down of Prox1 restored the anatomy of the atrioventricular node and His-Purkinje network both of which were severely hypoplastic in Nkx2.5(Cre/+) littermates. Steady state surface electrocardiography recordings and high-speed multiphoton imaging, to assess Ca(2+) handling, revealed atrioventricular conduction and excitation-contraction were also normalized by Prox1 haploinsufficiency, as was expression of conduction genes thought to act downstream of Nkx2.5. Chromatin immunoprecipitation on adult hearts, in combination with both gain and loss-of-function reporter assays in vitro, revealed that Prox1 recruits the corepressor HDAC3 to directly repress Nkx2.5 via a proximal upstream enhancer as a mechanism for regulating Nkx2.5 function in adult cardiac conduction. CONCLUSIONS: Here we identify Prox1 as a direct upstream modifier of Nkx2.5 in the maintenance of the adult conduction system and rescue of Nkx2.5 conduction disease phenotypes. This study is the first example of rescue of Nkx2.5 function and establishes a model for ensuring electrophysiological function within the adult heart alongside insight into a novel Prox1-HDAC3-Nkx2.5 signaling pathway for therapeutic targeting in conduction disease.

Empowering artificial intelligence in characterizing the human primary pacemaker of the heart at single cell resolution.

The sinus node (SN) serves as the primary pacemaker of the heart and is the first component of the cardiac conduction system. Due to its anatomical properties and sample scarcity, the cellular composition of the human SN has been historically challenging to study. Here, we employed a novel deep learning deconvolution method, namely Bulk2space, to characterise the cellular heterogeneity of the human SN using existing single-cell datasets of non-human species. As a proof of principle, we used Bulk2Space to profile the cells of the bulk human right atrium using publicly available mouse scRNA-Seq data as a reference. 18 human cell populations were identified, with cardiac myocytes being the most abundant. Each identified cell population correlated to its published experimental counterpart. Subsequently, we applied the deconvolution to the bulk transcriptome of the human SN and identified 11 cell populations, including a population of pacemaker cardiomyocytes expressing pacemaking ion channels (HCN1, HCN4, CACNA1D) and transcription factors (SHOX2 and TBX3). The connective tissue of the SN was characterised by adipocyte and fibroblast populations, as well as key immune cells. Our work unravelled the unique single cell composition of the human SN by leveraging the power of a novel machine learning method.

Morphology of human sinoatrial node and its surrounding right atrial muscle in the global obesity pandemic-does fat matter?

Introduction: The sinus node (SN) is the main pacemaker site of the heart, located in the upper right atrium at the junction of the superior vena cava and right atrium. The precise morphology of the SN in the human heart remains relatively unclear especially the SN microscopical anatomy in the hearts of aged and obese individuals. In this study, the histology of the SN with surrounding right atrial (RA) muscle was analyzed from young non-obese, aged non-obese, aged obese and young obese individuals. The impacts of aging and obesity on fibrosis, apoptosis and cellular hypertrophy were investigated in the SN and RA. Moreover, the impact of obesity on P wave morphology in ECG was also analyzed to determine the speed and conduction of the impulse generated by the SN. Methods: Human SN/RA specimens were dissected from 23 post-mortem hearts (preserved in 4% formaldehyde solution), under Polish local ethical rules. The SN/RA tissue blocks were embedded in paraffin and histologically stained with Masson's Trichrome. High and low-magnification images were taken, and analysis was done for appropriate statistical tests on Prism (GraphPad, USA). 12-lead ECGs from 14 patients under Polish local ethical rules were obtained. The P wave morphologies from lead II, lead III and lead aVF were analyzed. Results: Compared to the surrounding RA, the SN in all four groups has significantly more connective tissue (P ≤ 0.05) (young non-obese individuals, aged non-obese individuals, aged obese individuals and young obese individuals) and significantly smaller nodal cells (P ≤ 0.05) (young non-obese individuals, aged non-obese individuals, aged obese individuals, young obese individuals). In aging, overall, there was a significant increase in fibrosis, apoptosis, and cellular hypertrophy in the SN (P ≤ 0.05) and RA (P ≤ 0.05). Obesity did not further exacerbate fibrosis but caused a further increase in cellular hypertrophy (SN P ≤ 0.05, RA P ≤ 0.05), especially in young obese individuals. However, there was more infiltrating fat within the SN and RA bundles in obesity. Compared to the young non-obese individuals, the young obese individuals showed decreased P wave amplitude and P wave slope in aVF lead. Discussion: Aging and obesity are two risk factors for extensive fibrosis and cellular hypertrophy in SN and RA. Obesity exacerbates the morphological alterations, especially hypertrophy of nodal and atrial myocytes. These morphological alterations might lead to functional alterations and eventually cause cardiovascular diseases, such as SN dysfunction, atrial fibrillation, bradycardia, and heart failure.

Atrioventricular node dysfunction in pressure overload-induced heart failure-Involvement of the immune system and transcriptomic remodelling.

Heart failure is associated with atrioventricular (AV) node dysfunction, and AV node dysfunction in the setting of heart failure is associated with an increased risk of mortality and heart failure hospitalisation. This study aims to understand the causes of AV node dysfunction in heart failure by studying changes in the whole nodal transcriptome. The mouse transverse aortic constriction model of pressure overload-induced heart failure was studied; functional changes were assessed using electrocardiography and echocardiography and the transcriptome of the AV node was quantified using RNAseq. Heart failure was associated with a significant increase in the PR interval, indicating a slowing of AV node conduction and AV node dysfunction, and significant changes in 3,077 transcripts (5.6% of the transcriptome). Many systems were affected: transcripts supporting AV node conduction were downregulated and there were changes in transcripts identified by GWAS as determinants of the PR interval. In addition, there was evidence of remodelling of the sarcomere, a shift from fatty acid to glucose metabolism, remodelling of the extracellular matrix, and remodelling of the transcription and translation machinery. There was evidence of the causes of this widespread remodelling of the AV node: evidence of dysregulation of multiple intracellular signalling pathways, dysregulation of 109 protein kinases and 148 transcription factors, and an immune response with a proliferation of neutrophils, monocytes, macrophages and B lymphocytes and a dysregulation of 40 cytokines. In conclusion, inflammation and a widespread transcriptional remodelling of the AV node underlies AV node dysfunction in heart failure.

Remodelling and dysfunction of the sinus node in pulmonary arterial hypertension.

Patients with pulmonary arterial hypertension (PAH) have a high burden of arrhythmias, including arrhythmias arising from sinus node dysfunction, and the aim of this study was to investigate the effects of PAH on the sinus node. In the rat, PAH was induced by an injection of monocrotaline. Three weeks after injection, there was a decrease of the intrinsic heart rate (heart rate in the absence of autonomic tone) as well as the normal heart rate, evidence of sinus node dysfunction. In the sinus node of PAH rats, there was a significant downregulation of many ion channels and Ca2+-handling genes that could explain the dysfunction: HCN1 and HCN4 (responsible for pacemaker current, If), Cav1.2, Cav1.3 and Cav3.1 (responsible for L- and T-type Ca2+ currents, ICa,L and ICa,T), NCX1 (responsible for Na+-Ca2+ exchanger) and SERCA2 and RYR2 (Ca2+-handling molecules). In the sinus node of PAH rats, there was also a significant upregulation of many fibrosis genes that could also help explain the dysfunction: vimentin, collagen type 1, elastin, fibronectin and transforming growth factor ß1. In summary, in PAH, there is a remodelling of ion channel, Ca2+-handling and fibrosis genes in the sinus node that is likely to be responsible for the sinus node dysfunction. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'.

Postnatal development of transmural gradients in expression of ion channels and Ca²âº-handling proteins in the ventricle.

Transmural gradients in myocyte action potential duration (APD) and Ca(2+)-handling proteins are argued to be important for both the normal functioning of the ventricle and arrhythmogenesis. In rabbit, the transmural gradient in APD (left ventricular wedge preparation) is minimal in the neonate. During postnatal development, APD increases both in the epicardium and the endocardium, but the prolongation is more substantial in the endocardium leading to a significant transmural gradient. We have investigated changes in the expression of ion channels and also Ca(2+)-handling proteins in the subepicardial and subendocardial layers of the left ventricular free wall in neonatal (2-7 days of age) and adult male (~6 months of age) New Zealand White rabbits using quantitative PCR and also, when possible, in situ hybridisation and immunohistochemistry. In the adult, there were significant and substantial transmural gradients in Ca(v)1.2, KChIP2, ERG, K(v)LQT1, K(ir)2.1, NCX1, SERCA2a and RyR2 at the mRNA and, in some cases, protein level-in every case the mRNA or protein was more abundant in the epicardium than the endocardium. Of the eight transmural gradients seen in the adult, only three were observed in the neonate and, in two of these cases, the gradients were smaller than those in the adult. However, in the neonate there were also transmural gradients not observed in the adult: in HCN4, Na(v)1.5, minK, K(ir)3.1 and Cx40 mRNAs - in every case the mRNA was more abundant in the endocardium than the epicardium. If the postnatal changes in ion channel mRNAs are used to predict changes in ionic conductances, mathematical modelling predicts the changes in APD observed experimentally. It is concluded that many of the well known transmural gradients in the ventricle develop postnatally.

Visualization and quantification of whole rat heart laminar structure using high-spatial resolution contrast-enhanced MRI.

It has been shown by histology that cardiac myocytes are organized into laminae and this structure is important in function, both influencing the spread of electrical activation and enabling myocardial thickening in systole by laminar sliding. We have carried out high-spatial resolution three-dimensional MRI of the ventricular myolaminae of the entire volume of the isolated rat heart after contrast perfusion [dimeglumine gadopentate (Gd-DTPA)]. Four ex vivo rat hearts were perfused with Gd-DTPA and fixative and high-spatial resolution MRI was performed on a 9.4T MRI system. After MRI, cryosectioning followed by histology was performed. Images from MRI and histology were aligned, described, and quantitatively compared. In the three-dimensional MR images we directly show the presence of laminae and demonstrate that these are highly branching and are absent from much of the subepicardium. We visualized these MRI volumes to demonstrate laminar architecture and quantitatively demonstrated that the structural features observed are similar to those imaged in histology. We showed qualitatively and quantitatively that laminar architecture is similar in the four hearts. MRI can be used to image the laminar architecture of ex vivo hearts in three dimensions, and the images produced are qualitatively and quantitatively comparable with histology. We have demonstrated in the rat that: 1) laminar architecture is consistent between hearts; 2) myolaminae are absent from much of the subepicardium; and 3) although localized orthotropy is present throughout the myocardium, tracked myolaminae are branching structures and do not have a discrete identity.

Anatomical and molecular mapping of the left and right ventricular His-Purkinje conduction networks.

Functioning of the cardiac conduction system depends critically on its structure and its complement of ion channels. Therefore, the aim of this study was to document both the structure and ion channel expression of the left and right ventricular His-Purkinje networks, as we have previously done for the sinoatrial and atrioventricular nodes. A three-dimensional (3D) anatomical computer model of the His-Purkinje network of the rabbit heart was constructed after staining the network by immunoenzyme labelling of a marker protein, middle neurofilament. The bundle of His is a ribbon-like structure and the architecture of the His-Purkinje network differs between the left and right ventricles. The 3D model is able to explain the breakthrough points of the action potential on the ventricular epicardium during sinus rhythm. Using quantitative PCR, the expression levels of the major ion channels were measured in the free running left and right Purkinje fibres of the rabbit heart. Expression of ion channels differs from that of the working myocardium and can explain the specialised electrical activity of the Purkinje fibres as suggested by computer simulations; the expression profile of the left Purkinje fibres is more specialised than that of the right Purkinje fibres. The structure and ion channel expression of the Purkinje fibres are highly specialised and tailored to the functioning of the system. The His-Purkinje network in the left ventricle is more developed than that in the right ventricle and this may explain its greater clinical importance.

Changes in the expression of ion channels, connexins and Ca2+-handling proteins in the sino-atrial node during postnatal development.

There are important postnatal changes in the sino-atrial node (SAN), the pacemaker of the heart. Compared with the neonate, the adult has a slower intrinsic heart rate and a longer SAN action potential. These changes may be due to differences in ion channel expression. Consequently, we investigated postnatal developmental changes in the expression of ion channels and Ca(2+)-handling proteins in the SAN to see whether this is indeed the case. Using quantitative PCR, in situ hybridization and immunohistochemistry, we investigated the expression of ion channels, Ca(2+)-handling proteins and connexins in the SAN from neonatal (2-7 days of age) and adult (∼6 months of age) New Zealand White rabbits. The spontaneous beating rate of adult SAN preparations was 21% slower than that of neonatal preparations. During postnatal development, quantitative PCR revealed a significant decline in the SAN of the following mRNAs: HCN4 (major isoform responsible for I(f)), Na(V)1.5 (responsible for I(Na)), Ca(V)1.3 (in part responsible for I(Ca,L)) and NCX1 (responsible for inward I(NaCa)). These declines could be responsible for the slowing of the pacemaker during postnatal development. There was a significant decline during development in mRNA for delayed rectifier K(+) channel subunits (K(V)1.5, responsible for I(K,ur), K(V)LQT1 and minK, responsible for I(K,s), and ERG, responsible for I(K,r)) and this could explain the prolongation of the action potential. In situ hybridization confirmed the changes observed by quantitative PCR. In addition, immunohistochemistry revealed hypertrophy of nodal cells during postnatal development. Moreover, there were complex changes in the expression of Ca(2+)-handling proteins with age. In summary, there are significant postnatal changes in the expression of ion channels and Ca(2+)-handling proteins in the SAN that could explain the established changes in heart rate and action potential duration that occur during normal development.

Ageing-dependent remodelling of ion channel and Ca2+ clock genes underlying sino-atrial node pacemaking.

The function of the sino-atrial node (SAN), the pacemaker of the heart, is known to decline with age, resulting in pacemaker disease in the elderly. The aim of the study was to investigate the effects of ageing on the SAN by characterizing electrophysiological changes and determining whether changes in gene expression are involved. In young and old rats, SAN function was characterized in the anaesthetized animal, isolated heart and isolated right atrium using ECG and action potential recordings; gene expression was characterized using quantitative PCR. The SAN function declined with age as follows: the intrinsic heart rate declined by 18 ± 3%; the corrected SAN recovery time increased by 43 ± 13%; and the SAN action potential duration increased by 11 ± 3% (at 75% repolarization). Gene expression in the SAN changed considerably with age, e.g. there was an age-dependent decrease in the Ca(2+) clock gene, RYR2, and changes in many ion channels (e.g. increases in Na(v)1.5, Na(v)ß1 and Ca(v)1.2 and decreases in K(v)1.5 and HCN1). In conclusion, with age, there are changes in the expression of ion channel and Ca(2+) clock genes in the SAN, and the changes may provide a partial explanation for the age-dependent decline in pacemaker function.