Regulation of sinus node pacemaking and atrioventricular node conduction by HCN channels in health and disease.

The funny current, If, was first recorded in the heart 40 or more years ago by Dario DiFrancesco and others. Since then, we have learnt that If plays an important role in pacemaking in the sinus node, the innate pacemaker of the heart, and more recently evidence has accumulated to show that If may play an important role in action potential conduction through the atrioventricular (AV) node. Evidence has also accumulated to show that regulation of the transcription and translation of the underlying Hcn genes plays an important role in the regulation of sinus node pacemaking and AV node conduction under normal physiological conditions - in athletes, during the circadian rhythm, in pregnancy, and during postnatal development - as well as pathological states - ageing, heart failure, pulmonary hypertension, diabetes and atrial fibrillation. There may be yet more pathological conditions involving changes in the expression of the Hcn genes. Here, we review the role of If and the underlying HCN channels in physiological and pathological changes of the sinus and AV nodes and we begin to explore the signalling pathways (microRNAs, transcription factors, GIRK4, the autonomic nervous system and inflammation) involved in this regulation. This review is dedicated to Dario DiFrancesco on his retirement.

Characterization of a right atrial subsidiary pacemaker and acceleration of the pacing rate by HCN over-expression.

AIMS: Although the right atrium (RA contains subsidiary atrial pacemaker (SAP) tissue that can take over from the sinoatrial node (SAN) in sick sinus syndrome (SSS), SAP tissue is bradycardic. Little is known about SAP tissue and one aim of the study was to characterize ion channel expression to obtain insight into SAP pacemaker mechanisms. A second aim was to determine whether HCN over-expression (a 'biopacemaker'-like strategy) can accelerate the pacemaker rate producing a pacemaker that is similar in nature to the SAN. METHODS AND RESULTS: SAP tissue was isolated from the rat and the leading pacemaker site was characterized. Cell size at the leading pacemaker site in the SAP was smaller than in the RA and comparable to that in the SAN. mRNA levels showed the SAP to be similar to, but distinct from, the SAN. For example, in the SAN and SAP, expression of Tbx3 and HCN1 was higher and Nav1.5 and Cx43 lower than in the RA. Organ-cultured SAP tissue beat spontaneously, but at a slower rate than the SAN. Adenovirus-mediated gene transfer of HCN2 and the chimeric protein HCN212 significantly increased the pacemaker rate of the SAP close to that of the native SAN, but HCN4 was ineffective. CONCLUSION: SAP tissue near the inferior vena cava is bradycardic, but shares characteristics with the SAN. Pacing can be accelerated by the over-expression of HCN2 or HCN212. This provides proof of concept for the use of SAP tissue as a substrate for biopacemaking in the treatment of SSS.

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.

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.

Changes in ion channel gene expression underlying heart failure-induced sinoatrial node dysfunction.

BACKGROUND: Heart failure (HF) causes a decline in the function of the pacemaker of the heart-the sinoatrial node (SAN). The aim of the study was to investigate HF-induced changes in the expression of the ion channels and related proteins underlying the pacemaker activity of the SAN. METHODS AND RESULTS: HF was induced in rats by the ligation of the proximal left coronary artery. HF animals showed an increase in the left ventricular (LV) diastolic pressure (317%) and a decrease in the LV systolic pressure (19%) compared with sham-operated animals. They also showed SAN dysfunction wherein the intrinsic heart rate was reduced (16%) and the corrected SAN recovery time was increased (56%). Quantitative polymerase chain reaction was used to measure gene expression. Of the 91 genes studied during HF, 58% changed in the SAN, although only 1% changed in the atrial muscle. For example, there was an increase in the expression of ERG, K(v)LQT1, K(ir)2.4, TASK1, TWIK1, TWIK2, calsequestrin 2, and the A1 adenosine receptor in the SAN that could explain the slowing of the intrinsic heart rate. In addition, there was an increase in Na(+)-H(+) exchanger, and this could be the stimulus for the remodeling of the SAN. CONCLUSIONS: SAN dysfunction is associated with HF and is the result of an extensive remodeling of ion channels; gap junction channels; Ca(2+)-, Na(+)-, and H(+)-handling proteins; and receptors in the SAN.

Ion channel transcript expression at the rabbit atrioventricular conduction axis.

BACKGROUND: Little is known about the distribution of gap junctions and ion channels in the atrioventricular node, even though the physiology and pathology of the atrioventricular node is ultimately dependent on them. METHODS AND RESULTS: The abundance of 30 transcripts for markers, gap junctions, ion channels, and Ca(2+)-handling proteins in different regions of the rabbit atrioventricular node (nodal extension and proximal and distal penetrating bundle of His as well as atrial and ventricular muscle) was measured using a novel quantitative polymerase chain reaction technique and in situ hybridization. The expression profile of the nodal extension (slow pathway into penetrating bundle) was similar to that of the sinoatrial node. For example, in the nodal extension, in contrast to the atrial muscle and as expected for a slowly conducting tissue with pacemaker activity, there was no or reduced expression of Cx43, Na(v)1.5, Ca(v)1.2, K(v)1.4, KChIP2, and RYR3 and high expression of Ca(v)1.3 and HCN4. The expression profile of the penetrating bundle was less specialized. In situ hybridization revealed a transitional zone with reduced expression of Cx43, Na(v)1.5, and KChIP2 that may form the fast pathway into the penetrating bundle. CONCLUSIONS: At the atrioventricular node, the expression of gap junctions and ion channels in the nodal extension (slow pathway) and a transitional zone (putative fast pathway) as well as the penetrating bundle (output pathway) is specialized and heterogeneous and roughly matches the electrophysiology of the different regions.

Distribution of the pacemaker HCN4 channel mRNA and protein in the rabbit sinoatrial node.

Several studies of the pacemaker mechanisms in mammalian cells, most of which were carried out in cells isolated from the rabbit sinoatrial node (SAN), have highlighted the role of the I(f) current. While the distribution of Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels, the molecular correlates of f-channels, is known at the mRNA level, the identification of f-channel proteins in this tissue is still undetermined. Here we investigate HCN protein expression in the rabbit pacemaker region. We found that HCN4 is the main isoform, and set therefore to analyze its distribution within the SAN and surrounding areas with the aim of correlating protein expression and pacemaking function. The analysis was carried out in tissue slices and single cells of the intercaval area, which includes the crista terminalis (CT), the SAN, and the septum interatrialis (SI). Immunolabeling, in situ hybridization, qRT-PCR analysis, and electrophysiological recordings identified the SAN as a region characterized by high HCN4 signal and current levels, while the expression in the CT and in the SI was either negligible or absent. Detailed analysis of the central SAN area showed that cells are predominantly distributed in islets interconnected by cell prolongations, and single-cell HCN4 labeling suggested sites of channel clustering. Our data indicate that in the rabbit SAN, HCN4 proteins are major constituents of native f-channels, and their distribution matches closely the SAN as defined morphologically and electrophysiologically. Until recently, the SAN was identified as the region where Cx43 and atrial natriuretic peptide are not expressed; we propose here that expression of HCN4 is an appropriate tool to map and identify the cardiac SAN pacemaker region.

Computer three-dimensional reconstruction of the atrioventricular node.

Because of its complexity, the atrioventricular node (AVN), remains 1 of the least understood regions of the heart. The aim of the study was to construct a detailed anatomic model of the AVN and relate it to AVN function. The electric activity of a rabbit AVN preparation was imaged using voltage-dependent dye. The preparation was then fixed and sectioned. Sixty-five sections at 60- to 340-microm intervals were stained for histology and immunolabeled for neurofilament (marker of nodal tissue) and connexin43 (gap junction protein). This revealed multiple structures within and around the AVN, including transitional tissue, inferior nodal extension, penetrating bundle, His bundle, atrial and ventricular muscle, central fibrous body, tendon of Todaro, and valves. A 3D anatomically detailed mathematical model (approximately 13 million element array) of the AVN and surrounding atrium and ventricle, incorporating all cell types, was constructed. Comparison of the model with electric activity recorded in experiments suggests that the inferior nodal extension forms the slow pathway, whereas the transitional tissue forms the fast pathway into the AVN. In addition, it suggests the pacemaker activity of the atrioventricular junction originates in the inferior nodal extension. Computer simulation of the propagation of the action potential through the anatomic model shows how, because of the complex structure of the AVN, reentry (slow-fast and fast-slow) can occur. In summary, a mathematical model of the anatomy of the AVN has been generated that allows AVN conduction to be explored.

Distinguishing properties of cells from the myocardial sleeves of the pulmonary veins: a comparison of normal and abnormal pacemakers.

BACKGROUND: A common source of arrhythmogenic spontaneous activity instigating atrial fibrillation is the myocardial tissue, or sleeves, at the base of the pulmonary veins. This study compared the properties of cells from the myocardial sleeves of the pulmonary veins (PV(m)) with cells from the normal cardiac pacemaker (the sinoatrial node) and regions of the atria. Our objective was to identify key features of these cells that predispose them to becoming the focus of cardiac arrhythmias. METHODS AND RESULTS: Single cells were isolated from samples of rabbit PV(m), central and peripheral sinoatrial node, crista terminalis, and left and right atria. Detailed morphology of cells was assessed and intracellular calcium concentrations measured with the use of Fluo-3. Cells from the PV(m) were smaller than atrial cells and showed large elevations in diastolic calcium during activation at physiological rates, a feature the PV(m) cells shared with cells from the sinoatrial node. Unstimulated spontaneous activity was observed in a minority of cells from the PV(m), but numerous cells from this region showed spontaneous activity for a brief period immediately subsequent to stimulation at physiological rates. This was not observed in atrial cells. Assessment of calcium removal pathways showed sarcolemmal calcium extrusion in cells from the PV(m) to have a high reliance on "slow" extrusion pathways to maintain intracellular calcium homeostasis because of a low expression of sodium-calcium exchanger. CONCLUSIONS: We conclude that cells from the PV(m) share some features with cells from the sinoatrial node but also have distinctly unique features that predispose them to the development of spontaneous activity.

Differential expression of ion channel transcripts in atrial muscle and sinoatrial node in rabbit.

The aim of the study was to identify ion channel transcripts expressed in the sinoatrial node (SAN), the pacemaker of the heart. Functionally, the SAN can be divided into central and peripheral regions (center is adapted for pacemaking only, whereas periphery is adapted to protect center and drive atrial muscle as well as pacemaking) and the aim was to study expression in both regions. In rabbit tissue, the abundance of 30 transcripts (including transcripts for connexin, Na(+), Ca(2+), hyperpolarization-activated cation and K(+) channels, and related Ca(2+) handling proteins) was measured using quantitative PCR and the distribution of selected transcripts was visualized using in situ hybridization. Quantification of individual transcripts (quantitative PCR) showed that there are significant differences in the abundance of 63% of the transcripts studied between the SAN and atrial muscle, and cluster analysis showed that the transcript profile of the SAN is significantly different from that of atrial muscle. There are apparent isoform switches on moving from atrial muscle to the SAN center: RYR2 to RYR3, Na(v)1.5 to Na(v)1.1, Ca(v)1.2 to Ca(v)1.3 and K(v)1.4 to K(v)4.2. The transcript profile of the SAN periphery is intermediate between that of the SAN center and atrial muscle. For example, Na(v)1.5 messenger RNA is expressed in the SAN periphery (as it is in atrial muscle), but not in the SAN center, and this is probably related to the need of the SAN periphery to drive the surrounding atrial muscle.

Extended atrial conduction system characterised by the expression of the HCN4 channel and connexin45.

OBJECTIVE: In the heart, there are multiple supraventricular pacemakers involved in normal pacemaking as well as arrhythmias and the objective was to determine the distribution of HCN4 (major isoform underlying the pacemaker current, I(f)) in the atria. METHODS: In the atria of the rat, the localisation of HCN4 and connexins was determined using immunohistochemistry, and electrical activity was recorded using extracellular electrodes. RESULTS: As expected, HCN4 and Cx45 (but not Cx43) were expressed in the sinoatrial node extending from the superior vena cava down the crista terminalis. The same pattern of expression of HCN4 and connexins was observed in a novel tract of nodal-like cells extending from the superior vena cava down the interatrial groove. Although the sinoatrial node was usually the leading pacemaker site, the novel tract of HCN4-expressing cells was capable of pacemaking and could act as the leading pacemaker site; there was evidence of a hierarchy of pacemakers. The same pattern of expression of HCN4 and connexins was also observed in the atrioventricular ring bundle (including the atrioventricular node) encircling the tricuspid valve, but not in the atrioventricular ring bundle encircling the mitral valve. HCN4 was not expressed in the pulmonary veins. CONCLUSIONS: The widespread distribution of HCN4 can explain the widespread location of the leading pacemaker site during sinus rhythm, the extensive region of tissue that has to be ablated to stop sinus rhythm, and the widespread distribution of ectopic foci responsible for atrial tachycardia.