James O. Tellez

Molecular Architecture of the Human Sinus Node Insights Into the Function of the Cardiac Pacemaker

Background— Although we know much about the molecular makeup of the sinus node (SN) in small mammals, little is known about it in humans. The aims of the present study were to investigate the expression of ion channels in the human SN and to use the data to predict electrical activity. Methods and Results— Quantitative polymerase chain reaction, in situ hybridization, and immunofluorescence were used to analyze 6 human tissue samples. Messenger RNA (mRNA) for 120 ion channels (and some related proteins) was measured in the SN, a novel paranodal area, and the right atrium (RA). The results showed, for example, that in the SN compared with the RA, there was a lower expression of Nav1.5, Kv4.3, Kv1.5, ERG, Kir2.1, Kir6.2, RyR2, SERCA2a, Cx40, and Cx43 mRNAs but a higher expression of Cav1.3, Cav3.1, HCN1, and HCN4 mRNAs. The expression pattern of many ion channels in the paranodal area was intermediate between that of the SN and RA; however, compared with the SN and RA, the paranodal area showed greater expression of Kv4.2, Kir6.1, TASK1, SK2, and MiRP2. Expression of ion channel proteins was in agreement with expression of the corresponding mRNAs. The levels of mRNA in the SN, as a percentage of those in the RA, were used to estimate conductances of key ionic currents as a percentage of those in a mathematical model of human atrial action potential. The resulting SN model successfully produced pacemaking. Conclusions— Ion channels show a complex and heterogeneous pattern of expression in the SN, paranodal area, and RA in humans, and the expression pattern is appropriate to explain pacemaking.

Distribution and Functional Characterization of Equilibrative Nucleoside Transporter-4, a Novel Cardiac Adenosine Transporter Activated at Acidic pH

Adenosine plays multiple roles in the efficient functioning of the heart by regulating coronary blood flow, cardiac pacemaking, and contractility. Previous studies have implicated the equilibrative nucleoside transporter family member equilibrative nucleoside transporter-1 (ENT1) in the regulation of cardiac adenosine levels. We report here that a second member of this family, ENT4, is also abundant in the heart, in particular in the plasma membranes of ventricular myocytes and vascular endothelial cells but, unlike ENT1, is virtually absent from the sinoatrial and atrioventricular nodes. Originally described as a monoamine/organic cation transporter, we found that both human and mouse ENT4 exhibited a novel, pH-dependent adenosine transport activity optimal at acidic pH (apparent Km values 0.78 and 0.13 mmol/L, respectively, at pH 5.5) and absent at pH 7.4. In contrast, serotonin transport by ENT4 was relatively insensitive to pH. ENT4-mediated nucleoside transport was adenosine selective, sodium independent and only weakly inhibited by the classical inhibitors of equilibrative nucleoside transport, dipyridamole, dilazep, and nitrobenzylthioinosine. We hypothesize that ENT4, in addition to playing roles in cardiac serotonin transport, contributes to the regulation of extracellular adenosine concentrations, in particular under the acidotic conditions associated with ischemia.

Computer Three-Dimensional Reconstruction of the Sinoatrial Node

Background—There is an effort to build an anatomically and biophysically detailed virtual heart, and, although there are models for the atria and ventricles, there is no model for the sinoatrial node (SAN). For the SAN to show pacemaking and drive atrial muscle, theoretically, there should be a gradient in electrical coupling from the center to the periphery of the SAN and an interdigitation of SAN and atrial cells at the periphery. Any model should include such features. Methods and Results—Staining of rabbit SAN preparations for histology, middle neurofilament, atrial natriuretic peptide, and connexin (Cx) 43 revealed multiple cell types within and around the SAN (SAN and atrial cells, fibroblasts, and adipocytes). In contrast to atrial cells, all SAN cells expressed middle neurofilament (but not atrial natriuretic peptide) mRNA and protein. However, 2 distinct SAN cell types were observed: cells in the center (leading pacemaker site) were small, were organized in a mesh, and did not express Cx43. In contrast, cells in the periphery (exit pathway from the SAN) were large, were arranged predominantly in parallel, often expressed Cx43, and were mixed with atrial cells. An ≈2.5-million-element array model of the SAN and surrounding atrium, incorporating all cell types, was constructed. Conclusions—For the first time, a 3D anatomically detailed mathematical model of the SAN has been constructed, and this shows the presence of a specialized interface between the SAN and atrial muscle.

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+, Ca2+, hyperpolarization-activated cation and K+ channels, and related Ca2+ 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, Nav1.5 to Nav1.1, Cav1.2 to Cav1.3 and Kv1.4 to Kv4.2. The transcript profile of the SAN periphery is intermediate between that of the SAN center and atrial muscle. For example, Nav1.5 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.

Connexins in the Sinoatrial and Atrioventricular Nodes

The sinoatrial node (SAN) and the atrioventricular node (AVN) are specialized tissues in the heart: the SAN is specialized for pacemaking (it is the pacemaker of the heart), whereas the AVN is specialized for slow conduction of the action potential (to introduce a delay between atrial and ventricular activation during the cardiac cycle). These functions have special requirements regarding electrical coupling and, therefore, expression of connexin isoforms. Electrical coupling in the center of the SAN should be weak to protect it from the inhibitory electrotonic influence of the more hyperpolarized non-pacemaking atrial muscle surrounding the SAN. However, for the SAN to be able to drive the atrial muscle, electrical coupling should be strong in the periphery of the SAN. Consistent with this, in the center of the SAN there is no expression of Cx43 (the principal connexin of the working myocardium) and little expression of Cx40, but there is expression of Cx45 and Cx30.2, whereas in the periphery of the SAN Cx43 as well Cx45 is expressed. In the AVN, there is a similar pattern of expression of connexins as in the center of the SAN and this is likely to be in large part responsible for the slow conduction of the action potential.

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.

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.

Structural remodelling of the sinoatrial node in obese old rats

During ageing, the function of sinoatrial node (SAN), the pacemaker of the heart, declines, and the incidence of sick sinus syndrome increases markedly. The aim of the study was to investigate structural and functional remodelling of the SAN during ageing. Rats, 3 and 24 months old (equivalent to young adult and approximately 69-year-old humans), were studied. Extracellular potential recording from right atrial preparations showed that (as expected) the intrinsic heart rate was slower in the old animals. It also showed a shift of the leading pacemaker site towards the inferior vena cava in the old animals. Consistent with this, intracellular potential recording showed that slow pacemaker action potentials were more widespread and extended further towards the inferior vena cava in old animals. Immunohistochemistry demonstrated that SAN tissue expressing HCN4, but lacking the expression of Na(v)1.5 (lack of Na(v)1.5 explains why pacemaker action potential is slow), was also more widespread and extended further towards the inferior vena cava in the old animals. Immunolabelling of caveolin3 (expressed in cell membrane of cardiac myocytes) demonstrated that there was a hypertrophy of the SAN cells in the old animals. Histology, quantitative PCR, and immunohistochemistry revealed evidence of a substantial age-dependent remodelling of the extracellular matrix (e.g. approximately 79% downregulation of genes responsible for collagens 1 and 3 and approximately 52% downregulation of gene responsible for elastin). It is concluded that the age- (and/or obesity-) dependent decline in SAN function is associated with a structural remodelling of the SAN: an enlargement of the SAN, a hypertrophy of the SAN cells, and a remodelling of the extracellular matrix.

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 Ca2+ clock gene, RYR2, and changes in many ion channels (e.g. increases in Nav1.5, Navβ1 and Cav1.2 and decreases in Kv1.5 and HCN1). In conclusion, with age, there are changes in the expression of ion channel and Ca2+ 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, KvLQT1, Kir2.4, 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; Ca2+-, Na+-, and H+-handling proteins; and receptors in the SAN.