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.

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.

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.

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.

Left ventricle structural remodelling in the prediabetic Goto-Kakizaki rat.

This study tested the hypothesis that experimental prediabetes can elicit structural remodelling in the left ventricle (LV). Left ventricles isolated from 8-week-old male Goto-Kakizaki (GK) rats and age-matched male Wistar control rats were used to assess remodelling changes and underlying transforming growth factor ß1 (TGFß1) activity, prohypertrophic Akt-p70S6K1 signalling and gene expression profile of the extracellular matrix (ECM) using histological, immunohistochemical, immunoblotting and quantitative gene expression analyses. Prediabetes in GK rats was confirmed by impaired glucose tolerance and modestly elevated fasting blood glucose. Left ventricle remodelling in the GK rat presented with marked hypertrophy of cardiomyocytes and increased ECM deposition that together translated into increased heart size in the absence of ultrastructural changes or fibre disarray. Molecular derangements underlying this phenotype included recapitulation of the fetal gene phenotype markers B-type natriuretic peptide and α-skeletal muscle actin, activation of the Akt-p70S6K1 pathway and altered gene expression profile of key components (collagen 1α and fibronectin) and modulators of the ECM (matrix metalloproteinases 2 and 9 and connective tissue growth factor). These changes were correlated with parallel findings of increased TGFß1 transcription and activation in the LV and elevated active TGFß1 in plasma of GK rats compared with control animals (Student's t test, P < 0.05 versus age-matched Wistar control animals for all parameters). This is the first report to describe LV structural remodelling in experimental prediabetes. The results suggest that ventricular decompensation pathognomonic of advanced diabetic cardiomyopathy may have possible origins in profibrotic and prohypertrophic mechanisms triggered before the onset of type 2 diabetes mellitus.

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.

Further insights into the molecular complexity of the human sinus node - The role of 'novel' transcription factors and microRNAs.

RESEARCH PURPOSE: The sinus node (SN) is the heart's primary pacemaker. Key ion channels (mainly the funny channel, HCN4) and Ca2+-handling proteins in the SN are responsible for its function. Transcription factors (TFs) regulate gene expression through inhibition or activation and microRNAs (miRs) do this through inhibition. There is high expression of macrophages and mast cells within the SN connective tissue. 'Novel'/unexplored TFs and miRs in the regulation of ion channels and immune cells in the SN are not well understood. Using RNAseq and bioinformatics, the expression profile and predicted interaction of key TFs and cell markers with key miRs in the adult human SN vs. right atrial tissue (RA) were determined. PRINCIPAL RESULTS: 68 and 60 TFs significantly more or less expressed in the SN vs. RA respectively. Among those more expressed were ISL1 and TBX3 (involved in embryonic development of the SN) and 'novel' RUNX1-2, CEBPA, GLI1-2 and SOX2. These TFs were predicted to regulate HCN4 expression in the SN. Markers for different cells: fibroblasts (COL1A1), fat (FABP4), macrophages (CSF1R and CD209), natural killer (GZMA) and mast (TPSAB1) were significantly more expressed in the SN vs. RA. Interestingly, RUNX1-3, CEBPA and GLI1 also regulate expression of these cells. MiR-486-3p inhibits HCN4 and markers involved in immune response. MAJOR CONCLUSIONS: In conclusion, RUNX1-2, CSF1R, TPSAB1, COL1A1 and HCN4 are highly expressed in the SN but not miR-486-3p. Their complex interactions can be used to treat SN dysfunction such as bradycardia. Interestingly, another research group recently reported miR-486-3p is upregulated in blood samples from severe COVID-19 patients who suffer from bradycardia.

Structural and Functional Properties of Subsidiary Atrial Pacemakers in a Goat Model of Sinus Node Disease.

BACKGROUND: The sinoatrial/sinus node (SAN) is the primary pacemaker of the heart. In humans, SAN is surrounded by the paranodal area (PNA). Although the PNA function remains debated, it is thought to act as a subsidiary atrial pacemaker (SAP) tissue and become the dominant pacemaker in the setting of sinus node disease (SND). Large animal models of SND allow characterization of SAP, which might be a target for novel treatment strategies for SAN diseases. METHODS: A goat model of SND was developed (n = 10) by epicardially ablating the SAN and validated by mapping of emergent SAP locations through an ablation catheter and surface electrocardiogram (ECG). Structural characterization of the goat SAN and SAP was assessed by histology and immunofluorescence techniques. RESULTS: When the SAN was ablated, SAPs featured a shortened atrioventricular conduction, consistent with the location in proximity of atrioventricular junction. SAP recovery time showed significant prolongation compared to the SAN recovery time, followed by a decrease over a follow-up of 4 weeks. Like the SAN tissue, the SAP expressed the main isoform of pacemaker hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4) and Na+/Ca2+ exchanger 1 (NCX1) and no high conductance connexin 43 (Cx43). Structural characterization of the right atrium (RA) revealed that the SAN was located at the earliest activation [i.e., at the junction of the superior vena cava (SVC) with the RA] and was surrounded by the paranodal-like tissue, extending down to the inferior vena cava (IVC). Emerged SAPs were localized close to the IVC and within the thick band of the atrial muscle known as the crista terminalis (CT). CONCLUSIONS: SAN ablation resulted in the generation of chronic SAP activity in 60% of treated animals. SAP displayed development over time and was located within the previously discovered PNA in humans, suggesting its role as dominant pacemaker in SND. Therefore, SAP in goat constitutes a promising stable target for electrophysiological modification to construct a fully functioning pacemaker.

Remodeling of the Purkinje Network in Congestive Heart Failure in the Rabbit.

BACKGROUND: Purkinje fibers (PFs) control timing of ventricular conduction and play a key role in arrhythmogenesis in heart failure (HF) patients. We investigated the effects of HF on PFs. METHODS: Echocardiography, electrocardiography, micro-computed tomography, quantitative polymerase chain reaction, immunohistochemistry, volume electron microscopy, and sharp microelectrode electrophysiology were used. RESULTS: Congestive HF was induced in rabbits by left ventricular volume- and pressure-overload producing left ventricular hypertrophy, diminished fractional shortening and ejection fraction, and increased left ventricular dimensions. HF baseline QRS and corrected QT interval were prolonged by 17% and 21% (mean±SEMs: 303±6 ms HF, 249±11 ms control; n=8/7; P=0.0002), suggesting PF dysfunction and impaired ventricular repolarization. Micro-computed tomography imaging showed increased free-running left PF network volume and length in HF. mRNA levels for 40 ion channels, Ca2+-handling proteins, connexins, and proinflammatory and fibrosis markers were assessed: 50% and 35% were dysregulated in left and right PFs respectively, whereas only 12.5% and 7.5% changed in left and right ventricular muscle. Funny channels, Ca2+-channels, and K+-channels were significantly reduced in left PFs. Microelectrode recordings from left PFs revealed more negative resting membrane potential, reduced action potential upstroke velocity, prolonged duration (action potential duration at 90% repolarization: 378±24 ms HF, 249±5 ms control; n=23/38; P<0.0001), and arrhythmic events in HF. Similar electrical remodeling was seen at the left PF-ventricular junction. In the failing left ventricle, upstroke velocity and amplitude were increased, but action potential duration at 90% repolarization was unaffected. CONCLUSIONS: Severe volume- followed by pressure-overload causes rapidly progressing HF with extensive remodeling of PFs. The PF network is central to both arrhythmogenesis and contractile dysfunction and the pathological remodeling may increase the risk of fatal arrhythmias in HF patients.

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.

Identification of Key Small Non-Coding MicroRNAs Controlling Pacemaker Mechanisms in the Human Sinus Node.

Background The sinus node (SN) is the primary pacemaker of the heart. SN myocytes possess distinctive action potential morphology with spontaneous diastolic depolarization because of a unique expression of ion channels and Ca2+-handling proteins. MicroRNAs (miRs) inhibit gene expression. The role of miRs in controlling the expression of genes responsible for human SN pacemaking and conduction has not been explored. The aim of this study was to determine miR expression profile of the human SN as compared with that of non-pacemaker atrial muscle. Methods and Results SN and atrial muscle biopsies were obtained from donor or post-mortem hearts (n=10), histology/immunolabeling were used to characterize the tissues, TaqMan Human MicroRNA Arrays were used to measure 754 miRs, Ingenuity Pathway Analysis was used to identify miRs controlling SN pacemaker gene expression. Eighteen miRs were significantly more and 48 significantly less abundant in the SN than atrial muscle. The most interesting miR was miR-486-3p predicted to inhibit expression of pacemaking channels: HCN1 (hyperpolarization-activated cyclic nucleotide-gated 1), HCN4, voltage-gated calcium channel (Cav)1.3, and Cav3.1. A luciferase reporter gene assay confirmed that miR-486-3p can control HCN4 expression via its 3' untranslated region. In ex vivo SN preparations, transfection with miR-486-3p reduced the beating rate by ≈35±5% (P<0.05) and HCN4 expression (P<0.05). Conclusions The human SN possesses a unique pattern of expression of miRs predicted to target functionally important genes. miR-486-3p has an important role in SN pacemaker activity by targeting HCN4, making it a potential target for therapeutic treatment of SN disease such as sinus tachycardia.

Silencing miR-370-3p rescues funny current and sinus node function in heart failure.

Bradyarrhythmias are an important cause of mortality in heart failure and previous studies indicate a mechanistic role for electrical remodelling of the key pacemaking ion channel HCN4 in this process. Here we show that, in a mouse model of heart failure in which there is sinus bradycardia, there is upregulation of a microRNA (miR-370-3p), downregulation of the pacemaker ion channel, HCN4, and downregulation of the corresponding ionic current, If, in the sinus node. In vitro, exogenous miR-370-3p inhibits HCN4 mRNA and causes downregulation of HCN4 protein, downregulation of If, and bradycardia in the isolated sinus node. In vivo, intraperitoneal injection of an antimiR to miR-370-3p into heart failure mice silences miR-370-3p and restores HCN4 mRNA and protein and If in the sinus node and blunts the sinus bradycardia. In addition, it partially restores ventricular function and reduces mortality. This represents a novel approach to heart failure treatment.

The anatomy of the cardiac conduction system.

All the myocytes within the heart have the capacity to conduct the cardiac impulse. A population of myocytes is specialized so as to generate the cardiac impulse and then to conduct it from the atrial to the ventricular chambers. This population has become known as the conduction system. Anatomists who seek to demonstrate the location of the components of this system must contend with the fact that the components of the system cannot be distinguished from the working myocardial elements by gross dissection. In important presentations to the German Pathological Society in 1910, rules were suggested for the histological distinction of these conducting cells. These rules proposed that the myocytes, to be considered as part of the conduction system, should be histologically discrete, traceable from section to section in serially prepared material, and if to be considered as tracts, should be insulated by fibrous tissue from the adjacent myocytes. Immunohistochemical techniques have now been developed that better demonstrate the distinction between the cells specialized to conduct from working myocytes. These new techniques, for the most part, confirm the accuracy of the initial descriptions. They also reveal additional areas with the characteristics of conduction tissues. These additional areas are located in a paranodal area adjacent to the sinus node, in the vestibules of both atrioventricular valvar orifices, and in a partial ring around the aortic root. In this review, we describe all these features, emphasizing the relationship of the newly recognized components to the established parts of the cardiac conduction system, and how the new findings need to be assessed in the light of the old criteria.

Electrical Conduction System Remodeling in Streptozotocin-Induced Diabetes Mellitus Rat Heart.

Cardiovascular complications are common in type 1 diabetes mellitus (TIDM) and there is an increased risk of arrhythmias as a result of dysfunction of the cardiac conduction system (CCS). We have previously shown that, in vivo, there is a decrease in the heart rate and prolongation of the QRS complex in streptozotocin-induced type 1 diabetic rats indicating dysfunction of the CCS. The aim of this study was to investigate the function of the ex vivo CCS and key proteins that are involved in pacemaker mechanisms in TIDM. RR interval, PR interval and QRS complex duration were significantly increased in diabetic rats. The beating rate of the isolated sinoatrial node (SAN) preparation was significantly decreased in diabetic rats. The funny current density and cell capacitance were significantly decreased in diabetic nodal cells. Western blot showed that proteins involved in the function of the CCS were significantly decreased in diabetic rats, namely: HCN4, Cav1.3, Cav3.1, Cx45, and NCX1 in the SAN; RyR2 and NCX1 in the atrioventricular junction and Cx40, Cx43, Cx45, and RyR2 in the Purkinje network. We conclude that there are complex functional and cellular changes in the CCS in TIDM. The changes in the proteins involved in the function of this electrical system are expected to adversely affect action potential generation and propagation, and these changes are likely to be arrhythmogenic.

A sexy approach to pacemaking: differences in function and molecular make up of the sinoatrial node.

BACKGROUND: Functional properties of the sinoatrial node (SAN) are known to differ between sexes. Women have higher resting and intrinsic heart rates. Sex determines the risk of developing certain arrhythmias such as sick sinus syndrome, which occur more often in women. We believe that a major contributor to these differences is in gender specific ion channel expression. METHODS: qPCR was used to compare ion channel gene expression in the SAN and right atrium (RA) between male and female rats. Histology, immunohistochemistry and signal intensity analysis were used to locate the SAN and determine abundance of ion channels. The effect of nifedipine on extracellular potential recording was used to determine differences in beating rate between sexes. RESULTS: mRNAs for Cav1.3, Kir3.1, and Nkx2-5, as well as expression of the L-Type Ca²âº channel protein, were higher in the female SAN. Females had significantly higher intrinsic heart rates and the effect of nifedipine on isolated SAN preparations was significantly greater in male SAN. Computer modelling using a SAN cell model demonstrated a higher propensity of pacemaker-related arrhythmias in females. CONCLUSION: This study has identified key differences in the expression of Cav1.3, Kir3.1 and Nkx2-5 at mRNA and/or protein levels between male and female SAN. Cav1.3 plays an important role in the pacemaker function of the SAN, therefore the higher intrinsic heart rate of the female SAN could be caused by the higher expression of Cav1.3. The differences identified in this study advance our understanding of sex differences in cardiac electrophysiology and arrhythmias.

Organisation of the mouse sinoatrial node: structure and expression of HCN channels.

OBJECTIVE: To reveal the structural characteristics of the sinoatrial node (SAN) and the distribution of hyperpolarization-activated cyclic nucleotide-gated cation channels (HCN) in the SAN in the mouse. METHODS: The structure of the SAN and the distribution of HCN channels in the SAN in the mouse were studied by histology and immunolabelling of ANP, Cx43 and HCN channels. RESULTS: The mouse SAN is a comma-shaped structure with a length of approximately 1.5 mm parallel to the crista terminalis and is separated from atrial muscle by connective tissue at the border both with the crista terminalis and the atrial septum. A unique compact nodal structure with densely-packed nodal cells was identified at the head of the comma-shaped SAN. Cell size and fibre orientation vary regionally in the SAN: the cells in the compact node are small and are orientated perpendicular to the crista terminalis, whereas the cells in the more inferior part are larger and more loosely-packed and are orientated parallel to the crista terminalis. All SAN cells exhibited labelling of HCN4, but no cell exhibited detectable labelling of HCN1, HCN2, ANP and Cx43, while surrounding atrial cells exhibited labelling of ANP and Cx43, but not HCN1, HCN2 and HCN4. A specialised interface between the SAN and surrounding atrial muscle was also identified: strands of HCN4-positive nodal cells protrude into the atrial muscle and strands of Cx43-positive atrial cells protrude into the SAN; thus, there are interdigitations between the SAN and atrial muscle. CONCLUSIONS: In the mouse, (i) the SAN is structurally complex with a densely-packed head and loosely-packed tail; (ii) HCN4 is the only HCN isoform detectable and is present throughout the SAN; and (iii) there is a specialised interface between the SAN and surrounding atrium that may be necessary for the SAN to drive the more hyperpolarized atrial muscle.

Structural and functional remodeling of the atrioventricular node with aging in rats: The role of hyperpolarization-activated cyclic nucleotide-gated and ryanodine 2 channels.

BACKGROUND: Aging is associated with an increased incidence of atrioventricular nodal (AVN) dysfunction. OBJECTIVE: The aim of this study was to investigate the structural and functional remodeling in the atrioventricular junction (AVJ) with aging. METHODS: Electrophysiology, histology, and immunohistochemistry experiments on male Wistar Hannover rats aged 3 months (n = 24) and 2 years (n = 15) were performed. Atrio-His (AH) interval, Wenkebach cycle length (WBCL), and AVN effective refractory period (AVNERP) were measured. Cesium (2 mM) was used to block hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, while ryanodine (2 µM) was used to block ryanodine 2 (RyR2) channels. Protein expression from different regions of the AVJ was studied using immunofluorescence. The expression of connexins (connexin 43 and connexin 40), ion channels (Hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4), voltage sensitive sodium channel (Nav1.5), and L-Type calcium channel (Cav1.3)), and calcium handling proteins (RyR2 and sarco/endoplasmic reticulum calcium ATPaset type 2a (SERCA2a)) were measured. Morphological characteristics were studied with histology. RESULTS: Without drugs to block HCN and RyR2 channels, there was prolongation of the AH interval, WBCL, and AVNERP (P < .05) with aging. In young rats only, cesium prolonged the AH interval, WBCL, and AVNERP (P < .01). Ryanodine prolonged the AH interval and WBCL (P < .01) in both young and old rats. Immunofluorescence revealed that with aging, connexin 43, HCN4, Nav1.5, and RyR2 downregulate in the regions of the AVJ and connexin 40, SERCA2a, and Cav1.3 upregulate (P < .05). Aging results in cellular hypertrophy, loosely packed cells, a decrease in the number of nuclei, and an increase in collagen content. CONCLUSION: Heterogeneous ion channel expression changes were observed in the AVJ with aging. For the first time, we have shown that HCN and RyR2 play an important role in AVN dysfunction with aging.