Sunil Logantha

Exercise training reduces resting heart rate via downregulation of the funny channel HCN4

Endurance athletes exhibit sinus bradycardia, that is a slow resting heart rate, associated with a higher incidence of sinus node (pacemaker) disease and electronic pacemaker implantation. Here we show that training-induced bradycardia is not a consequence of changes in the activity of the autonomic nervous system but is caused by intrinsic electrophysiological changes in the sinus node. We demonstrate that training-induced bradycardia persists after blockade of the autonomous nervous system in vivo in mice and in vitro in the denervated sinus node. We also show that a widespread remodelling of pacemaker ion channels, notably a downregulation of HCN4 and the corresponding ionic current, If. Block of If abolishes the difference in heart rate between trained and sedentary animals in vivo and in vitro. We further observe training-induced downregulation of Tbx3 and upregulation of NRSF and miR-1 (transcriptional regulators) that explains the downregulation of HCN4. Our findings provide a molecular explanation for the potentially pathological heart rate adaptation to exercise training.

Structure, function and clinical relevance of the cardiac conduction system, including the atrioventricular ring and outflow tract tissues.

It is now over 100years since the discovery of the cardiac conduction system, consisting of three main parts, the sinus node, the atrioventricular node and the His-Purkinje system. The system is vital for the initiation and coordination of the heartbeat. Over the last decade, immense strides have been made in our understanding of the cardiac conduction system and these recent developments are reviewed here. It has been shown that the system has a unique embryological origin, distinct from that of the working myocardium, and is more extensive than originally thought with additional structures: atrioventricular rings, a third node (so called retroaortic node) and pulmonary and aortic sleeves. It has been shown that the expression of ion channels, intracellular Ca(2+)-handling proteins and gap junction channels in the system is specialised (different from that in the ordinary working myocardium), but appropriate to explain the functioning of the system, although there is continued debate concerning the ionic basis of pacemaking. We are beginning to understand the mechanisms (fibrosis and remodelling of ion channels and related proteins) responsible for dysfunction of the system (bradycardia, heart block and bundle branch block) associated with atrial fibrillation and heart failure and even athletic training. Equally, we are beginning to appreciate how naturally occurring mutations in ion channels cause congenital cardiac conduction system dysfunction. Finally, current therapies, the status of a new therapeutic strategy (use of a specific heart rate lowering drug) and a potential new therapeutic strategy (biopacemaking) are reviewed.

Anger, emotion, and arrhythmias: from brain to heart.

Strong emotion and mental stress are now recognized as playing a significant role in severe and fatal ventricular arrhythmias. The mechanisms, although incompletely understood, include central processing at the cortical and brain stem level, the autonomic nerves and the electrophysiology of the myocardium. Each of these is usually studied separately by investigators from different disciplines. However, many are regulatory processes which incorporate interactive feedforward and feedback mechanisms. In this review we consider the whole as an integrated interactive brain–heart system.

Functional, Anatomical, and Molecular Investigation of the Cardiac Conduction System and Arrhythmogenic Atrioventricular Ring Tissue in the Rat Heart

Background The cardiac conduction system consists of the sinus node, nodal extensions, atrioventricular (AV) node, penetrating bundle, bundle branches, and Purkinje fibers. Node‐like AV ring tissue also exists at the AV junctions, and the right and left rings unite at the retroaortic node. The study aims were to (1) construct a 3‐dimensional anatomical model of the AV rings and retroaortic node, (2) map electrical activation in the right ring and study its action potential characteristics, and (3) examine gene expression in the right ring and retroaortic node. Methods and Results Three‐dimensional reconstruction (based on magnetic resonance imaging, histology, and immunohistochemistry) showed the extent and organization of the specialized tissues (eg, how the AV rings form the right and left nodal extensions into the AV node). Multiextracellular electrode array and microelectrode mapping of isolated right ring preparations revealed robust spontaneous activity with characteristic diastolic depolarization. Using laser microdissection gene expression measured at the mRNA level (using quantitative PCR) and protein level (using immunohistochemistry and Western blotting) showed that the right ring and retroaortic node, like the sinus node and AV node but, unlike ventricular muscle, had statistically significant higher expression of key transcription factors (including Tbx3, Msx2, and Id2) and ion channels (including HCN4, Cav3.1, Cav3.2, Kv1.5, SK1, Kir3.1, and Kir3.4) and lower expression of other key ion channels (Nav1.5 and Kir2.1). Conclusions The AV rings and retroaortic node possess gene expression profiles similar to that of the AV node. Ion channel expression and electrophysiological recordings show the AV rings could act as ectopic pacemakers and a source of atrial tachycardia.

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.

Spontaneous and electrically evoked Ca2+ transients in cardiomyocytes of the rat pulmonary vein

The pulmonary vein is surrounded by an external sleeve of cardiomyocytes that are widely recognised to play an important role in atrial fibrillation. While intracellular Ca(2+) is thought to influence the electrical activity of cardiomyocytes, there have been relatively few studies examining Ca(2+) signalling in these cells. Therefore, using fluo-4 and fluorescence imaging microscopy, we have investigated Ca(2+) signalling in an intact section of the rat pulmonary vein. Under resting conditions cardiomyocytes displayed spontaneous Ca(2+) transients, which were variable in amplitude and had a frequency of 1.6±0.03Hz. The Ca(2+) transients were asynchronous amongst neighbouring cardiomyocytes and tended to propagate throughout the cell as a wave. Removing extracellular Ca(2+) produced a slight reduction in the amplitude and frequency of the spontaneous Ca(2+) transients; however, ryanodine (20μM) had a much greater effect on the amplitude and reduced the frequency by 94±2%. Blocking IP(3) receptors with 2-aminoethoxydiphenyl borate (20μM) also reduced the amplitude and frequency (by 73±11%) of these events, indicating the importance of Ca(2+) release from the SR. Electrical field stimulation of the pulmonary vein produced Ca(2+) transients in cardiomyocytes that were significantly reduced by either voltage-gated Ca(2+) channel blockers or ryanodine.

Atrioventricular Node Dysfunction and Ion Channel Transcriptome in Pulmonary Hypertension

Background—Heart block is associated with pulmonary hypertension, and the aim of the study was to test the hypothesis that the heart block is the result of a change in the ion channel transcriptome of the atrioventricular (AV) node. Methods and Results—The most commonly used animal model of pulmonary hypertension, the monocrotaline-injected rat, was used. The functional consequences of monocrotaline injection were determined by echocardiography, ECG recording, and electrophysiological experiments on the Langendorff-perfused heart and isolated AV node. The ion channel transcriptome was measured by quantitative PCR, and biophysically detailed computer modeling was used to explore the changes observed. After monocrotaline injection, echocardiography revealed the pattern of pulmonary artery blood flow characteristic of pulmonary hypertension and right-sided hypertrophy and failure; the Langendorff-perfused heart and isolated AV node revealed dysfunction of the AV node (eg, 50% incidence of heart block in isolated AV node); and quantitative PCR revealed a widespread downregulation of ion channel and related genes in the AV node (eg, >50% downregulation of Cav1.2/3 and HCN1/2/4 channels). Computer modeling predicted that the changes in the transcriptome if translated into protein and function would result in heart block. Conclusions—Pulmonary hypertension results in a derangement of the ion channel transcriptome in the AV node, and this is the likely cause of AV node dysfunction in this disease.

Ca2+-clock-dependent pacemaking in the sinus node is impaired in mice with a cardiac specific reduction in SERCA2 abundance

Background: The sarcoplasmic reticulum Ca2+-ATPase (SERCA2) pump is an important component of the Ca2+-clock pacemaker mechanism that provides robustness and flexibility to sinus node pacemaking. We have developed transgenic mice with reduced cardiac SERCA2 abundance (Serca2 KO) as a model for investigating SERCA2s role in sinus node pacemaking. Methods and Results: In Serca2 KO mice, ventricular SERCA2a protein content measured by Western blotting was 75% (P < 0.05) lower than that in control mice (Serca2 FF) tissue. Immunofluorescent labeling of SERCA2a in ventricular, atrial, sinus node periphery and center tissue sections revealed 46, 45, 55, and 34% (all P < 0.05 vs. Serca2 FF) lower labeling, respectively and a mosaic pattern of expression. With telemetric ECG surveillance, we observed no difference in basal heart rate, but the PR-interval was prolonged in Serca2 KO mice: 49 ± 1 vs. 40 ± 1 ms (P < 0.001) in Serca2 FF. During exercise, heart rate in Serca2 KO mice was elevated to 667 ± 22 bpm, considerably less than 780 ± 17 bpm (P < 0.01) in Serca2 FF. In isolated sinus node preparations, 2 mM Cs+ caused bradycardia that was equally pronounced in Serca2 KO and Serca2 FF (32 ± 4% vs. 29 ± 5%), indicating no change in the pacemaker current, If. Disabling the Ca2+-clock with 2 μM ryanodine induced bradycardia that was less pronounced in Serca2 KO preparations (9 ± 1% vs. 20 ± 3% in Serca2 FF; P < 0.05), suggesting a disrupted Ca2+-clock. Mathematical modeling was used to dissect the effects of membrane- and Ca2+-clock components on Serca2 KO mouse heart rate and sinus node action potential. Computer modeling predicted a slowing of heart rate with SERCA2 downregulation and the heart rate slowing was pronounced at >70% reduction in SERCA2 activity. Conclusions: Serca2 KO mice show a disrupted Ca2+-clock-dependent pacemaker mechanism contributing to impaired sinus node and atrioventricular node function.

Molecular Basis of Arrhythmias Associated with the Cardiac Conduction System

The cardiac conduction system is responsible for the initiation and coordination of the heartbeat. It consists of three central components: the sinus node, the atrioventricular node, and the His-Purkinje system. Since the discovery of the sinus node in 1907, the cardiac conduction system has been a topic of immense interest to basic science and clinical researchers investigating the function/dysfunction of the heart. In the last 10 years, our understanding of the system has been immensely enriched. We now know that the system has specialized (different from that in the working myocardium) expression profile of ion channels, intracellular Ca2+-handling proteins, and gap junction channels that are appropriate for its functioning, although there is continued debate concerning the ionic mechanisms underlying pacemaking. We are beginning to understand the mechanisms responsible for cardiac conduction system dysfunction in disease and appreciate how naturally occurring ion channel mutations cause congenital cardiac conduction system dysfunction. In this chapter we present the molecular basis of arrhythmias associated with the cardiac conduction system, with particular emphasis on recent developments in the field.

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.