Cardiac GR Mediates the Diurnal Rhythm in Ventricular Arrhythmia Susceptibility.

BACKGROUND: Ventricular arrhythmias (VAs) demonstrate a prominent day-night rhythm, commonly presenting in the morning. Transcriptional rhythms in cardiac ion channels accompany this phenomenon, but their role in the morning vulnerability to VAs and the underlying mechanisms are not understood. We investigated the recruitment of transcription factors that underpins transcriptional rhythms in ion channels and assessed whether this mechanism was pertinent to the heart's intrinsic diurnal susceptibility to VA. METHODS AND RESULTS: Assay for transposase-accessible chromatin with sequencing performed in mouse ventricular myocyte nuclei at the beginning of the animals' inactive (ZT0) and active (ZT12) periods revealed differentially accessible chromatin sites annotating to rhythmically transcribed ion channels and distinct transcription factor binding motifs in these regions. Notably, motif enrichment for the glucocorticoid receptor (GR; transcriptional effector of corticosteroid signaling) in open chromatin profiles at ZT12 was observed, in line with the well-recognized ZT12 peak in circulating corticosteroids. Molecular, electrophysiological, and in silico biophysically-detailed modeling approaches demonstrated GR-mediated transcriptional control of ion channels (including Scn5a underlying the cardiac Na+ current, Kcnh2 underlying the rapid delayed rectifier K+ current, and Gja1 responsible for electrical coupling) and their contribution to the day-night rhythm in the vulnerability to VA. Strikingly, both pharmacological block of GR and cardiomyocyte-specific genetic knockout of GR blunted or abolished ion channel expression rhythms and abolished the ZT12 susceptibility to pacing-induced VA in isolated hearts. CONCLUSIONS: Our study registers a day-night rhythm in chromatin accessibility that accompanies diurnal cycles in ventricular myocytes. Our approaches directly implicate the cardiac GR in the myocyte excitability rhythm and mechanistically link the ZT12 surge in glucocorticoids to intrinsic VA propensity at this time.

Immediate and sustained increases in the activity of vagal preganglionic neurons during exercise and after exercise training.

AIMS: The brain controls the heart by dynamic recruitment and withdrawal of cardiac parasympathetic (vagal) and sympathetic activity. Autonomic control is essential for the development of cardiovascular responses during exercise, however, the patterns of changes in the activity of the two autonomic limbs, and their functional interactions in orchestrating physiological responses during exercise, are not fully understood. The aim of this study was to characterize changes in vagal parasympathetic drive in response to exercise and exercise training by directly recording the electrical activity of vagal preganglionic neurons in experimental animals (rats). METHODS AND RESULTS: Single unit recordings were made using carbon-fibre microelectrodes from the populations of vagal preganglionic neurons of the nucleus ambiguus (NA) and the dorsal vagal motor nucleus of the brainstem. It was found that (i) vagal preganglionic neurons of the NA and the dorsal vagal motor nucleus are strongly activated during bouts of acute exercise, and (ii) exercise training markedly increases the resting activity of both populations of vagal preganglionic neurons and augments the excitatory responses of NA neurons during exercise. CONCLUSIONS: These data show that central vagal drive increases during exercise and provide the first direct neurophysiological evidence that exercise training increases vagal tone. The data argue against the notion of exercise-induced central vagal withdrawal during exercise. We propose that robust increases in the activity of vagal preganglionic neurons during bouts of exercise underlie activity-dependent plasticity, leading to higher resting vagal tone that confers multiple health benefits associated with regular exercise.

Non-canonical role of the sympathetic nervous system in the day-night rhythm in heart rate.

Although, for many decades, the day-night rhythm in resting heart rate has been attributed to the parasympathetic branch of the autonomic nervous system (high vagal tone during sleep), recently we have shown that there is a circadian clock in the cardiac pacemaker, the sinus node, and the day-night rhythm in heart rate involves an intrinsic rhythmic transcriptional remodelling of pacemaker ion channels, particularly Hcn4. We have now investigated the role of the sympathetic branch of the autonomic nervous system in this and shown it to have a non-canonical role. In mice, sustained long-term block of cardiac ß-adrenergic receptors by propranolol administered in the drinking water abolished the day-night rhythm in pacemaking in the isolated sinus node. Concomitant with this, there was a loss of the normal day-night rhythm in many pacemaker ion channel transcripts. However, there was little or no change in the local circadian clock, indicating that the well-known day-night rhythm in sympathetic nerve activity is directly involved in pacemaker ion channel transcription. The day-night rhythm in pacemaking helps explain the occurrence of clinically significant bradyarrhythmias during sleep, and this study improves our understanding of this pathology. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'.

Proteomic Analysis of Cardiac Adaptation to Exercise by High Resolution Mass Spectrometry.

Regular exercise has many health benefits, among which is a significant reduction of cardiovascular risk. Although many beneficial effects of exercise are well described, the exact mechanisms by which exercise confers cardiovascular benefits are yet to be fully understood. In the current study, we have used high resolution mass spectrometry to determine the proteomic responses of the heart to exercise training in mice. The impact of exercise-induced oxidative stress on modifications of cardiomyocyte proteins with lipid peroxidation biomarker 4-hydroxynonenal (4-HNE) was examined as well. Fourteen male mice were randomized into the control (sedentary) group and the exercise group that was subjected to a swim exercise training program for 5 days a week for 5 months. Proteins were isolated from the left ventricular tissue, fractionated and digested for shotgun proteomics. Peptides were separated by nanoliquid chromatography and analyzed on an Orbitrap Fusion mass spectrometer using high-energy collision-induced dissociation and electron transfer dissociation fragmentation. We identified distinct ventricular protein signatures established in response to exercise training. Comparative proteomics identified 23 proteins that were upregulated and 37 proteins that were downregulated with exercise, in addition to 65 proteins that were identified only in ventricular tissue samples of exercised mice. Most of the proteins specific to exercised mice are involved in respiratory electron transport and/or implicated in glutathione conjugation. Additionally, 10 proteins were found to be modified with 4-HNE. This study provides new data on the effects of exercise on the cardiac proteome and contributes to our understanding of the molecular mechanisms underlying the beneficial effects of exercise on the heart.

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.

Intrinsic Electrical Remodeling Underlies Atrioventricular Block in Athletes.

[Figure: see text].

RNAseq shows an all-pervasive day-night rhythm in the transcriptome of the pacemaker of the heart.

Physiological systems vary in a day-night manner anticipating increased demand at a particular time. Heart is no exception. Cardiac output is primarily determined by heart rate and unsurprisingly this varies in a day-night manner and is higher during the day in the human (anticipating increased day-time demand). Although this is attributed to a day-night rhythm in post-translational ion channel regulation in the heart's pacemaker, the sinus node, by the autonomic nervous system, we investigated whether there is a day-night rhythm in transcription. RNAseq revealed that ~ 44% of the sinus node transcriptome (7134 of 16,387 transcripts) has a significant day-night rhythm. The data revealed the oscillating components of an intrinsic circadian clock. Presumably this clock (or perhaps the master circadian clock in the suprachiasmatic nucleus) is responsible for the rhythm observed in the transcriptional machinery, which in turn is responsible for the rhythm observed in the transcriptome. For example, there is a rhythm in transcripts responsible for the two principal pacemaker mechanisms (membrane and Ca2+ clocks), transcripts responsible for receptors and signalling pathways known to control pacemaking, transcripts from genes identified by GWAS as determinants of resting heart rate, and transcripts from genes responsible for familial and acquired sick sinus syndrome.

A circadian clock in the sinus node mediates day-night rhythms in Hcn4 and heart rate.

BACKGROUND: Heart rate follows a diurnal variation, and slow heart rhythms occur primarily at night. OBJECTIVE: The lower heart rate during sleep is assumed to be neural in origin, but here we tested whether a day-night difference in intrinsic pacemaking is involved. METHODS: In vivo and in vitro electrocardiographic recordings, vagotomy, transgenics, quantitative polymerase chain reaction, Western blotting, immunohistochemistry, patch clamp, reporter bioluminescence recordings, and chromatin immunoprecipitation were used. RESULTS: The day-night difference in the average heart rate of mice was independent of fluctuations in average locomotor activity and persisted under pharmacological, surgical, and transgenic interruption of autonomic input to the heart. Spontaneous beating rate of isolated (ie, denervated) sinus node (SN) preparations exhibited a day-night rhythm concomitant with rhythmic messenger RNA expression of ion channels including hyperpolarization-activated cyclic nucleotide-gated potassium channel 4 (HCN4). In vitro studies demonstrated 24-hour rhythms in the human HCN4 promoter and the corresponding funny current. The day-night heart rate difference in mice was abolished by HCN block, both in vivo and in the isolated SN. Rhythmic expression of canonical circadian clock transcription factors, for example, Brain and muscle ARNT-Like 1 (BMAL1) and Cryptochrome (CRY) was identified in the SN and disruption of the local clock (by cardiomyocyte-specific knockout of Bmal1) abolished the day-night difference in Hcn4 and intrinsic heart rate. Chromatin immunoprecipitation revealed specific BMAL1 binding sites on Hcn4, linking the local clock with intrinsic rate control. CONCLUSION: The circadian variation in heart rate involves SN local clock-dependent Hcn4 rhythmicity. Data reveal a novel regulator of heart rate and mechanistic insight into bradycardia during sleep.

Genetic Ablation of G Protein-Gated Inwardly Rectifying K+ Channels Prevents Training-Induced Sinus Bradycardia.

Background: Endurance athletes are prone to bradyarrhythmias, which in the long-term may underscore the increased incidence of pacemaker implantation reported in this population. Our previous work in rodent models has shown training-induced sinus bradycardia to be due to microRNA (miR)-mediated transcriptional remodeling of the HCN4 channel, leading to a reduction of the "funny" (I f) current in the sinoatrial node (SAN). Objective: To test if genetic ablation of G-protein-gated inwardly rectifying potassium channel, also known as I KACh channels prevents sinus bradycardia induced by intensive exercise training in mice. Methods: Control wild-type (WT) and mice lacking GIRK4 (Girk4 -/-), an integral subunit of I KACh were assigned to trained or sedentary groups. Mice in the trained group underwent 1-h exercise swimming twice a day for 28 days, 7 days per week. We performed electrocardiogram recordings and echocardiography in both groups at baseline, during and after the training period. At training cessation, mice were euthanized and SAN tissues were isolated for patch clamp recordings in isolated SAN cells and molecular profiling by quantitative PCR (qPCR) and western blotting. Results: At swimming cessation trained WT mice presented with a significantly lower resting HR that was reversible by acute I KACh block whereas Girk4 -/- mice failed to develop a training-induced sinus bradycardia. In line with HR reduction, action potential rate, density of I f, as well as of T- and L-type Ca2+ currents (I CaT and I CaL ) were significantly reduced only in SAN cells obtained from WT-trained mice. I f reduction in WT mice was concomitant with downregulation of HCN4 transcript and protein, attributable to increased expression of corresponding repressor microRNAs (miRs) whereas reduced I CaL in WT mice was associated with reduced Cav1.3 protein levels. Strikingly, I KACh ablation suppressed all training-induced molecular remodeling observed in WT mice. Conclusion: Genetic ablation of cardiac I KACh in mice prevents exercise-induced sinus bradycardia by suppressing training induced remodeling of inward currents I f, I CaT and I CaL due in part to the prevention of miR-mediated transcriptional remodeling of HCN4 and likely post transcriptional remodeling of Cav1.3. Strategies targeting cardiac I KACh may therefore represent an alternative to pacemaker implantation for bradyarrhythmias seen in some veteran athletes.