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Abstract Tomato pomace (TP) is an underutilized source of bioactive compounds with potential application in the food sector. A factorial experiment was designed to compare three culinary techniques, Thermomix®, Roner®, and traditional pan-frying, for the preparation of tomato sauces, enriched or not with TP, applying two temperatures and two cooking times. A multivariate analysis was performed on all the results obtained for the metabolites. The addition of TP significantly increased the content of bioactive compounds, especially phenolic compounds. OPLS-DA models were generated using cooking technique, temperature, and time as discriminant factors. The cooking technique had a greater effect on the phenolic content than cooking temperature or time. Thermomix® released bioactive compounds from the tomato into the sauce to a similar extent as pan-frying. Roner® proved to be effective in preserving the volatile fraction of the sauce. The Thermomix® significantly increased the amount of bioactive compounds, while the Roner® increased the volatile compounds.

Abstract L-type voltage-gated calcium channels are involved in multiple physiological functions. Currently available antagonists do not discriminate between L-type channel isoforms. Importantly, no selective blocker is available to dissect the role of L-type isoforms Cav1.2 and Cav1.3 that are concomitantly co-expressed in the heart, neuroendocrine and neuronal cells. Here we show that calciseptine, a snake toxin purified from mamba venom, selectively blocks Cav1.2 -mediated L-type calcium currents (ICaL) at concentrations leaving Cav1.3-mediated ICaL unaffected in both native cardiac myocytes and HEK-293T cells expressing recombinant Cav1.2 and Cav1.3 channels. Functionally, calciseptine potently inhibits cardiac contraction without altering the pacemaker activity in sino-atrial node cells, underscoring differential roles of Cav1.2− and Cav1.3 in cardiac contractility and automaticity. In summary, calciseptine is a selective L-type Cav1.2 Ca2+ channel blocker and should be a valuable tool to dissect the role of these L-channel isoforms.

Abstract Microcebus murinus, or gray mouse lemur (GML), is one of the smallest primates known, with a size in between mice and rats. The small size, genetic proximity to humans and prolonged senescence, make this lemur an emerging model for neurodegenerative diseases. For the same reasons, it could help understand how aging affects cardiac activity. Here, we provide the first characterization of sinoatrial (SAN) pacemaker activity and of the effect of aging on GML heart rate (HR). According to GML size, its heartbeat and intrinsic pacemaker frequencies lie in between those of mice and rats. To sustain this fast automaticity the GML SAN expresses funny and Ca2+ currents (I f , I Ca,L and I Ca,T ) at densities similar to that of small rodents. SAN automaticity was also responsive to β-adrenergic and cholinergic pharmacological stimulation, showing a consequent shift in the localization of the origin of pacemaker activity. We found that aging causes decrease of basal HR and atrial remodeling in GML. We also estimated that, over 12 years of a lifetime, GML generates about 3 billion heartbeats, thus, as many as humans and three times more than rodents of equivalent size. In addition, we estimated that the high number of heartbeats per lifetime is a characteristic that distinguishes primates from rodents or other eutherian mammals, independently from body size. Thus, cardiac endurance could contribute to the exceptional longevity of GML and other primates, suggesting that GML’s heart sustains a workload comparable to that of humans in a lifetime. In conclusion, despite the fast HR, GML replicates some of the cardiac deficiencies reported in old people, providing a suitable model to study heart rhythm impairment in aging. Moreover, we estimated that, along with humans and other primates, GML presents a remarkable cardiac longevity, enabling longer life span than other mammals of equivalent size.

BackgroundAcute myocardial infarction (AMI) is the major cause of cardiovascular mortality worldwide. Most ischemic episodes are triggered by an increase in heart rate, which induces an imbalance between myocardial oxygen delivery and consumption. Developing drugs that selectively reduce heart rate by inhibiting ion channels involved in heart rate control could provide more clinical benefits. The Cav1.3-mediated L-type Ca2+ current (ICav1.3) play important roles in the generation of heart rate. Therefore, they can constitute relevant targets for selective control of heart rate and cardioprotection during AMI.ObjectiveWe aimed to investigate the relationship between heart rate and infarct size using mouse strains knockout for Cav1.3 (Cav1.3−/−) L-type calcium channel and of the cardiac G protein gated potassium channel (Girk4−/−) in association with the funny (f)-channel inhibitor ivabradine.MethodsWild-type (WT), Cav1.3+/−, Cav1.3−/− and Girk4−/− mice were used as models of respectively normal heart rate, moderate heart rate reduction, bradycardia, and mild tachycardia, respectively. Mice underwent a surgical protocol of myocardial IR (40 min ischemia and 60 min reperfusion). Heart rate was recorded by one-lead surface ECG recording, and infarct size measured by triphenyl tetrazolium chloride staining. In addition, Cav1.3−/− and WT hearts perfused on a Langendorff system were subjected to the same ischemia-reperfusion protocol ex vivo, without or with atrial pacing, and the coronary flow was recorded.ResultsCav1.3−/− mice presented reduced infarct size (−29%), while Girk4−/− displayed increased infarct size (+30%) compared to WT mice. Consistently, heart rate reduction in Cav1.3+/− or by the f-channel blocker ivabradine was associated with significant decrease in infarct size (−27% and −32%, respectively) in comparison to WT mice.ConclusionOur results show that decreasing heart rate allows to protect the myocardium against IR injury in vivo and reveal a close relationship between basal heart rate and IR injury. In addition, this study suggests that targeting Cav1.3 channels could constitute a relevant target for reducing infarct size, since maximal heart rate dependent cardioprotective effect is already observed in Cav1.3+/− mice.

Bradyarrhythmias including sinus bradycardia and atrioventricular (AV) block are frequently encountered in endurance athletes especially at night. While these are well tolerated by the young athlete, there is evidence that generally from the fifth decade of life onward, such arrhythmias can degenerate into pathological symptomatic bradycardia requiring pacemaker therapy. For many years, athletic bradycardia and AV block have been attributed to high vagal tone, but work from our group has questioned this widely held assumption and demonstrated a role for intrinsic electrophysiological remodeling of the sinus node and the AV node. In this article, we argue that bradyarrhythmias in the veteran athlete arise from the cumulative effects of exercise training, the circadian rhythm and aging on the electrical activity of the nodes. We consider contemporary strategies for the treatment of symptomatic bradyarrhythmias in athletes and highlight potential therapies resulting from our evolving mechanistic understanding of this phenomenon. [Display omitted] [ABSTRACT FROM AUTHOR]

The understanding of the electrophysiological mechanisms that underlie mechanosensitivity of the sinoatrial node (SAN), the primary pacemaker of the heart, has been evolving over the past century. The heart is constantly exposed to a dynamic mechanical environment; as such, the SAN has numerous canonical and emerging mechanosensitive ion channels and signaling pathways that govern its ability to respond to both fast (within second or on beat-to-beat manner) and slow (minutes) timescales. This review summarizes the effects of mechanical loading on the SAN activity and reviews putative candidates, including fast mechanoactivated channels (Piezo, TREK, and BK) and slow mechanoresponsive ion channels [including volume-regulated chloride channels and transient receptor potential (TRP)], as well as the components of mechanochemical signal transduction, which may contribute to SAN mechanosensitivity. Furthermore, we examine the structural foundation for both mechano-electrical and mechanochemical signal transduction and discuss the role of specialized membrane nanodomains, namely, caveolae, in mechanical regulation of both membrane and calcium clock components of the so-called coupled-clock pacemaker system responsible for SAN automaticity. Finally, we emphasize how these mechanically activated changes contribute to the pathophysiology of SAN dysfunction and discuss controversial areas necessitating future investigations. Though the exact mechanisms of SAN mechanosensitivity are currently unknown, identification of such components, their impact into SAN pacemaking, and pathological remodeling may provide new therapeutic targets for the treatment of SAN dysfunction and associated rhythm abnormalities.

The pacemaker cells of the cardiac sinoatrial node (SAN) are essential for normal cardiac automaticity. Dysfunction in cardiac pacemaking results in human sinoatrial node dysfunction (SND). SND more generally occurs in the elderly population and is associated with impaired pacemaker function causing abnormal heart rhythm. Individuals with SND have a variety of symptoms including sinus bradycardia, sinus arrest, SAN block, bradycardia/tachycardia syndrome, and syncope. Importantly, individuals with SND report chronotropic incompetence in response to stress and/or exercise. SND may be genetic or secondary to systemic or cardiovascular conditions. Current management of patients with SND is limited to the relief of arrhythmia symptoms and pacemaker implantation if indicated. Lack of effective therapeutic measures that target the underlying causes of SND renders management of these patients challenging due to its progressive nature and has highlighted a critical need to improve our understanding of its underlying mechanistic basis of SND. This review focuses on current information on the genetics underlying SND, followed by future implications of this knowledge in the management of individuals with SND.

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” (If) current in the sinoatrial node (SAN).Objective: To test if genetic ablation of G-protein-gated inwardly rectifying potassium channel, also known as IKACh 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 IKACh 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 IKACh block whereas Girk4–/– mice failed to develop a training-induced sinus bradycardia. In line with HR reduction, action potential rate, density of If, as well as of T- and L-type Ca2+ currents (ICaT and ICaL) were significantly reduced only in SAN cells obtained from WT-trained mice. If reduction in WT mice was concomitant with downregulation of HCN4 transcript and protein, attributable to increased expression of corresponding repressor microRNAs (miRs) whereas reduced ICaL in WT mice was associated with reduced Cav1.3 protein levels. Strikingly, IKACh ablation suppressed all training-induced molecular remodeling observed in WT mice.Conclusion: Genetic ablation of cardiac IKACh in mice prevents exercise-induced sinus bradycardia by suppressing training induced remodeling of inward currents If, ICaT and ICaL due in part to the prevention of miR-mediated transcriptional remodeling of HCN4 and likely post transcriptional remodeling of Cav1.3. Strategies targeting cardiac IKACh may therefore represent an alternative to pacemaker implantation for bradyarrhythmias seen in some veteran athletes.

Background: Sinoatrial node cells (SANC) automaticity is generated by functional association between the activity of plasmalemmal ion channels and local diastolic intracellular Ca2+ release (LCR) from ryanodine receptors. Strikingly, most isolated SANC exhibit a “dormant” state, whereas only a fraction shows regular firing as observed in intact SAN. Recent studies showed that β-adrenergic stimulation can initiate spontaneous firing in dormant SANC, though this mechanism is not entirely understood. Methods: To investigate the role of L-type Cav1.3 Ca2+ channels in the adrenergic regulation of automaticity in dormant SANC, we used a knock-in mouse strain in which the sensitivity of L-type Cav1.2 α1 subunits to dihydropyridines (DHPs) was inactivated (Cav1.2DHP−/−), enabling the selective pharmacological inhibition of Cav1.3 by DHPs. Results: In dormant SANC, β-adrenergic stimulation with isoproterenol (ISO) induced spontaneous action potentials (AP) and Ca2+ transients, which were completely arrested with concomitant perfusion of the DHP nifedipine. In spontaneously firing SANC at baseline, Cav1.3 inhibition completely reversed the effect of β-adrenergic stimulation on AP and the frequency of Ca2+ transients. Confocal calcium imaging of SANC showed that the β-adrenergic-induced synchronization of LCRs is regulated by the activity of Cav1.3 channels. Conclusions: Our study shows a novel role of Cav1.3 channels in initiating and maintaining automaticity in dormant SANC upon β-adrenergic stimulation.

Questo contributo si propone di condividere i risultati di un percorso di Ricerca Azione svolto nell’ambito del Master di Secondo Livello in Progettazione avanzata dell’insegnamento della lingua e cultura italiana a stranieri (Itals) dell’Università di Venezia. Il percorso – svoltosi nell’ambito della mia attività didattica di docente di Italiano L3 alla Scuola Europea di Bruxelles II – è stato dedicato a valutare l’efficacia di un’analisi del testo letterario affine a quella solitamente proposta in Italiano L1, a partire dall’ipotesi che la qualità approfondita dell’analisi potenziasse la produzione scritta, ossia mettesse in atto un processo virtuoso di “trascinamento” della qualità della lingua scritta ad essa sottesa. Keywords: italiano L3, analisi del testo letterario, figure retoriche, fluency, produzione scritta

Abstract The spontaneous activity of sinoatrial node (SAN) pacemaker cells is generated by a functional interplay between the activity of ionic currents of the plasma membrane and intracellular Ca2+ dynamics. The molecular correlate of a dihydropyridine (DHP)-sensitive sustained inward Na+ current (I st), a key player in SAN automaticity, is still unknown. Here we show that I st and the L-type Ca2+ current (I Ca,L) share CaV1.3 as a common molecular determinant. Patch-clamp recordings of mouse SAN cells showed that I st is activated in the diastolic depolarization range, and displays Na+ permeability and minimal inactivation and sensitivity to I Ca,L activators and blockers. Both CaV1.3-mediated I Ca,L and I st were abolished in CaV1.3-deficient (CaV1.3−/−) SAN cells but the CaV1.2-mediated I Ca,L current component was preserved. In SAN cells isolated from mice expressing DHP-insensitive CaV1.2 channels (CaV1.2DHP−/−), I st and CaV1.3-mediated I Ca,L displayed overlapping sensitivity and concentration–response relationships to the DHP blocker nifedipine. Consistent with the hypothesis that CaV1.3 rather than CaV1.2 underlies I st, a considerable fraction of I Ca,L was resistant to nifedipine inhibition in CaV1.2DHP−/− SAN cells. These findings identify CaV1.3 channels as essential molecular components of the voltage-dependent, DHP-sensitive I st Na+ current in the SAN.

Binding of TRIP8b to the cyclic nucleotide binding domain (CNBD) of mammalian hyperpolarization-activated cyclic nucleotide-gated (HCN) channels prevents their regulation by cAMP. Since TRIP8b is expressed exclusively in the brain, we envisage that it can be used for orthogonal control of HCN channels beyond the central nervous system. To this end, we have identified by rational design a 40-aa long peptide (TRIP8bnano) that recapitulates affinity and gating effects of TRIP8b in HCN isoforms (hHCN1, mHCN2, rbHCN4) and in the cardiac current If in rabbit and mouse sinoatrial node cardiomyocytes. Guided by an NMR-derived structural model that identifies the key molecular interactions between TRIP8bnano and the HCN CNBD, we further designed a cell-penetrating peptide (TAT-TRIP8bnano) which successfully prevented β-adrenergic activation of mouse If leaving the stimulation of the L-type calcium current (ICaL) unaffected. TRIP8bnano represents a novel approach to selectively control HCN activation, which yields the promise of a more targeted pharmacology compared to pore blockers.

Circadian clocks drive biological rhythms with a period of approximately 24 hours and keep in time with the outside world through daily resetting by environmental cues. While this external entrainment has been extensively investigated in the suprachiasmatic nuclei (SCN), the role of internal systemic rhythms, including daily fluctuations in core temperature or circulating hormones remains debated. Here, we show that lactating mice, which exhibit dampened systemic rhythms, possess normal molecular clockwork but impaired rhythms in both heat shock response gene expression and electrophysiological output in their SCN. This suggests that body rhythms regulate SCN activity downstream of the clock. Mathematical modeling predicts that systemic feedback upon the SCN functions as an internal oscillator that accounts for in vivo and ex vivo observations. Thus we are able to propose a new bottom-up hierarchical organization of circadian timekeeping in mammals, based on the interaction in the SCN between clock-dependent and system-driven oscillators.

This study concerns the effects of a weak static magnetic field (MF) at 10 μT oriented downward, combined with a 16-Hz sinusoidal MF (10 μT), on in vitro pollen germination of kiwifruit (Actinidia deliciosa). Extremely low frequency magnetic field (ELF-MF) exposure was carried out by a signal generator unit connected to a copper wire solenoid, inside which samples where placed. Two different kinds of treatment were performed: direct and indirect. In the direct treatment, pollen samples were directly exposed during rehydration, germination, or both. In the indirect treatment, the pollen growth medium was prepared with water aliquots (at standard temperature of 20°C and pH = 6.74) that were exposed before use for 8 or 24 h. The main purpose of our research was to identify a biological marker (in vitro pollen germination in a stressing growth medium without Ca2+) susceptible to the effects of direct or indirect ELF-MF exposure. The working variable was the pollen germination rate, as detected blind after 3 h 30 min by an Axioplan microscope. A directionally consistent recovery of germination percentage was observed both for direct exposure (during germination and both rehydration and germination phases) and water-mediated exposure (with water exposed for 24 h and immediately used). Our results suggest that the ELF-MF treatment might partially remove the inhibitory effect caused by the lack of Ca2+ in the culture medium, inducing a release of internal Ca2+ stored in the secretory vesicles of pollen plasma membrane. Although preliminary, findings seem to indicate the in vitro pollen performance as adequate to study the effects of ELF-MFs on living matter.

The mechanisms underlying heart rate (HR) acceleration by catecholamines are incompletely understood. It is widely accepted that cAMP-dependent regulation of HCN4 channels and the activation of the Na+/Ca2+exchanger by ryanodine receptors mediated Ca2+ release are the predominant mechanisms of HR acceleration. However, experimental evidence demonstrates that none of these mechanisms is essential for sympathetic regulation of HR, suggesting that other ones could be involved in the positive chronotropic effect of β-adrenoceptors. The sinoatrial node expresses two distinct L-type Ca2+ channel isoforms, Cav1.2, which controls myocardial contractility and Cav1.3. We previously demonstrated that Cav1.3 channels play a major role in the generation of sinoatrial pacemaker activity and impulse conduction. Here we study the role of Cav1.3 in the acceleration of HR by catecholamines. We crossed mice in which Cav1.2 channels are rendered insensitive to dihydropyridines (DHP) (Cav1.2DHP-/-), with mice lacking cAMP dependent regulation of HCN4 (HCN4-CNBD). Cav1.2DHP-/-/HCN4-CNBD mice enable selective inhibition of Cav1.3 by DHP amlodipine (3 μM) or nifedipine (3 μM), or activation by BayK8644 (0.5 μM), in the absence of any possible HCN4 modulation by cAMP. We then studied HR regulation ex-vivo using the isolated Langendorff-perfused heart. In n = 8 Cav1.2DHP-/-/HCN4-CNBD hearts, adrenaline (ADR) failed to significantly increase HR after selective Cav1.3 inhibition by amlodipine (207 ± 19bpm vs 220 ± 17bpm n = 8 P > 0.05 respectively), showing that inhibition of Cav1.3 in the absence of cAMP-dependent regulation of HCN4 channels prevented the positive chronotropic response of the heart and suggesting that catecholaminergic regulation of HR was reliant on Cav1.3 in this genotype. Consistently, activation of CaV1.3 by BayK8644 in Cav1.2DHP-/-/HCN4-CNBD hearts increased HR similarly to ADR in the same hearts (+33.1 ± 7.4% vs. +28.6 ± 4.9% n = 8 P > 0.05), suggesting that in the absence of cAMP-dependent regulation of HCN4, β-adrenergic regulation of Cav1.3 channels is sufficient to explain the increase in HR by catecholamines. Our study thus identifies, for the first time, Cav1.3 channels as key effectors of β-adrenergic regulation of heart rate acceleration. [ABSTRACT FROM AUTHOR]

In everyday life, cardiac automaticity is generated in the sino-atrial node (SAN) by specialised myocytes called pacemaker cells. Failure to generate a normal SAN impulse causes Sinus Node Dysfunction (SND). SND may be inherited (primary SND) or secondary to systemic or cardiovascular conditions (secondary SND). We have previously demonstrated that genetic ablation or pharmacologic inhibition of G protein gated K+ GIRK4 channels abrogates bradyarrhythmia in models of primary and secondary SND, but the mechanisms underlying rescuing of heart rhythm are not fully elucidated. In this context, a decrease in intrinsic electrical coupling due to progressive tissue fibrosis has been proposed as a primary factor in age-related SND, but its role in other forms of SND is currently unknown. To test if genetic inhibition of Girk4 channel rescues cardiac conduction defects and fibrosis in Cav1.3-/- mouse model of primary SND. We performed telemetric electrocardiogram recordings in control, Girk4-/-, Cav1.3-/- and GIRK4 -/-/Cav1.3-/- mice. Animals were then euthanized, and SAN tissues were isolated for investigations of pacemaker activity by optical and electrical mapping. Immunohistochemistry (IHC) staining was performed to study fibrosis in SAN tissues. P-wave duration, PR and RR intervals were significantly longer in Cav1.3-/- than in WT and GIRK4 mice, suggesting conduction troubles and enhanced fibrotic tissue in Cav1.3-/- animals. Genetic ablation of Girk4 channels in Cav1.3-/- animals significantly shorten P-wave duration (in ms: 17.7 ± 0.8 Cav1.3-/- vs. 14.3 ± 0.2** GIRK4-/-/Cav1.3-/-) and both PR (in ms: 59 ± 1 Cav1.3-/- vs. 44 ± 1**** GIRK4-/-/Cav1.3-/-) and RR intervals (172 ± 12 Cav1.3-/- vs. 137 ± 5* GIRK4-/-/Cav1.3-/-) to WT values. Shift of the leading pacemaker site (defined by optical mapping experiments) and defects of conduction (quantified by fractionation index in MEA electrograms) recorded in Cav1.3-/- SAN isolated tissue were restored to control condition by genetic ablation of Girk4 in SND tissue. IHC experiments showed more 8% of fibrotic tissue (Sirius red histology) in Cav1.3-/- than in WT SAN tissue. GIRK4-/-/Cav1.3-/- SAN tissue fibrosis was strongly ameliorated. In the context of SND generated by Cav1.3 ablation, concomitant inhibition of Girk4 normalises heart rate by restoring normal cardiac conduction pathway by limiting fibrosis in SAN tissue. [ABSTRACT FROM AUTHOR]

Human induced pluripotent stem cells derived-cardiomyocytes (hiPSC-CMs) are widely recognized as a model to study several cardiac disorders. In this perspective, differentiation of hiPSCs into sinoatrial node (SAN)-like cells may represent a significant advance in providing an in vitro platform for exploring SAN dysfunction (SND) and evaluate new therapeutic approaches. Recently, hiPSC-CMs derived from patient carrying mutation in the cardiac type 2 ryanodine receptor (RyR2-H29D) have been linked to catecholaminergic polymorphic ventricular tachycardia (CPVT), in association with SND-dependent severe bradycardia. We aimed to obtain hiPSCs-derived cardiac pacemaker cells (SAN-hiPSCs) from RyR2-H29D mutated patient and characterize these cells at electrophysiological level. hiPSCs derived from patients with CPVT and healthy subjects were differentiated into SAN-hiPSCs using a 2D matrix sandwich method, modulating Wnt signaling. Moreover, cells were treated with triiodothyronine, dexamethasone and intracellular cyclic AMP (DTA) "cocktail" to improve cell maturity, including intracellular Ca2+ handling. At day 40 post-differentiation, electrical activity was assessed by patch-clamp. Significant decrease in action potential (AP) frequency was observed in SAN-hiPSCs derived from CPVT patients compared to control (Ctrl) ones (Ctrl: 157.5 ± 9.9 bpm; CPVT: 38.4 ± 13.6 bpm). RyR2-H29D mutation did not affect pacemaker "funny" current (If) (@–135 mV Ctrl: –34.9 ± 16.7 pA/pF; CPVT: –21.8 ± 2.8 pA/pF). SAN-hiPSCs derived from CPVT patient present a bradycardic phenotype compared to SAN-hiPSCs derived from healthy patient and consistent with patient clinical feature. Bradycardia is not related to alteration of If. Nevertheless, G protein-gated inwardly rectifying potassium current (IKACh), L- and T-type Ca2+ currents as well as intracellular Ca2+ handling, involved in cardiac automaticity, remain to be evaluated. Finally, recent studies have demonstrated that the genetic ablation or pharmacological inhibition of IKACh prevents SND-dependent bradycardia. In the future, this therapeutic strategy could be tested to rescue bradycardia in our model of SAN-hiPSCs derived from CPVT patient. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]