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1.
Int J Mol Sci ; 25(2)2024 Jan 12.
Artículo en Inglés | MEDLINE | ID: mdl-38256054

RESUMEN

Caveolae constitute membrane microdomains where receptors and ion channels functionally interact. Caveolin-3 (cav-3) is the key structural component of muscular caveolae. Mutations in CAV3 lead to caveolinopathies, which result in both muscular dystrophies and cardiac diseases. In cardiomyocytes, cav-1 participates with cav-3 to form caveolae; skeletal myotubes and adult skeletal fibers do not express cav-1. In the heart, the absence of cardiac alterations in the majority of cases may depend on a conserved organization of caveolae thanks to the expression of cav-1. We decided to focus on three specific cav-3 mutations (Δ62-64YTT; T78K and W101C) found in heterozygosis in patients suffering from skeletal muscle disorders. We overexpressed both the WT and mutated cav-3 together with ion channels interacting with and modulated by cav-3. Patch-clamp analysis conducted in caveolin-free cells (MEF-KO), revealed that the T78K mutant is dominant negative, causing its intracellular retention together with cav-3 WT, and inducing a significant reduction in current densities of all three ion channels tested. The other cav-3 mutations did not cause significant alterations. Mathematical modelling of the effects of cav-3 T78K would impair repolarization to levels incompatible with life. For this reason, we decided to compare the effects of this mutation in other cell lines that endogenously express cav-1 (MEF-STO and CHO cells) and to modulate cav-1 expression with an shRNA approach. In these systems, the membrane localization of cav-3 T78K was rescued in the presence of cav-1, and the current densities of hHCN4, hKv1.5 and hKir2.1 were also rescued. These results constitute the first evidence of a compensatory role of cav-1 in the heart, justifying the reduced susceptibility of this organ to caveolinopathies.


Asunto(s)
Caveolina 1 , Caveolina 3 , Adulto , Animales , Cricetinae , Humanos , Caveolina 1/genética , Caveolina 3/genética , Cricetulus , Mutación , Células CHO , Canales Iónicos
2.
Pflugers Arch ; 473(7): 1009-1021, 2021 07.
Artículo en Inglés | MEDLINE | ID: mdl-33934225

RESUMEN

Properties of the funny current (If) have been studied in several animal and cellular models, but so far little is known concerning its properties in human pacemaker cells. This work provides a detailed characterization of If in human-induced pluripotent stem cell (iPSC)-derived pacemaker cardiomyocytes (pCMs), at different time points. Patch-clamp analysis showed that If density did not change during differentiation; however, after day 30, it activates at more negative potential and with slower time constants. These changes are accompanied by a slowing in beating rate. If displayed the voltage-dependent block by caesium and reversed (Erev) at - 22 mV, compatibly with the 3:1 K+/Na+ permeability ratio. Lowering [Na+]o (30 mM) shifted the Erev to - 39 mV without affecting conductance. Increasing [K+]o (30 mM) shifted the Erev to - 15 mV with a fourfold increase in conductance. pCMs express mainly HCN4 and HCN1 together with the accessory subunits CAV3, KCR1, MiRP1, and SAP97 that contribute to the context-dependence of If. Autonomic agonists modulated the diastolic depolarization, and thus rate, of pCMs. The adrenergic agonist isoproterenol induced rate acceleration and a positive shift of If voltage-dependence (EC50 73.4 nM). The muscarinic agonists had opposite effects (Carbachol EC50, 11,6 nM). Carbachol effect was however small but it could be increased by pre-stimulation with isoproterenol, indicating low cAMP levels in pCMs. In conclusion, we demonstrated that pCMs display an If with the physiological properties expected by pacemaker cells and may thus represent a suitable model for studying human If-related sinus arrhythmias.


Asunto(s)
Potenciales de Acción/fisiología , Relojes Biológicos/fisiología , Células Madre Pluripotentes Inducidas/fisiología , Miocitos Cardíacos/fisiología , Potenciales de Acción/efectos de los fármacos , Relojes Biológicos/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Línea Celular , Electrofisiología/métodos , Atrios Cardíacos/efectos de los fármacos , Atrios Cardíacos/metabolismo , Atrios Cardíacos/fisiopatología , Humanos , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Isoproterenol/farmacología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Técnicas de Placa-Clamp/métodos , Nodo Sinoatrial/efectos de los fármacos , Nodo Sinoatrial/metabolismo , Nodo Sinoatrial/fisiología
3.
Neurobiol Dis ; 118: 55-63, 2018 10.
Artículo en Inglés | MEDLINE | ID: mdl-29936235

RESUMEN

The causes of genetic epilepsies are unknown in the majority of patients. HCN ion channels have a widespread expression in neurons and increasing evidence demonstrates their functional involvement in human epilepsies. Among the four known isoforms, HCN1 is the most expressed in the neocortex and hippocampus and de novo HCN1 point mutations have been recently associated with early infantile epileptic encephalopathy. So far, HCN1 mutations have not been reported in patients with idiopathic epilepsy. Using a Next Generation Sequencing approach, we identified the de novo heterozygous p.Leu157Val (c.469C > G) novel mutation in HCN1 in an adult male patient affected by genetic generalized epilepsy (GGE), with normal cognitive development. Electrophysiological analysis in heterologous expression model (CHO cells) and in neurons revealed that L157V is a loss-of-function, dominant negative mutation causing reduced HCN1 contribution to net inward current and responsible for an increased neuronal firing rate and excitability, potentially predisposing to epilepsy. These data represent the first evidence that autosomal dominant missense mutations of HCN1 can also be involved in GGE, without the characteristics of epileptic encephalopathy reported previously. It will be important to include HCN1 screening in patients with GGE, in order to extend the knowledge of the genetic causes of idiopathic epilepsies, thus paving the way for the identification of innovative therapeutic strategies.


Asunto(s)
Epilepsia Generalizada/diagnóstico , Epilepsia Generalizada/genética , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/genética , Mutación/genética , Neuronas/fisiología , Canales de Potasio/genética , Potenciales de Acción/fisiología , Secuencia de Aminoácidos , Animales , Animales Recién Nacidos , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Epilepsia Generalizada/fisiopatología , Femenino , Humanos , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/química , Masculino , Linaje , Canales de Potasio/química , Estructura Secundaria de Proteína , Ratas , Adulto Joven
4.
Nat Chem Biol ; 10(6): 457-62, 2014 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-24776929

RESUMEN

cAMP mediates autonomic regulation of heart rate by means of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which underlie the pacemaker current If. cAMP binding to the C-terminal cyclic nucleotide binding domain enhances HCN open probability through a conformational change that reaches the pore via the C-linker. Using structural and functional analysis, we identified a binding pocket in the C-linker of HCN4. Cyclic dinucleotides, an emerging class of second messengers in mammals, bind the C-linker pocket (CLP) and antagonize cAMP regulation of the channel. Accordingly, cyclic dinucleotides prevent cAMP regulation of If in sinoatrial node myocytes, reducing heart rate by 30%. Occupancy of the CLP hence constitutes an efficient mechanism to hinder ß-adrenergic stimulation on If. Our results highlight the regulative role of the C-linker and identify a potential drug target in HCN4. Furthermore, these data extend the signaling scope of cyclic dinucleotides in mammals beyond their first reported role in innate immune system.


Asunto(s)
AMP Cíclico/metabolismo , GMP Cíclico/análogos & derivados , Fosfatos de Dinucleósidos/metabolismo , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Activación del Canal Iónico/fisiología , Proteínas Musculares/metabolismo , Canales de Potasio/metabolismo , Animales , Sitios de Unión , Western Blotting , Cristalografía por Rayos X , GMP Cíclico/química , GMP Cíclico/metabolismo , Fosfatos de Dinucleósidos/química , Células HEK293 , Ensayos Analíticos de Alto Rendimiento , Humanos , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/genética , Activación del Canal Iónico/efectos de los fármacos , Ligandos , Ratones , Ratones Endogámicos C57BL , Simulación del Acoplamiento Molecular , Estructura Molecular , Proteínas Musculares/genética , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Técnicas de Placa-Clamp , Canales de Potasio/genética , Nodo Sinoatrial/citología , Nodo Sinoatrial/efectos de los fármacos , Nodo Sinoatrial/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Transfección
5.
Circ Res ; 113(4): 389-98, 2013 Aug 02.
Artículo en Inglés | MEDLINE | ID: mdl-23753573

RESUMEN

RATIONALE: A cell-based biological pacemaker is based on the differentiation of stem cells and the selection of a population displaying the molecular and functional properties of native sinoatrial node (SAN) cardiomyocytes. So far, such selection has been hampered by the lack of proper markers. CD166 is specifically but transiently expressed in the mouse heart tube and sinus venosus, the prospective SAN. OBJECTIVE: We have explored the possibility of using CD166 expression for isolating SAN progenitors from differentiating embryonic stem cells. METHODS AND RESULTS: We found that in embryonic day 10.5 mouse hearts, CD166 and HCN4, markers of the pacemaker tissue, are coexpressed. Sorting embryonic stem cells for CD166 expression at differentiation day 8 selects a population of pacemaker precursors. CD166+ cells express high levels of genes involved in SAN development (Tbx18, Tbx3, Isl-1, Shox2) and function (Cx30.2, HCN4, HCN1, CaV1.3) and low levels of ventricular genes (Cx43, Kv4.2, HCN2, Nkx2.5). In culture, CD166+ cells form an autorhythmic syncytium composed of cells morphologically similar to and with the electrophysiological properties of murine SAN myocytes. Isoproterenol increases (+57%) and acetylcholine decreases (-23%) the beating rate of CD166-selected cells, which express the ß-adrenergic and muscarinic receptors. In cocultures, CD166-selected cells are able to pace neonatal ventricular myocytes at a rate faster than their own. Furthermore, CD166+ cells have lost pluripotency genes and do not form teratomas in vivo. CONCLUSIONS: We demonstrated for the first time the isolation of a nonteratogenic population of cardiac precursors able to mature and form a fully functional SAN-like tissue.


Asunto(s)
Molécula de Adhesión Celular del Leucocito Activado/metabolismo , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Miocitos Cardíacos/citología , Nodo Sinoatrial/citología , Células Madre/citología , Acetilcolina/farmacología , Animales , Biomarcadores/metabolismo , Cardiotónicos/farmacología , Diferenciación Celular/fisiología , Línea Celular , Proliferación Celular , Técnicas de Cocultivo , Células Madre Embrionarias/efectos de los fármacos , Ventrículos Cardíacos/citología , Ventrículos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/metabolismo , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Isoproterenol/farmacología , Ratones , Modelos Animales , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Nodo Sinoatrial/efectos de los fármacos , Nodo Sinoatrial/metabolismo , Células Madre/efectos de los fármacos , Células Madre/metabolismo
6.
Proc Natl Acad Sci U S A ; 108(4): 1705-10, 2011 Jan 25.
Artículo en Inglés | MEDLINE | ID: mdl-21220308

RESUMEN

Cardiac pacemaking generation and modulation rely on the coordinated activity of several processes. Although a wealth of evidence indicates a relevant role of the I(f) ("funny," or pacemaker) current, whose molecular constituents are the hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels and particularly HCN4, work with mice where Hcn genes were knocked out, or functionally modified, has challenged this view. However, no previous studies used a cardiac-specific promoter to induce HCN4 ablation in adult mice. We report here that, in an inducible and cardiac-specific HCN4 knockout (ciHCN4-KO) mouse model, ablation of HCN4 consistently leads to progressive development of severe bradycardia (∼50% reduction of original rate) and AV block, eventually leading to heart arrest and death in about 5 d. In vitro analysis of sinoatrial node (SAN) myocytes isolated from ciHCN4-KO mice at the mean time of death revealed a strong reduction of both the I(f) current (by ∼70%) and of the spontaneous rate (by ∼60%). In agreement with functional results, immunofluorescence and Western blot analysis showed reduced expression of HCN4 protein in SAN tissue and cells. In ciHCN4-KO animals, the residual I(f) was normally sensitive to ß-adrenergic receptor (ß-AR) modulation, and the permanence of rate response to ß-AR stimulation was observed both in vivo and in vitro. Our data show that cardiac HCN4 channels are essential for normal heart impulse generation and conduction in adult mice and support the notion that dysfunctional HCN4 channels can be a direct cause of rhythm disorders. This work contributes to identifying the molecular mechanism responsible for cardiac pacemaking.


Asunto(s)
Bradicardia/fisiopatología , Canales Catiónicos Regulados por Nucleótidos Cíclicos/fisiología , Bloqueo Cardíaco/fisiopatología , Corazón/fisiopatología , Potenciales de Acción/efectos de los fármacos , Animales , Western Blotting , Conservadores de la Densidad Ósea/farmacología , Bradicardia/genética , Canales Catiónicos Regulados por Nucleótidos Cíclicos/genética , Electrocardiografía , Femenino , Técnica del Anticuerpo Fluorescente , Corazón/efectos de los fármacos , Bloqueo Cardíaco/genética , Frecuencia Cardíaca/efectos de los fármacos , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Miocardio/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/fisiología , Receptores Adrenérgicos beta/metabolismo , Nodo Sinoatrial/efectos de los fármacos , Nodo Sinoatrial/metabolismo , Nodo Sinoatrial/fisiología , Tamoxifeno/farmacología
8.
Acta Physiol (Oxf) ; 239(2): e13981, 2023 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-37186371

RESUMEN

AIMS: Nfix is a transcription factor belonging to the Nuclear Factor I (NFI) family comprising four members (Nfia, b, c, x). Nfix plays important roles in the development and function of several organs. In muscle development, Nfix controls the switch from embryonic to fetal myogenesis by promoting fast twitching fibres. In the adult muscle, following injury, lack of Nfix impairs regeneration, inducing higher content of slow-twitching fibres. Nfix is expressed also in the heart, but its function has been never investigated before. We studied Nfix role in this organ. METHODS: Using Nfix-null and wild type (WT) mice we analyzed: (1) the expression pattern of Nfix during development by qPCR and (2) the functional alterations caused by its absence, by in vivo telemetry and in vitro patch clamp analysis. RESULTS AND CONCLUSIONS: Nfix expression start in the heart from E12.5. Adult hearts of Nfix-null mice show a hearts morphology and sarcomeric proteins expression similar to WT. However, Nfix-null animals show tachycardia that derives form an intrinsic higher beating rate of the sinus node (SAN). Molecular and functional analysis revealed that sinoatrial cells of Nfix-null mice express a significantly larger L-type calcium current (Cacna1d + Cacna1c). Interestingly, downregulation of Nfix by sh-RNA in primary cultures of neonatal rat ventricular cardiomyocytes induced a similar increase in their spontaneous beating rate and in ICaL current. In conclusion, our data provide the first demonstration of a role of Nfix that, increasing the L-type calcium current, modulates heart rate.

9.
Front Physiol ; 14: 1250951, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-38028792

RESUMEN

Atrial fibrillation (AF) is the most common cardiac arrhythmia worldwide; however, the underlying causes of AF initiation are still poorly understood, particularly because currently available models do not allow in distinguishing the initial causes from maladaptive remodeling that induces and perpetuates AF. Lately, the genetic background has been proven to be important in the AF onset. iPSC-derived cardiomyocytes, being patient- and mutation-specific, may help solve this diatribe by showing the initial cell-autonomous changes underlying the development of the disease. Transcription factor paired-like homeodomain 2 (PITX2) has been identified as a key regulator of atrial development/differentiation, and the PITX2 genomic locus has the highest association with paroxysmal AF. PITX2 influences mitochondrial activity, and alterations in either its expression or function have been widely associated with AF. In this work, we investigate the activity of mitochondria in iPSC-derived atrial cardiomyocytes (aCMs) obtained from a young patient (24 years old) with paroxysmal AF, carrying a gain-of-function mutation in PITX2 (rs138163892) and from its isogenic control (CTRL) in which the heterozygous point mutation has been reverted to WT. PITX2 aCMs show a higher mitochondrial content, increased mitochondrial activity, and superoxide production under basal conditions when compared to CTRL aCMs. However, increasing mitochondrial workload by FCCP or ß-adrenergic stimulation allows us to unmask mitochondrial defects in PITX2 aCMs, which are incapable of responding efficiently to the higher energy demand, determining ATP deficiency.

10.
J Neurosci ; 31(48): 17327-37, 2011 Nov 30.
Artículo en Inglés | MEDLINE | ID: mdl-22131395

RESUMEN

The hyperpolarization-activated I(h) current, coded for by hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels, controls synaptic integration and intrinsic excitability in many brain areas. Because of their role in pacemaker function, defective HCN channels are natural candidates for contributing to epileptogenesis. Indeed, I(h) is pathologically altered after experimentally induced seizures, and several independent data indicate a link between dysfunctional HCN channels and different forms of epilepsy. However, direct evidence for functional changes of defective HCN channels correlating with the disease in human patients is still elusive. By screening families with epilepsy for mutations in Hcn1 and Hcn2 genes, we found a recessive loss-of-function point mutation in the gene coding for the HCN2 channel in a patient with sporadic idiopathic generalized epilepsy. Of 17 screened members of the same family, the proband was the only one affected and homozygous for the mutation. The mutation (E515K) is located in the C-linker, a region known to affect channel gating. Functional analysis revealed that homomeric mutant, but not heteromeric wild-type/mutant channels, have a strongly inhibited function caused by a large negative shift of activation range and slowed activation kinetics, effectively abolishing the HCN2 contribution to activity. After transfection into acutely isolated newborn rat cortical neurons, homomeric mutant, but not heteromeric wild type/mutant channels, lowered the threshold of action potential firing and strongly increased cell excitability and firing frequency when compared with wild-type channels. This is the first evidence in humans for a single-point, homozygous loss-of-function mutation in HCN2 potentially associated with generalized epilepsy with recessive inheritance.


Asunto(s)
Epilepsia/genética , Canales Iónicos/genética , Mutación , Neuronas/fisiología , Potenciales de Acción/genética , Adulto , Alelos , Animales , Células Cultivadas , Corteza Cerebral/fisiología , Niño , Epilepsia/metabolismo , Epilepsia/fisiopatología , Humanos , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización , Activación del Canal Iónico/genética , Canales Iónicos/metabolismo , Masculino , Linaje , Polimorfismo de Nucleótido Simple , Canales de Potasio , Ratas , Transfección
11.
Elife ; 112022 03 22.
Artículo en Inglés | MEDLINE | ID: mdl-35315774

RESUMEN

Tongmai Yangxin (TMYX) is a complex compound of the Traditional Chinese Medicine (TCM) used to treat several cardiac rhythm disorders; however, no information regarding its mechanism of action is available. In this study we provide a detailed characterization of the effects of TMYX on the electrical activity of pacemaker cells and unravel its mechanism of action. Single-cell electrophysiology revealed that TMYX elicits a reversible and dose-dependent (2/6 mg/ml) slowing of spontaneous action potentials rate (-20.8/-50.2%) by a selective reduction of the diastolic phase (-50.1/-76.0%). This action is mediated by a negative shift of the If activation curve (-6.7/-11.9 mV) and is caused by a reduction of the cyclic adenosine monophosphate (cAMP)-induced stimulation of pacemaker channels. We provide evidence that TMYX acts by directly antagonizing the cAMP-induced allosteric modulation of the pacemaker channels. Noticeably, this mechanism functionally resembles the pharmacological actions of muscarinic stimulation or ß-blockers, but it does not require generalized changes in cytoplasmic cAMP levels thus ensuring a selective action on rate. In agreement with a competitive inhibition mechanism, TMYX exerts its maximal antagonistic action at submaximal cAMP concentrations and then progressively becomes less effective thus ensuring a full contribution of If to pacemaker rate during high metabolic demand and sympathetic stimulation.


Asunto(s)
AMP Cíclico , Sistemas de Mensajero Secundario , Potenciales de Acción , Animales , China , AMP Cíclico/metabolismo , Miocitos Cardíacos/metabolismo , Conejos
12.
Front Neurosci ; 15: 617698, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34084126

RESUMEN

OBJECTIVE: The aim of this study was to assess age-related changes in cardiac autonomic modulation and heart rate variability (HRV) and their association with spontaneous and pharmacologically induced vulnerability to cardiac arrhythmias, to verify the translational relevance of mouse models for further in-depth evaluation of the link between autonomic changes and increased arrhythmic risk with advancing age. METHODS: Heart rate (HR) and time- and frequency-domain indexes of HRV were calculated from Electrocardiogram (ECG) recordings in two groups of conscious mice of different ages (4 and 19 months old) (i) during daily undisturbed conditions, (ii) following peripheral ß-adrenergic (atenolol), muscarinic (methylscopolamine), and ß-adrenergic + muscarinic blockades, and (iii) following ß-adrenergic (isoprenaline) stimulation. Vulnerability to arrhythmias was evaluated during daily undisturbed conditions and following ß-adrenergic stimulation. RESULTS: HRV analysis and HR responses to autonomic blockades revealed that 19-month-old mice had a lower vagal modulation of cardiac function compared with 4-month-old mice. This age-related autonomic effect was not reflected in changes in HR, since intrinsic HR was lower in 19-month-old compared with 4-month-old mice. Both time- and frequency-domain HRV indexes were reduced following muscarinic, but not ß-adrenergic blockade in younger mice, and to a lesser extent in older mice, suggesting that HRV is largely modulated by vagal tone in mice. Finally, 19-month-old mice showed a larger vulnerability to both spontaneous and isoprenaline-induced arrhythmias. CONCLUSION: The present study combines HRV analysis and selective pharmacological autonomic blockades to document an age-related impairment in cardiac vagal modulation in mice which is consistent with the human condition. Given their short life span, mice could be further exploited as an aged model for studying the trajectory of vagal decline with advancing age using HRV measures, and the mechanisms underlying its association with proarrhythmic remodeling of the senescent heart.

13.
Prog Biophys Mol Biol ; 166: 189-204, 2021 11.
Artículo en Inglés | MEDLINE | ID: mdl-34400215

RESUMEN

Discovered some 40 years ago, the If current has since been known as the "pacemaker" current due to its role in the initiation and modulation of the heartbeat and of neuronal excitability. But this is not all, the funny current keeps entertaining the researchers; indeed, several data discovering novel and uncanonical roles of f/HCN channel are quickly accumulating. In the present review, we provide an overview of the expression and cellular functions of HCN/f channels in a variety of systems/organs, and particularly in sour taste transduction, hormones secretion, activation of astrocytes and microglia, inhibition of osteoclastogenesis, renal ammonium excretion, and peristalsis in the gastrointestinal and urine systems. We also analyzed the role of HCN channels in sustaining cellular respiration in mitochondria and their participation to mitophagy under specific conditions. The relevance of HCN currents in undifferentiated cells, and specifically in the control of stem cell cycle and in bioelectrical signals driving left/right asymmetry during zygote development, is also considered. Finally, we present novel data concerning the expression of HCN mRNA in human leukocytes. We can thus conclude that the emerging evidence presented in this review clearly points to an increasing interest and importance of the "funny" current that goes beyond its role in cardiac sinoatrial and neuronal excitability regulation.


Asunto(s)
Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización , Canales de Potasio , Corazón , Frecuencia Cardíaca , Humanos , Neuronas
14.
Prog Biophys Mol Biol ; 166: 61-85, 2021 11.
Artículo en Inglés | MEDLINE | ID: mdl-34197836

RESUMEN

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.


Asunto(s)
Fibrilación Atrial , Nodo Atrioventricular , Potenciales de Acción , Frecuencia Cardíaca , Humanos , Nodo Sinoatrial
15.
Heart Rhythm ; 18(5): 801-810, 2021 05.
Artículo en Inglés | MEDLINE | ID: mdl-33278629

RESUMEN

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.


Asunto(s)
Bradicardia/genética , Relojes Circadianos/fisiología , Electrocardiografía/métodos , Regulación de la Expresión Génica , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/genética , ARN/genética , Nodo Sinoatrial/fisiopatología , Animales , Bradicardia/metabolismo , Bradicardia/fisiopatología , Modelos Animales de Enfermedad , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/biosíntesis , Ratones
16.
J Mol Cell Cardiol ; 48(1): 55-64, 2010 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-19591835

RESUMEN

In mammals cardiac rate is determined by the duration of the diastolic depolarization of sinoatrial node (SAN) cells which is mainly determined by the pacemaker I(f) current. f-channels are encoded by four members of the hyperpolarization-activated cyclic nucleotide-gated gene (HCN1-4) family. HCN4 is the most abundant isoform in the SAN, and its relevance to pacemaking has been further supported by the discovery of four loss-of-function mutations in patients with mild or severe forms of cardiac rate disturbances. Due to its selective contribution to pacemaking, the I(f) current is also the pharmacological target of a selective heart rate-reducing agent (ivabradine) currently used in the clinical practice. Albeit to a minor extent, the I(f) current is also present in other spontaneously active myocytes of the cardiac conduction system (atrioventricular node and Purkinje fibres). In working atrial and ventricular myocytes f-channels are expressed at a very low level and do not play any physiological role; however in certain pathological conditions over-expression of HCN proteins may represent an arrhythmogenic mechanism. In this review some of the most recent findings on f/HCN channels contribution to pacemaking are described.


Asunto(s)
Canales Catiónicos Regulados por Nucleótidos Cíclicos/metabolismo , Frecuencia Cardíaca/fisiología , Miocardio/metabolismo , Animales , Canales Catiónicos Regulados por Nucleótidos Cíclicos/genética , Frecuencia Cardíaca/genética , Humanos , Modelos Biológicos , Nodo Sinoatrial/metabolismo
17.
J Physiol ; 587(Pt 7): 1513-25, 2009 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-19171659

RESUMEN

The pacemaker current, mediated by hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, contributes to the initiation and regulation of cardiac rhythm. Previous experiments creating HCN-based biological pacemakers in vivo found that an engineered HCN2/HCN1 chimeric channel (HCN212) resulted in significantly faster rates than HCN2, interrupted by 1-5 s pauses. To elucidate the mechanisms underlying the differences in HCN212 and HCN2 in vivo functionality as biological pacemakers, we studied newborn rat ventricular myocytes over-expressing either HCN2 or HCN212 channels. The HCN2- and HCN212-over-expressing myocytes manifest similar voltage dependence, current density and sensitivity to saturating cAMP concentrations, but HCN212 has faster activation/deactivation kinetics. Compared with HCN2, myocytes expressing HCN212 exhibit a faster spontaneous rate and greater incidence of irregular rhythms (i.e. periods of rapid spontaneous rate followed by pauses). To explore these rhythm differences further, we imposed consecutive pacing and found that activation kinetics of the two channels are slower at faster pacing frequencies. As a result, time-dependent HCN current flowing during diastole decreases for both constructs during a train of stimuli at a rapid frequency, with the effect more pronounced for HCN2. In addition, the slower deactivation kinetics of HCN2 contributes to more pronounced instantaneous current at a slower frequency. As a result of the frequency dependence of both instantaneous and time-dependent current, HCN2 exhibits more robust negative feedback than HCN212, contributing to the maintenance of a stable pacing rhythm. These results illustrate the benefit of screening HCN constructs in spontaneously active myocyte cultures and may provide the basis for future optimization of HCN-based biological pacemakers.


Asunto(s)
Relojes Biológicos , Canales Catiónicos Regulados por Nucleótidos Cíclicos/metabolismo , Frecuencia Cardíaca , Canales Iónicos/metabolismo , Contracción Miocárdica , Miocitos Cardíacos/metabolismo , Canales de Potasio/metabolismo , Potasio/metabolismo , Potenciales de Acción , Animales , Animales Recién Nacidos , Estimulación Cardíaca Artificial , Células Cultivadas , Simulación por Computador , AMP Cíclico/metabolismo , Canales Catiónicos Regulados por Nucleótidos Cíclicos/genética , Ventrículos Cardíacos/metabolismo , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización , Canales Iónicos/genética , Cinética , Modelos Cardiovasculares , Canales de Potasio/genética , Ratas , Ratas Wistar , Transducción Genética
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