Assuntos
Fibrilação Atrial , Mutação com Ganho de Função , Proteína Homeobox PITX2 , Proteínas de Homeodomínio , Fatores de Transcrição , Fibrilação Atrial/genética , Fibrilação Atrial/fisiopatologia , Fibrilação Atrial/diagnóstico , Humanos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas de Homeodomínio/genética , Mitocôndrias Cardíacas/genética , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/patologia , Animais , Potencial da Membrana Mitocondrial/genética , Predisposição Genética para Doença , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , FenótipoAssuntos
Síndrome de Brugada , Mutação , Canal de Sódio Disparado por Voltagem NAV1.5 , Fenótipo , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Síndrome de Brugada/genética , Síndrome de Brugada/fisiopatologia , Síndrome de Brugada/metabolismo , Humanos , Potenciais de Ação , Potenciais da Membrana , AnimaisAssuntos
Proteína Adaptadora de Sinalização NOD1 , Nó Sinoatrial , Animais , Nó Sinoatrial/metabolismo , Nó Sinoatrial/fisiopatologia , Camundongos , Proteína Adaptadora de Sinalização NOD1/metabolismo , Proteína Adaptadora de Sinalização NOD1/genética , Potenciais de Ação , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Camundongos Endogâmicos C57BLRESUMO
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.
Assuntos
Caveolina 1 , Caveolina 3 , Adulto , Animais , Cricetinae , Humanos , Caveolina 1/genética , Caveolina 3/genética , Cricetulus , Mutação , Células CHO , Canais IônicosRESUMO
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.
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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.
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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.
Assuntos
AMP Cíclico , Sistemas do Segundo Mensageiro , Potenciais de Ação , Animais , China , AMP Cíclico/metabolismo , Miócitos Cardíacos/metabolismo , CoelhosRESUMO
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.
Assuntos
Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Canais de Potássio , Coração , Frequência Cardíaca , Humanos , NeurôniosRESUMO
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.
Assuntos
Fibrilação Atrial , Nó Atrioventricular , Potenciais de Ação , Frequência Cardíaca , Humanos , Nó SinoatrialRESUMO
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.
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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.
Assuntos
Potenciais de Ação/fisiologia , Relógios Biológicos/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Miócitos Cardíacos/fisiologia , Potenciais de Ação/efeitos dos fármacos , Relógios Biológicos/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Linhagem Celular , Eletrofisiologia/métodos , Átrios do Coração/efeitos dos fármacos , Átrios do Coração/metabolismo , Átrios do Coração/fisiopatologia , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Isoproterenol/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Técnicas de Patch-Clamp/métodos , Nó Sinoatrial/efeitos dos fármacos , Nó Sinoatrial/metabolismo , Nó Sinoatrial/fisiologiaRESUMO
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.
Assuntos
Bradicardia/genética , Relógios Circadianos/fisiologia , Eletrocardiografia/métodos , Regulação da Expressão Gênica , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , RNA/genética , Nó Sinoatrial/fisiopatologia , Animais , Bradicardia/metabolismo , Bradicardia/fisiopatologia , Modelos Animais de Doenças , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/biossíntese , CamundongosRESUMO
HCN channels are highly expressed and functionally relevant in neurons and increasing evidence demonstrates their involvement in the etiology of human epilepsies. Among HCN isoforms, HCN4 is important in cardiac tissue, where it underlies pacemaker activity. Despite being expressed also in deep structures of the brain, mutations of this channel functionally shown to be associated with epilepsy have not been reported yet. Using Next Generation Sequencing for the screening of patients with idiopathic epilepsy, we identified the p.Arg550Cys (c.1648C>T) heterozygous mutation on HCN4 in two brothers affected by benign myoclonic epilepsy of infancy. Functional characterization in heterologous expression system and in neurons showed that the mutation determines a loss of function of HCN4 contribution to activity and an increase of neuronal discharge, potentially predisposing to epilepsy. Expressed in cardiomyocytes, mutant channels activate at slightly more negative voltages than wild-type (WT), in accordance with borderline bradycardia. While HCN4 variants have been frequently associated with cardiac arrhythmias, these data represent the first experimental evidence that functional alteration of HCN4 can also be involved in human epilepsy through a loss-of-function effect and associated increased neuronal excitability. Since HCN4 appears to be highly expressed in deep brain structures only early during development, our data provide a potential explanation for a link between dysfunctional HCN4 and infantile epilepsy. These findings suggest that it may be useful to include HCN4 screening to extend the knowledge of the genetic causes of infantile epilepsies, potentially paving the way for the identification of innovative therapeutic strategies.
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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 ß-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.
Assuntos
AMP Cíclico/química , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/química , Miócitos Cardíacos/efeitos dos fármacos , Peptídeos/farmacologia , Canais de Potássio/química , Animais , Sítios de Ligação , Canais de Cálcio Tipo L/química , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/metabolismo , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/genética , Peptídeos Penetradores de Células/metabolismo , AMP Cíclico/metabolismo , Expressão Gênica , Células HEK293 , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Técnicas de Patch-Clamp , Peptídeos/síntese química , Peroxinas/química , Peroxinas/genética , Peroxinas/metabolismo , Canais de Potássio/genética , Canais de Potássio/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Coelhos , Nó Sinoatrial/citologia , Nó Sinoatrial/efeitos dos fármacos , Nó Sinoatrial/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
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.469Câ¯>â¯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.
Assuntos
Epilepsia Generalizada/diagnóstico , Epilepsia Generalizada/genética , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Mutação/genética , Neurônios/fisiologia , Canais de Potássio/genética , Potenciais de Ação/fisiologia , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Epilepsia Generalizada/fisiopatologia , Feminino , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/química , Masculino , Linhagem , Canais de Potássio/química , Estrutura Secundária de Proteína , Ratos , Adulto JovemRESUMO
AMPK is a conserved serine/threonine kinase whose activity maintains cellular energy homeostasis. Eukaryotic AMPK exists as αßγ complexes, whose regulatory γ subunit confers energy sensor function by binding adenine nucleotides. Humans bearing activating mutations in the γ2 subunit exhibit a phenotype including unexplained slowing of heart rate (bradycardia). Here, we show that γ2 AMPK activation downregulates fundamental sinoatrial cell pacemaker mechanisms to lower heart rate, including sarcolemmal hyperpolarization-activated current (I f) and ryanodine receptor-derived diastolic local subsarcolemmal Ca2+ release. In contrast, loss of γ2 AMPK induces a reciprocal phenotype of increased heart rate, and prevents the adaptive intrinsic bradycardia of endurance training. Our results reveal that in mammals, for which heart rate is a key determinant of cardiac energy demand, AMPK functions in an organ-specific manner to maintain cardiac energy homeostasis and determines cardiac physiological adaptation to exercise by modulating intrinsic sinoatrial cell behavior.
Assuntos
Proteínas Quinases Ativadas por AMP/genética , Bradicardia/genética , Cálcio/metabolismo , Frequência Cardíaca/genética , Sarcolema/metabolismo , Nó Sinoatrial/metabolismo , Adulto , Animais , Bradicardia/metabolismo , Eletrocardiografia Ambulatorial , Exercício Físico , Coração/diagnóstico por imagem , Humanos , Imagem Cinética por Ressonância Magnética , Espectroscopia de Ressonância Magnética , Camundongos , Microscopia Eletrônica de Transmissão , Mutação , Miocárdio/metabolismo , Miocárdio/patologia , Miocárdio/ultraestrutura , Condicionamento Físico Animal , Resistência Física , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Nó Sinoatrial/patologiaRESUMO
AIMS: Caveolinopathies are a family of genetic disorders arising from alterations of the caveolin-3 (cav-3) gene. The T78M cav-3 variant has been associated with both skeletal and cardiac muscle pathologies but its functional contribution, especially to cardiac diseases, is still controversial. Here, we evaluated the effect of the T78M cav-3 variant on cardiac ion channel function and membrane excitability. METHODS AND RESULTS: We transfected either the wild type (WT) or T78M cav-3 in caveolin-1 knock-out mouse embryonic fibroblasts and found by immunofluorescence and electron microscopy that both are expressed at the plasma membrane and form caveolae. Two ion channels known to interact and co-immunoprecipitate with the cav-3, hKv1.5 and hHCN4, interact also with T78M cav-3 and reside in lipid rafts. Electrophysiological analysis showed that the T78M cav-3 causes hKv1.5 channels to activate and inactivate at more hyperpolarized potentials and the hHCN4 channels to activate at more depolarized potentials, in a dominant way. In spontaneously beating neonatal cardiomyocytes, the expression of the T78M cav-3 significantly increased action potential peak-to-peak variability without altering neither the mean rate nor the maximum diastolic potential. We also found that in a small cohort of patients with supraventricular arrhythmias, the T78M cav-3 variant is more frequent than in the general population. Finally, in silico analysis of both sinoatrial and atrial cell models confirmed that the T78M-dependent changes are compatible with a pro-arrhythmic effect. CONCLUSION: This study demonstrates that the T78M cav-3 induces complex modifications in ion channel function that ultimately alter membrane excitability. The presence of the T78M cav-3 can thus generate a susceptible substrate that, in concert with other structural alterations and/or genetic mutations, may become arrhythmogenic.
Assuntos
Potenciais de Ação , Caveolina 3/genética , Caveolina 3/metabolismo , Fibroblastos/metabolismo , Mutação , Miócitos Cardíacos/metabolismo , Células 3T3 , Animais , Arritmias Cardíacas/genética , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/fisiopatologia , Cavéolas/metabolismo , Caveolina 1/deficiência , Caveolina 1/genética , Simulação por Computador , Fibroblastos/ultraestrutura , Frequência Cardíaca , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Ativação do Canal Iônico , Cinética , Canal de Potássio Kv1.5/genética , Canal de Potássio Kv1.5/metabolismo , Camundongos , Camundongos Knockout , Modelos Cardiovasculares , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Contração Miocárdica , Miócitos Cardíacos/ultraestrutura , Canais de Potássio/genética , Canais de Potássio/metabolismo , Ratos Sprague-Dawley , TransfecçãoAssuntos
Mutação com Ganho de Função/genética , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Proteínas Musculares/genética , Canais de Potássio/genética , Taquicardia Sinusal/genética , AMP Cíclico/fisiologia , Feminino , Células HEK293 , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/química , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/fisiologia , Masculino , Proteínas Mutantes/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/fisiologia , Linhagem , Recidiva , Síncope/genéticaRESUMO
The pathogenic role of SOD1 mutations in amyotrophic lateral sclerosis (ALS) was investigated using a zebrafish disease model stably expressing the ALS-linked G93R mutation. In addition to the main pathological features of ALS shown by adult fish, we found remarkably precocious alterations in the development of motor nerve circuitry and embryo behavior, and suggest that these alterations are prompted by interneuron and motor neuron hyperexcitability triggered by anomalies in the persistent pacemaker sodium current INaP. The riluzole-induced modulation of INaP reduced spinal neuron excitability, reverted the behavioral phenotypes and improved the deficits in motor nerve circuitry development, thus shedding new light on the use of riluzole in the management of ALS. Our findings provide a valid phenotype-based tool for unbiased in vivo drug screening that can be used to develop new therapies.