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1.
Sci Rep ; 9(1): 11781, 2019 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-31409881

RESUMO

In adult mammalian hearts, atrioventricular rings (AVRs) surround the atrial orifices of atrioventricular valves and are hotbed of ectopic activity in patients with focal atrial tachycardia. Experimental data offering mechanistic insights into initiation and maintenance of ectopic foci is lacking. We aimed to characterise AVRs in structurally normal rat hearts, identify arrhythmia predisposition and investigate mechanisms underlying arrhythmogenicity. Extracellular potential mapping and intracellular action potential recording techniques were used for electrophysiology, qPCR for gene and, Western blot and immunohistochemistry for protein expression. Conditions favouring ectopic foci were assessed by simulations. In right atrial preparations, sinus node (SN) was dominant and AVRs displayed 1:1 impulse conduction. Detaching SN unmasked ectopic pacemaking in AVRs and pacemaker action potentials were SN-like. Blocking pacemaker current If, and disrupting intracellular Ca2+ release, prolonged spontaneous cycle length in AVRs, indicating a role for SN-like pacemaker mechanisms. AVRs labelled positive for HCN4, and SERCA2a was comparable to SN. Pacemaking was potentiated by isoproterenol and abolished with carbachol and AVRs had abundant sympathetic nerve endings. ß2-adrenergic and M2-muscarinic receptor mRNA and ß2-receptor protein were comparable to SN. In computer simulations of a sick SN, ectopic foci in AVR were unmasked, causing transient suppression of SN pacemaking.

2.
Front Physiol ; 10: 826, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31338036

RESUMO

Cardiovascular complications are common in type 1 diabetes mellitus (TIDM) and there is an increased risk of arrhythmias as a result of dysfunction of the cardiac conduction system (CCS). We have previously shown that, in vivo, there is a decrease in the heart rate and prolongation of the QRS complex in streptozotocin-induced type 1 diabetic rats indicating dysfunction of the CCS. The aim of this study was to investigate the function of the ex vivo CCS and key proteins that are involved in pacemaker mechanisms in TIDM. RR interval, PR interval and QRS complex duration were significantly increased in diabetic rats. The beating rate of the isolated sinoatrial node (SAN) preparation was significantly decreased in diabetic rats. The funny current density and cell capacitance were significantly decreased in diabetic nodal cells. Western blot showed that proteins involved in the function of the CCS were significantly decreased in diabetic rats, namely: HCN4, Cav1.3, Cav3.1, Cx45, and NCX1 in the SAN; RyR2 and NCX1 in the atrioventricular junction and Cx40, Cx43, Cx45, and RyR2 in the Purkinje network. We conclude that there are complex functional and cellular changes in the CCS in TIDM. The changes in the proteins involved in the function of this electrical system are expected to adversely affect action potential generation and propagation, and these changes are likely to be arrhythmogenic.

3.
Nat Commun ; 10(1): 2889, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253831

RESUMO

The sinus node is a collection of highly specialised cells constituting the heart's pacemaker. The molecular underpinnings of its pacemaking abilities are debated. Using high-resolution mass spectrometry, we here quantify >7,000 proteins from sinus node and neighbouring atrial muscle. Abundances of 575 proteins differ between the two tissues. By performing single-nucleus RNA sequencing of sinus node biopsies, we attribute measured protein abundances to specific cell types. The data reveal significant differences in ion channels responsible for the membrane clock, but not in Ca2+ clock proteins, suggesting that the membrane clock underpins pacemaking. Consistently, incorporation of ion channel expression differences into a biophysically-detailed atrial action potential model result in pacemaking and a sinus node-like action potential. Combining our quantitative proteomics data with computational modeling, we estimate ion channel copy numbers for sinus node myocytes. Our findings provide detailed insights into the unique molecular make-up of the cardiac pacemaker.


Assuntos
Relógios Biológicos/fisiologia , Peptídeos/química , Peptídeos/metabolismo , Proteômica , Nó Sinoatrial/metabolismo , Transcriptoma , Potenciais de Ação , Animais , Cromatografia Líquida , Regulação da Expressão Gênica/fisiologia , Concentração de Íons de Hidrogênio , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/genética , Espectrometria de Massas em Tandem
4.
Histol Histopathol ; 34(11): 1255-1268, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30968943

RESUMO

BACKGROUND: Functional properties of the sinoatrial node (SAN) are known to differ between sexes. Women have higher resting and intrinsic heart rates. Sex determines the risk of developing certain arrhythmias such as sick sinus syndrome, which occur more often in women. We believe that a major contributor to these differences is in gender specific ion channel expression. METHODS: qPCR was used to compare ion channel gene expression in the SAN and right atrium (RA) between male and female rats. Histology, immunohistochemistry and signal intensity analysis were used to locate the SAN and determine abundance of ion channels. The effect of nifedipine on extracellular potential recording was used to determine differences in beating rate between sexes. RESULTS: mRNAs for Cav1.3, Kir3.1, and Nkx2-5, as well as expression of the L-Type Ca²âº channel protein, were higher in the female SAN. Females had significantly higher intrinsic heart rates and the effect of nifedipine on isolated SAN preparations was significantly greater in male SAN. Computer modelling using a SAN cell model demonstrated a higher propensity of pacemaker-related arrhythmias in females. CONCLUSION: This study has identified key differences in the expression of Cav1.3, Kir3.1 and Nkx2-5 at mRNA and/or protein levels between male and female SAN. Cav1.3 plays an important role in the pacemaker function of the SAN, therefore the higher intrinsic heart rate of the female SAN could be caused by the higher expression of Cav1.3. The differences identified in this study advance our understanding of sex differences in cardiac electrophysiology and arrhythmias.

5.
Heart Rhythm ; 15(5): 752-760, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29288034

RESUMO

BACKGROUND: Aging is associated with an increased incidence of atrioventricular nodal (AVN) dysfunction. OBJECTIVE: The aim of this study was to investigate the structural and functional remodeling in the atrioventricular junction (AVJ) with aging. METHODS: Electrophysiology, histology, and immunohistochemistry experiments on male Wistar Hannover rats aged 3 months (n = 24) and 2 years (n = 15) were performed. Atrio-His (AH) interval, Wenkebach cycle length (WBCL), and AVN effective refractory period (AVNERP) were measured. Cesium (2 mM) was used to block hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, while ryanodine (2 µM) was used to block ryanodine 2 (RyR2) channels. Protein expression from different regions of the AVJ was studied using immunofluorescence. The expression of connexins (connexin 43 and connexin 40), ion channels (Hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4), voltage sensitive sodium channel (Nav1.5), and L-Type calcium channel (Cav1.3)), and calcium handling proteins (RyR2 and sarco/endoplasmic reticulum calcium ATPaset type 2a (SERCA2a)) were measured. Morphological characteristics were studied with histology. RESULTS: Without drugs to block HCN and RyR2 channels, there was prolongation of the AH interval, WBCL, and AVNERP (P < .05) with aging. In young rats only, cesium prolonged the AH interval, WBCL, and AVNERP (P < .01). Ryanodine prolonged the AH interval and WBCL (P < .01) in both young and old rats. Immunofluorescence revealed that with aging, connexin 43, HCN4, Nav1.5, and RyR2 downregulate in the regions of the AVJ and connexin 40, SERCA2a, and Cav1.3 upregulate (P < .05). Aging results in cellular hypertrophy, loosely packed cells, a decrease in the number of nuclei, and an increase in collagen content. CONCLUSION: Heterogeneous ion channel expression changes were observed in the AVJ with aging. For the first time, we have shown that HCN and RyR2 play an important role in AVN dysfunction with aging.


Assuntos
Envelhecimento , Nó Atrioventricular/fisiologia , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Rianodina/farmacologia , Animais , Nó Atrioventricular/citologia , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/efeitos dos fármacos , Imuno-Histoquímica , Masculino , Modelos Animais , Técnicas de Patch-Clamp , Ratos , Ratos Wistar , Canal de Liberação de Cálcio do Receptor de Rianodina/efeitos dos fármacos
6.
Circ Res ; 121(9): 1058-1068, 2017 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-28821541

RESUMO

RATIONALE: Downregulation of the pacemaking ion channel, HCN4 (hyperpolarization-activated cyclic nucleotide gated channel 4), and the corresponding ionic current, If, underlies exercise training-induced sinus bradycardia in rodents. If this occurs in humans, it could explain the increased incidence of bradyarrhythmias in veteran athletes, and it will be important to understand the underlying processes. OBJECTIVE: To test the role of HCN4 in the training-induced bradycardia in human athletes and investigate the role of microRNAs (miRs) in the repression of HCN4. METHODS AND RESULTS: As in rodents, the intrinsic heart rate was significantly lower in human athletes than in nonathletes, and in all subjects, the rate-lowering effect of the HCN selective blocker, ivabradine, was significantly correlated with the intrinsic heart rate, consistent with HCN repression in athletes. Next-generation sequencing and quantitative real-time reverse transcription polymerase chain reaction showed remodeling of miRs in the sinus node of swim-trained mice. Computational predictions highlighted a prominent role for miR-423-5p. Interaction between miR-423-5p and HCN4 was confirmed by a dose-dependent reduction in HCN4 3'-untranslated region luciferase reporter activity on cotransfection with precursor miR-423-5p (abolished by mutation of predicted recognition elements). Knockdown of miR-423-5p with anti-miR-423-5p reversed training-induced bradycardia via rescue of HCN4 and If. Further experiments showed that in the sinus node of swim-trained mice, upregulation of miR-423-5p (intronic miR) and its host gene, NSRP1, is driven by an upregulation of the transcription factor Nkx2.5. CONCLUSIONS: HCN remodeling likely occurs in human athletes, as well as in rodent models. miR-423-5p contributes to training-induced bradycardia by targeting HCN4. This work presents the first evidence of miR control of HCN4 and heart rate. miR-423-5p could be a therapeutic target for pathological sinus node dysfunction in veteran athletes.


Assuntos
Bradicardia/metabolismo , Exercício/fisiologia , Marcação de Genes/métodos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , MicroRNAs/metabolismo , Proteínas Musculares/metabolismo , Condicionamento Físico Animal/fisiologia , Canais de Potássio/metabolismo , Adolescente , Adulto , Animais , Bradicardia/genética , Bradicardia/fisiopatologia , Técnicas de Silenciamento de Genes/métodos , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Proteínas Musculares/genética , Condicionamento Físico Animal/métodos , Canais de Potássio/genética , Nó Sinoatrial/metabolismo , Nó Sinoatrial/fisiopatologia , Adulto Jovem
7.
Artigo em Inglês | MEDLINE | ID: mdl-27979911

RESUMO

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


Assuntos
Nó Atrioventricular/metabolismo , Bloqueio Cardíaco/metabolismo , Hipertensão Pulmonar/metabolismo , Canais Iônicos/metabolismo , Transcriptoma , Animais , Nó Atrioventricular/fisiopatologia , Modelos Animais de Doenças , Regulação para Baixo , Ecocardiografia , Eletrocardiografia , Técnicas Eletrofisiológicas Cardíacas , Bloqueio Cardíaco/etiologia , Bloqueio Cardíaco/fisiopatologia , Hipertensão Pulmonar/complicações , Hipertensão Pulmonar/fisiopatologia , Canais Iônicos/genética , Masculino , Monocrotalina , Reação em Cadeia da Polimerase , Ratos , Ratos Wistar
8.
Front Physiol ; 7: 197, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27313537

RESUMO

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

9.
Nat Commun ; 5: 3775, 2014 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-24825544

RESUMO

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


Assuntos
Bradicardia/genética , Frequência Cardíaca/genética , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Condicionamento Físico Animal , RNA Mensageiro/metabolismo , Nó Sinoatrial/metabolismo , Adaptação Fisiológica/genética , Animais , Bradicardia/metabolismo , Regulação para Baixo , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Técnicas In Vitro , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Ratos , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Regulação para Cima
10.
J Am Heart Assoc ; 2(6): e000246, 2013 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-24356527

RESUMO

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


Assuntos
Sistema de Condução Cardíaco/metabolismo , Potenciais de Ação/fisiologia , Animais , Nó Atrioventricular/anatomia & histologia , Nó Atrioventricular/metabolismo , Nó Atrioventricular/fisiologia , Fascículo Atrioventricular/anatomia & histologia , Fascículo Atrioventricular/metabolismo , Fascículo Atrioventricular/fisiologia , Sistema de Condução Cardíaco/anatomia & histologia , Sistema de Condução Cardíaco/fisiologia , Modelos Anatômicos , Proteoma , Ramos Subendocárdicos/anatomia & histologia , Ramos Subendocárdicos/metabolismo , Ramos Subendocárdicos/fisiologia , Ratos , Nó Sinoatrial/anatomia & histologia , Nó Sinoatrial/metabolismo , Nó Sinoatrial/fisiologia , Transcriptoma
11.
Cell Calcium ; 48(2-3): 150-60, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20817251

RESUMO

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


Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Potenciais Evocados/fisiologia , Miócitos Cardíacos/metabolismo , Veias Pulmonares/metabolismo , Veias Pulmonares/fisiologia , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Estimulação Elétrica/métodos , Potenciais Evocados/efeitos dos fármacos , Átrios do Coração/citologia , Átrios do Coração/efeitos dos fármacos , Átrios do Coração/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/antagonistas & inibidores , Receptores de Inositol 1,4,5-Trifosfato/fisiologia , Masculino , Miócitos Cardíacos/efeitos dos fármacos , Veias Pulmonares/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Canal de Liberação de Cálcio do Receptor de Rianodina/fisiologia
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