RESUMO
KEY POINTS: The sinoatrial node (SAN) is the primary pacemaker of the heart. SAN dysfunction, or 'sick sinus syndrome', can cause excessively slow heart rates and pauses, leading to exercise limitation and syncope, currently treated by implantation of an electronic pacemaker. 'Biopacemaking' utilises gene therapy to restore pacemaker activity by manipulating gene expression. Overexpressing the HCN pacemaker ion channel has been widely used with limited success. We utilised bradycardic rat subsidiary atrial pacemaker tissue to evaluate alternative gene targets: the Na+ /Ca2+ exchanger NCX1, and the transcription factors TBX3 and TBX18 known to be involved in SAN embryonic development. TBX18 overexpression restored normal SAN function, as assessed by increased rate, improved heart rate stability and restoration of isoprenaline response. TBX3 and NCX1 were not effective in accelerating the rate of subsidiary atrial pacemaker tissue. Gene therapy targeting TBX18 could therefore have the potential to restore pacemaker function in human sick sinus syndrome obviating electronic pacemakers. ABSTRACT: The sinoatrial node (SAN) is the primary pacemaker of the heart. Disease of the SAN, sick sinus syndrome, causes heart rate instability in the form of bradycardia and pauses, leading to exercise limitation and syncope. Biopacemaking aims to restore pacemaker activity by manipulating gene expression, and approaches utilising HCN channel overexpression have been widely used. We evaluated alternative gene targets for biopacemaking to restore normal SAN pacemaker physiology within bradycardic subsidiary atrial pacemaker (SAP) tissue, using the Na+ /Ca2+ exchanger NCX1, and the transcription factors TBX3 and TBX18. TBX18 expression in SAP tissue restored normal SAN function, as assessed by increased rate (SAN 267.5 ± 13.6 bpm, SAP 144.1 ± 8.6 bpm, SAP-TBX18 214.4 ± 14.4 bpm; P < 0.001), improved heart rate stability (standard deviation of RR intervals fell from 39.3 ± 7.2 ms to 6.9 ± 0.8 ms, P < 0.01; root mean square of successive differences of RR intervals fell from 41.7 ± 8.2 ms to 6.1 ± 1.2 ms, P < 0.01; standard deviation of points perpendicular to the line of identity of Poincaré plots (SD1) fell from 29.5 ± 5.8 ms to 7.9 ± 2.0 ms, P < 0.05) and restoration of isoprenaline response (increases in rates of SAN 65.5 ± 1.3%, SAP 28.4 ± 3.4% and SAP-TBX18 103.3 ± 10.2%; P < 0.001). These changes were driven by a TBX18-induced switch in the dominant HCN isoform in SAP tissue, with a significant upregulation of HCN2 (from 1.01 × 10-5 ± 2.2 × 10-6 to 2.8 × 10-5 ± 4.3 × 10-6 arbitrary units, P < 0.001). Biophysically detailed computer modelling incorporating isoform-specific HCN channel electrophysiology confirmed that the measured changes in HCN abundance could account for the observed changes in beating rates. TBX3 and NCX1 were not effective in accelerating the rate of SAP tissue.
Assuntos
Sistema de Condução Cardíaco/metabolismo , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Síndrome do Nó Sinusal/terapia , Nó Sinoatrial/fisiologia , Proteínas com Domínio T/metabolismo , Animais , Simulação por Computador , Regulação da Expressão Gênica , Átrios do Coração , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Masculino , Modelos Biológicos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratos , Trocador de Sódio e Cálcio/metabolismo , Proteínas com Domínio T/genética , Técnicas de Cultura de TecidosRESUMO
The sinus node is an intensively researched structure in terms of anatomical, histological, electrophysiological, molecular and genetic approach. For postmortem diagnosis it is still difficult to investigate due to a still reduced accessibility. In this study we tried and succeed to apply molecular biology techniques on postmortem tissues in order to widen the range of postmortem forensic investigation and provide information related to the diagnostic of cardiac arrhythmia. We described the stages of this investigation, with dissection, preservation and analysis that included classical histology, immunohistochemistry, confocal microscope, microdissection, RIN testing, mRNA expression obtaining a precise morphofunctional location of the sinus node.
Assuntos
Nó Sinoatrial , Autopsia , Imuno-Histoquímica , Mudanças Depois da MorteRESUMO
In this study, a fixation protocol using a 10% neutral buffered formalin (FA) solution and another protocol using a methanol (MeOH) solution were compared for detection of ion channels, Kv1.5, Kv4.2, Cav1.2, Kir6.2, Nav1.5 and Nav1.1 in rat myocytes by immunolabelling. Kv1.5 and Kv4.2 at intercalated discs and Cav1.2 at transverse tubules were not detected by FA but were detected by MeOH. Kir6.2 at transverse tubules and Nav1.5 at sarcolemma were detected by FA but not by MeOH. It is suggested that both FA and MeOH fixation protocols should be used for the detection of cardiac ion channels by immunolabelling.
RESUMO
BACKGROUND: Recent clinical reports have emphasized the clinical significance of the left ventricular summit (LVS), a specific triangular epicardial area, as the source of ventricular arrhythmias where radiofrequency ablation is of great difficulty. MATERIALS AND METHODS: The macroscopic morphology of the LVS has been assessed in 80 autopsied and 48 angio-computed tomography (CT) human hearts. According to Yamada's equation, the size was calculated based on the distance to the first, most prominent septal perforator. RESULTS: The size of the LVS varies from 33.69 to 792.2 mm2, is highly variable, and does not correlate with body mass index, sex, or age in general. The mean size of the LVS was 287.38 ± 144.95 mm2 in autopsied and angio-CT (p = 0.44). LVS is mostly disproportionately bisected by cardiac coronary veins to superior-inaccessible and inferior-accessible areas. The superior aspect dominates over the inferior in both groups (p = 0.04). The relation between superior and inferior groups determines three possible arrangements: the most common type is superior domination (50.2%), then inferior domination (26.6%), and finally, equal distribution (17.2%). In 10.9%, the inferior aspect is absent. Only 16.4% of the LVS were empty, without additional trespassing coronary arteries. CONCLUSIONS: The difference in size and content of the LVS is significant, with no correlation to any variable. The size depends on the anatomy of the most prominent septal perforator artery. The superior, inaccessible aspect dominates, and the LVS is seldom free from additional coronary vessels, thus making this region hazardous for electrophysiological procedures.
Assuntos
Ablação por Cateter , Ablação por Radiofrequência , Taquicardia Ventricular , Humanos , Taquicardia Ventricular/cirurgia , Ablação por Cateter/métodos , Resultado do Tratamento , Ventrículos do Coração/diagnóstico por imagem , EletrocardiografiaRESUMO
PURPOSE: The left ventricular summit (LVS) is a source of difficult-to-treat arrhythmias because of anatomical limitations. The aim of this study was to perform detailed research of the left atrial appendage (LAA) anatomy of cadaveric hearts to analyze their complex anatomy and coverage of the LVS. METHODS AND RESULTS: Eighty human formalin fixed hearts (mean age 44.4 ± 15.5, 27.5% females) were investigated. Each LAA size, type, and its relationship to the LVS were analyzed, as well as possible access sites for mapping/ablating electrode. Four types of LAA were observed over two LVS sites that are either accessible or not. The highest coverage over an inaccessible LVS area was observed in the Broccoli type, followed by the Windsock then the Chicken Wing and finally the Cactus types; over the accessible area of the LVS was observed in the Windsock, then in the Chicken Wing, then in the Cactus, and finally in the Broccoli types. The attainable coverage for electrode access is diminished from 25 to 65% because of the complex pectinate muscles and sharp angles. The highest density of the LAA floor made by pectinate muscles can be found in the Broccoli type (p < 0.005), while the Chicken Wing had the highest number of paper-thin-like pouches. CONCLUSIONS: The LAA appears to be a promising entry for ablation-qualified patients with the LV summit originate arrhythmias. The complex internal structure of the LAA may complicate ablation procedures. More prominent appendages are promising in more extensive mapping areas over the LVS.
Assuntos
Apêndice Atrial , Fibrilação Atrial , Ablação por Cateter , Feminino , Humanos , Masculino , Apêndice Atrial/cirurgia , Átrios do Coração/cirurgia , Ventrículos do Coração/diagnóstico por imagem , Ventrículos do Coração/cirurgia , Ablação por Cateter/métodosRESUMO
The atrioventricular conduction axis, located in the septal component of the atrioventricular junctions, is arguably the most complex structure in the heart. It fulfils a multitude of functions, including the introduction of a delay between atrial and ventricular systole and backup pacemaking. Like any other multifunctional tissue, complexity is a key feature of this specialised tissue in the heart, and this complexity is both anatomical and electrophysiological, with the two being inextricably linked. We used quantitative PCR, histology and immunohistochemistry to analyse the axis from six human subjects. mRNAs for ~50 ion and gap junction channels, Ca(2+)-handling proteins and markers were measured in the atrial muscle (AM), a transitional area (TA), inferior nodal extension (INE), compact node (CN), penetrating bundle (PB) and ventricular muscle (VM). When compared to the AM, we found a lower expression of Na(v)1.5, K(ir)2.1, Cx43 and ANP mRNAs in the CN for example, but a higher expression of HCN1, HCN4, Ca(v)1.3, Ca(v)3.1, K(ir)3.4, Cx40 and Tbx3 mRNAs. Expression of some related proteins was in agreement with the expression of the corresponding mRNAs. There is a complex and heterogeneous pattern of expression of ion and gap junction channels and Ca(2+)-handling proteins in the human atrioventricular conduction axis that explains the function of this crucial pathway.
Assuntos
Nó Atrioventricular/citologia , Nó Atrioventricular/metabolismo , Sistema de Condução Cardíaco/citologia , Sistema de Condução Cardíaco/metabolismo , Arritmias Cardíacas/metabolismo , Canais de Cálcio Tipo T/metabolismo , Caveolina 3/metabolismo , Conexina 43/metabolismo , Conexinas/metabolismo , Eletrofisiologia , Junções Comunicantes/metabolismo , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Canais Iônicos/metabolismo , Proteínas Musculares/metabolismo , Miocárdio/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Canais de Sódio/metabolismoRESUMO
A 6-month-old female cat presented with respiratory distress. Physical examination showed a grade 5/6 holosystolic murmur with prominent precordial impulse over the left cranial chest wall. Echocardiography revealed bilateral hypertrophy of the ventricular walls, a dilated ascending aorta overriding the interventricular septum, a membranous ventricular septal defect and no obvious pulmonary trunk or pulmonary artery branches. Turbulent blood flow was detected around the ventricular septal defect and ascending aorta. Follow-up assessment, 12 months later, revealed marked and progressive biatrial dilation and biventricular hypertrophy. Four months after that, the cat died of severe congestive heart failure. To make a definitive postmortem diagnosis, we performed contrast enhanced micro-computed tomography (CT) on the ex vivo heart with micron-scale spatial resolution imaging and three-dimensional reconstruction. Micro-computed tomography analysis confirmed a common arterial trunk that bifurcated into the left pulmonary artery and aorta 5-mm distally from the truncal valve. The pulmonary trunk was absent. Slightly distal to the first branching, the common arterial trunk further branched into the right pulmonary artery and ascending aorta, indicating the aortic dominant form. Although CT angiography would be a preferred imaging modality for living animals, micro-computed tomography is a valuable tool for the ex vivo diagnosis of complex cardiac anomaly, such as presented in this cat.
Assuntos
Doenças do Gato , Cardiopatias Congênitas , Comunicação Interventricular , Persistência do Tronco Arterial , Animais , Doenças do Gato/diagnóstico por imagem , Gatos , Ecocardiografia/veterinária , Feminino , Cardiopatias Congênitas/veterinária , Comunicação Interventricular/veterinária , Artéria Pulmonar/diagnóstico por imagem , Persistência do Tronco Arterial/veterinária , Microtomografia por Raio-XRESUMO
During ageing, the function of sinoatrial node (SAN), the pacemaker of the heart, declines, and the incidence of sick sinus syndrome increases markedly. The aim of the study was to investigate structural and functional remodelling of the SAN during ageing. Rats, 3 and 24 months old (equivalent to young adult and approximately 69-year-old humans), were studied. Extracellular potential recording from right atrial preparations showed that (as expected) the intrinsic heart rate was slower in the old animals. It also showed a shift of the leading pacemaker site towards the inferior vena cava in the old animals. Consistent with this, intracellular potential recording showed that slow pacemaker action potentials were more widespread and extended further towards the inferior vena cava in old animals. Immunohistochemistry demonstrated that SAN tissue expressing HCN4, but lacking the expression of Na(v)1.5 (lack of Na(v)1.5 explains why pacemaker action potential is slow), was also more widespread and extended further towards the inferior vena cava in the old animals. Immunolabelling of caveolin3 (expressed in cell membrane of cardiac myocytes) demonstrated that there was a hypertrophy of the SAN cells in the old animals. Histology, quantitative PCR, and immunohistochemistry revealed evidence of a substantial age-dependent remodelling of the extracellular matrix (e.g. approximately 79% downregulation of genes responsible for collagens 1 and 3 and approximately 52% downregulation of gene responsible for elastin). It is concluded that the age- (and/or obesity-) dependent decline in SAN function is associated with a structural remodelling of the SAN: an enlargement of the SAN, a hypertrophy of the SAN cells, and a remodelling of the extracellular matrix.
Assuntos
Obesidade/fisiopatologia , Nó Sinoatrial/patologia , Envelhecimento , Animais , Modelos Animais de Doenças , Matriz Extracelular/metabolismo , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Imuno-Histoquímica/métodos , Masculino , Proteínas Musculares/metabolismo , Miócitos Cardíacos/citologia , Canal de Sódio Disparado por Voltagem NAV1.5 , Canais de Potássio/metabolismo , Ratos , Ratos Wistar , Canais de Sódio/metabolismo , Fatores de Tempo , Veia Cava Inferior/patologiaRESUMO
Abnormal QT prolongation with the associated arrhythmias is a significant predictor of mortality in diabetic patients. Gap junctional intercellular communication allows electrical coupling between heart muscle cells. The effects of streptozotocin (STZ)-induced diabetes mellitus on the expression and distribution of connexin 43 (Cx43) in ventricular muscle have been investigated. Cx43 mRNA expression was measured in ventricular muscle by quantitative PCR. The distribution of total Cx43, phosphorylated Cx43 (at serine 368) and non-phosphorylated Cx43 was measured in ventricular myocytes and ventricular muscle by immunocytochemistry and confocal microscopy. There was no significant difference in Cx43 mRNA between diabetic rat ventricle and controls. Total and phosphorylated Cx43 were significantly increased in ventricular myocytes and ventricular muscle and dephosphorylated Cx43 was not significantly altered in ventricular muscle from diabetic rat hearts compared to controls. Disturbances in gap junctional intercellular communication, which in turn may be attributed to alterations in balance between total, phosphorylated and dephosporylated Cx43, might partly underlie prolongation of QRS and QT intervals in diabetic heart.
Assuntos
Conexina 43/biossíntese , Diabetes Mellitus Experimental/metabolismo , Regulação da Expressão Gênica , Proteínas Musculares/biossíntese , Miocárdio/metabolismo , RNA Mensageiro/biossíntese , Animais , Diabetes Mellitus Experimental/patologia , Junções Comunicantes/metabolismo , Junções Comunicantes/patologia , Ventrículos do Coração/metabolismo , Ventrículos do Coração/patologia , Masculino , Miocárdio/patologia , Fosforilação , Ratos , Ratos WistarRESUMO
BACKGROUND: In vivo experiments in Goto-Kakizaki (GK) type 2 diabetic rats have demonstrated reductions in heart rate from a young age. The expression of genes encoding more than 70 proteins that are associated with the generation and conduction of electrical activity in the GK sinoatrial node (SAN) have been evaluated to further clarify the molecular basis of the low heart rate. MATERIALS AND METHODS: Heart rate and expression of genes were evaluated with an extracellular electrode and real-time RT-PCR, respectively. Rats aged 12-13 months were employed in these experiments. RESULTS: Isolated spontaneous heart rate was reduced in GK heart (161 ± 12 bpm) compared to controls (229 ± 11 bpm). There were many differences in expression of mRNA, and some of these differences were of particular interest. Compared to control SAN, expression of some genes were downregulated in GK-SAN: gap junction, Gja1 (Cx43), Gja5 (Cx40), Gjc1 (Cx45), and Gjd3 (Cx31.9); cell membrane transport, Trpc1 (TRPC1) and Trpc6 (TRPC6); hyperpolarization-activated cyclic nucleotide-gated channels, Hcn1 (HCN1) and Hcn4 (HCN4); calcium channels, Cacna1d (Cav1.3), Cacna1g (Cav3.1), Cacna1h (Cav3.2), Cacna2d1 (Cavα2δ1), Cacna2d3 (Cavα2δ3), and Cacng4 (Cav γ 4); and potassium channels, Kcna2 (Kv1.2), Kcna4 (Kv1.4), Kcna5 (Kv1.5), Kcnb1 (Kv2.1), Kcnd3 (Kv4.3), Kcnj2 (Kir2.1), Kcnk1 (TWIK1), Kcnk5 (K2P5.1), Kcnk6 (TWIK2), and Kcnn2 (SK2) whilst others were upregulated in GK-SAN: Ryr2 (RYR2) and Nppb (BNP). CONCLUSIONS: This study provides new insight into the changing expression of genes in the sinoatrial node of diabetic heart.
Assuntos
Arritmias Cardíacas/genética , Diabetes Mellitus Tipo 2/genética , Cardiomiopatias Diabéticas/genética , RNA Mensageiro/genética , Nó Sinoatrial/metabolismo , Potenciais de Ação , Animais , Arritmias Cardíacas/etiologia , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/fisiopatologia , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/metabolismo , Cardiomiopatias Diabéticas/etiologia , Cardiomiopatias Diabéticas/metabolismo , Cardiomiopatias Diabéticas/fisiopatologia , Modelos Animais de Doenças , Regulação da Expressão Gênica , Frequência Cardíaca/genética , Preparação de Coração Isolado , Masculino , RNA Mensageiro/metabolismo , Ratos Wistar , Nó Sinoatrial/fisiopatologiaRESUMO
BACKGROUND: There is an effort to build an anatomically and biophysically detailed virtual heart, and, although there are models for the atria and ventricles, there is no model for the sinoatrial node (SAN). For the SAN to show pacemaking and drive atrial muscle, theoretically, there should be a gradient in electrical coupling from the center to the periphery of the SAN and an interdigitation of SAN and atrial cells at the periphery. Any model should include such features. METHODS AND RESULTS: Staining of rabbit SAN preparations for histology, middle neurofilament, atrial natriuretic peptide, and connexin (Cx) 43 revealed multiple cell types within and around the SAN (SAN and atrial cells, fibroblasts, and adipocytes). In contrast to atrial cells, all SAN cells expressed middle neurofilament (but not atrial natriuretic peptide) mRNA and protein. However, 2 distinct SAN cell types were observed: cells in the center (leading pacemaker site) were small, were organized in a mesh, and did not express Cx43. In contrast, cells in the periphery (exit pathway from the SAN) were large, were arranged predominantly in parallel, often expressed Cx43, and were mixed with atrial cells. An approximately 2.5-million-element array model of the SAN and surrounding atrium, incorporating all cell types, was constructed. CONCLUSIONS: For the first time, a 3D anatomically detailed mathematical model of the SAN has been constructed, and this shows the presence of a specialized interface between the SAN and atrial muscle.
Assuntos
Simulação por Computador , Imageamento Tridimensional , Modelos Cardiovasculares , Nó Sinoatrial/anatomia & histologia , Nó Sinoatrial/citologia , Animais , Modelos Teóricos , Miocárdio , Proteínas de Neurofilamentos/análise , Proteínas de Neurofilamentos/genética , CoelhosRESUMO
The sinoatrial node (SAN) and the atrioventricular node (AVN) are specialized tissues in the heart: the SAN is specialized for pacemaking (it is the pacemaker of the heart), whereas the AVN is specialized for slow conduction of the action potential (to introduce a delay between atrial and ventricular activation during the cardiac cycle). These functions have special requirements regarding electrical coupling and, therefore, expression of connexin isoforms. Electrical coupling in the center of the SAN should be weak to protect it from the inhibitory electrotonic influence of the more hyperpolarized non-pacemaking atrial muscle surrounding the SAN. However, for the SAN to be able to drive the atrial muscle, electrical coupling should be strong in the periphery of the SAN. Consistent with this, in the center of the SAN there is no expression of Cx43 (the principal connexin of the working myocardium) and little expression of Cx40, but there is expression of Cx45 and Cx30.2, whereas in the periphery of the SAN Cx43 as well Cx45 is expressed. In the AVN, there is a similar pattern of expression of connexins as in the center of the SAN and this is likely to be in large part responsible for the slow conduction of the action potential.
Assuntos
Nó Atrioventricular/fisiologia , Conexinas/fisiologia , Nó Sinoatrial/fisiologia , Potenciais de Ação/fisiologia , Animais , Conexina 43/metabolismo , Conexina 43/fisiologia , Conexinas/metabolismo , Junções Comunicantes/fisiologia , Humanos , Taquicardia Supraventricular/fisiopatologia , Regulação para Cima/fisiologia , Proteína alfa-5 de Junções ComunicantesRESUMO
The distribution of atrial natriuretic peptide (ANP) in blood plasma and cardiac muscle and its effects on ventricular myocyte contraction and intracellular free calcium concentration [Ca2+]i in the streptozotocin (STZ)-induced diabetic rat have been investigated. Blood plasma concentration and heart atrial and ventricular contents of ANP were significantly increased in STZ-treated rats compared to age-matched controls. STZ treatment increased the number of ventricular myocytes immunolabeled with antibodies against ANP. In control myocytes the percentage of cells that labeled positively and negatively were 17% versus 83%, respectively. However, in myocytes from STZ-treated rat the percentages were 52% versus 53%. Time to peak (TPK) shortening was significantly and characteristically prolonged in myocytes from STZ-treated rats (360+/-5 ms) compared to controls (305+/-5 ms). Amplitude of the Ca2+ transient was significantly increased in myocytes from STZ-treated rats compared to controls (0.39+/-0.02 versus 0.29+/-0.02 fura-2 RU in controls) and treatment with ANP reduced the amplitude of the Ca2+ transient to control levels. ANP may have a protective role in STZ-induced diabetic rat heart.
Assuntos
Fator Natriurético Atrial/farmacologia , Fator Natriurético Atrial/fisiologia , Diabetes Mellitus Experimental/fisiopatologia , Ventrículos do Coração/fisiopatologia , Células Musculares/fisiologia , Contração Miocárdica/fisiologia , Animais , Fator Natriurético Atrial/sangue , Diabetes Mellitus Experimental/patologia , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/patologia , Masculino , Células Musculares/efeitos dos fármacos , Contração Miocárdica/efeitos dos fármacos , Ratos , Ratos Wistar , Estreptozocina/toxicidadeRESUMO
The functionally important effects on the heart of ACh released from vagal nerves are principally mediated by the muscarinic K+ channel. The aim of this study was to determine the abundance and cellular location of the muscarinic K+ channel subunits Kir3.1 and Kir3.4 in different regions of heart. Western blotting showed a very low abundance of Kir3.1 in rat ventricle, although Kir3.1 was undetectable in guinea pig and ferret ventricle. Although immunofluorescence on tissue sections showed no labeling of Kir3.1 in rat, guinea pig, and ferret ventricle and Kir3.4 in rat ventricle, immunofluorescence on single ventricular cells from rat showed labeling in t-tubules of both Kir3.1 and Kir3.4. Kir3.1 was abundant in the atrium of the three species, as shown by Western blotting and immunofluorescence, and Kir3.4 was abundant in the atrium of rat, as shown by immunofluorescence. Immunofluorescence showed Kir3.1 expression in SA node from the three species and Kir3.4 expression in the SA node from rat. The muscarinic K+ channel is activated by ACh via the m2 muscarinic receptor and, in atrium and SA node from ferret, Kir3.1 labeling was co-localized with m2 muscarinic receptor labeling throughout the outer cell membrane.
Assuntos
Átrios do Coração/metabolismo , Ventrículos do Coração/metabolismo , Miocárdio/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização , Canais de Potássio/metabolismo , Receptores Muscarínicos/metabolismo , Nó Sinoatrial/metabolismo , Animais , Western Blotting , Furões , Imunofluorescência , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G , Cobaias , Microscopia Confocal , Especificidade de Órgãos , Ratos , Receptor Muscarínico M2 , Especificidade da EspécieRESUMO
Adenosine exerts multiple receptor-mediated effects in the heart, including a negative chronotropic effect on the sinoatrial node. The aim of this study was to investigate the distribution of the equilibrative nucleoside transporter rENT1 in rat sinoatrial node and atrial muscle. Immunocytochemistry and/or immunoblotting revealed abundant expression of this protein in plasma membranes of sinoatrial node and in atrial and ventricular cells. Because rENT1-mediated transport is likely to regulate the local concentrations of adenosine in the sinoatrial node and other parts of the heart, it represents a potential pharmacological target that might be exploited to ameliorate ischemic damage during heart surgery.
Assuntos
Proteínas de Transporte/análise , Transportador Equilibrativo 1 de Nucleosídeo , Imuno-Histoquímica , Nó Sinoatrial/química , Animais , Conexina 43/análise , Proteínas de Transporte de Nucleosídeo Equilibrativas , Feminino , Átrios do Coração/química , Immunoblotting , Masculino , Microscopia Confocal , Ratos , Distribuição TecidualRESUMO
The pacemaker of the heart, the sinoatrial (SA) node, is characterized by unique electrical coupling properties. To investigate the contribution of gap junction organization and composition to these properties, the spatial pattern of expression of three gap junctional proteins, connexin45 (Cx45), connexin40 (Cx40), and connexin43 (Cx43), was investigated by immunocytochemistry combined with confocal microscopy. The SA nodal regions of rabbits were dissected and rapidly frozen. Serial cryosections were double labeled for Cx45 and Cx43 and for Cx40 and Cx43, using pairs of antibody probes raised in different species. Dual-channel scanning confocal microscopy was applied to allow simultaneous visualization of the different connexins. Cx45 and Cx40, but not Cx43, were expressed in the central SA node. The major part of the SA nodal-crista terminalis border revealed a sharply demarcated boundary between Cx43-expressing myocytes of the crista terminalis and Cx45/Cx40-expressing myocytes of the node. On the endocardial side, however, a transitional zone between the crista terminalis and the periphery of the node was detected in which Cx43 and Cx45 expression merged. These distinct patterns of connexin compartmentation and merger identified suggest a morphological basis for minimization of contact between the tissues, thereby restricting the hyperpolarizing influence of the atrial muscle on the SA node while maintaining a communication route for directed exit of the impulse into the crista terminalis.
Assuntos
Conexina 43/biossíntese , Conexinas/biossíntese , Átrios do Coração/metabolismo , Nó Sinoatrial/metabolismo , Animais , Imuno-Histoquímica , Microscopia Confocal , Coelhos , Proteína alfa-5 de Junções ComunicantesRESUMO
The aim of this study was to establish, using immunolabeling, whether the Kv1.5 K(+) channel is present in the pacemaker of the heart, the sinoatrial (SA) node. In the atrial muscle surrounding the SA node and in the SA node itself (from guinea pig and ferret), Western blotting analysis showed a major band of the expected molecular weight, approximately 64 kD. Confocal microscopy and immunofluorescence labeling showed Kv1.5 labeling clustered in atrial muscle but punctate in the SA node. In atrial muscle, Kv1.5 labeling was closely associated with labeling of Cx43 (gap junction protein) and DPI/II (desmosomal protein), whereas in SA node Kv1.5 labeling was closely associated with labeling of DPI/II but not labeling of Cx43 (absent in the SA node) or Cx45 (another gap junction protein present in the SA node). Electron microscopy and immunogold labeling showed that the Kv1.5 labeling in atrial muscle is preferentially associated with desmosomes rather than gap junctions.
Assuntos
Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio/análise , Nó Sinoatrial/química , Animais , Western Blotting/métodos , Bovinos , Conexina 43/análise , Conexinas/análise , Proteínas do Citoesqueleto/análise , Desmoplaquinas , Feminino , Furões , Técnica Indireta de Fluorescência para Anticorpo , Cobaias , Humanos , Canal de Potássio Kv1.5 , Masculino , Camundongos , Frações SubcelularesAssuntos
Relógios Biológicos/fisiologia , Canais de Cálcio/metabolismo , Cálcio/metabolismo , Nó Sinoatrial/fisiologia , Canais de Cátion TRPC/metabolismo , Animais , Relógios Biológicos/efeitos dos fármacos , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/efeitos dos fármacos , Humanos , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Sarcolema/metabolismo , Nó Sinoatrial/efeitos dos fármacos , Canais de Cátion TRPC/antagonistas & inibidoresRESUMO
Voltage-dependent sodium (Na(+)) channels are heterogeneously distributed through the pacemaker of the heart, the sinoatrial node (SA node). The measured sodium channel current (i(Na)) density is higher in the periphery but low or zero in the center of the SA node. The functional roles of i(Na) in initiation and conduction of cardiac pacemaker activity remain uncertain. We evaluated the functional roles of i(Na) by computer modeling. A gradient model of the intact SA node and atrium of the rabbit heart was developed that incorporates both heterogeneities of the SA node electrophysiology and histological structure. Our computations show that a large i(Na) in the periphery helps the SA node to drive the atrial muscle. Removal i(Na) from the SA node slows down the pacemaking rate and increases the sinoatrial node-atrium conduction time. In some cases, reduction of the SA node i(Na) results in impairment of impulse initiation and conduction that leads to the SA node-atrium conduction exit block. Decrease in active SA node cell population has similar effects. Combined actions of reduced cell population and removal of i(Na) from the SA node have greater impacts on weakening the ability of the SA node to pace and drive the atrium.