Localization of Na+ channel isoforms at the atrioventricular junction and atrioventricular node in the rat.

BACKGROUND: The electrical activity of the atrioventricular node (AVN) is functionally heterogeneous, but how this relates to distinct cell types and the 3-dimensional structure of the AVN is unknown. To address this, we have studied the expression of Na(V)1.5 and other Na+ channel isoforms in the AVN. METHODS AND RESULTS: The rat AVN was identified by Masson's trichrome staining together with immunolabeling of marker proteins: connexin40, connexin43, desmoplakin, atrial natriuretic peptide, and hyperpolarization-activated and cyclic nucleotide-gated channel 4. Na+ channel expression was investigated with immunohistochemistry with isoform-specific Na+ channel antibodies. Na(V)1.1 was distributed in a similar manner to Na(V)1.5. Na(V)1.2 was not detected. Na(V)1.3 labeling was present in nerve fibers and cell bodies (but not myocytes) and was abundant in the penetrating atrioventricular (AV) bundle and the common bundle but was much less abundant in other regions. Na(V)1.5 labeling was abundant in the atrial and ventricular myocardium and the left bundle branch. Na(V)1.5 labeling was absent in the open node, penetrating AV bundle, AV ring bundle, and common bundle but present at a reduced level in the inferior nodal extension and transitional zone. Na(V)1.6 was not detected. CONCLUSIONS: Our findings provide molecular evidence of multiple electrophysiological cell types at the AV junction. Impaired AV conduction as a result of mutations in or loss of Na(V)1.5 must be the result of impaired conduction in the AVN inputs (inferior nodal extension and transitional zone) or output (bundle branches) rather than the AVN itself (open node and penetrating AV bundle).

Three-dimensional reconstruction of the rabbit atrioventricular conduction axis by combining histological, desmin, and connexin mapping data.

BACKGROUND: The 3D structure of the atrioventricular conduction axis incorporating detailed cellular and molecular composition, especially that relating to gap-junctional proteins, is still unclear, impeding mechanistic understanding of cardiac rhythmic disorders. METHODS AND RESULTS: A 3D model of the rabbit atrioventricular conduction axis was reconstructed by combining histological and immunofluorescence staining on serial sections. The exact cellular boundaries, especially those between transitional cells and atrial myocardium, were demarcated by a dense and irregular desmin-labeling pattern in conductive myocardium. The model demonstrates that the atrioventricular conduction axis is segregated into 2 connecting compartments, 1 predominantly expressing connexin45 (compact node and transitional cells) and the other predominantly coexpressing connexin43 and connexin45 (His bundle, lower nodal cells, and posterior nodal extension). The transitional zone shows unique features of spatial complexity, including a bridging bilayer structure (a deep transitional zone connecting with a superficial atrial-transitional overlay) and asymmetrical continuity (wider atrial-transitional interfaces and shorter atrial-axial distances in the hisian portion than in the ostial portion). In the latter compartment, the His bundle, lower nodal cells, and posterior nodal extension form a continual axis and longitudinal transitional-axial interface. CONCLUSIONS: Key findings of the present study are the demonstration of a distinct anatomical border between transitional and atrial cells, connection between transitional cells and both lower nodal cells and posterior nodal extension, and distinctive connexin expression patterns in different compartments of the rabbit atrioventricular conduction axis. These features, synthesized in a novel 3D model, provide a structural framework for the interpretation of nodal function.

Connexin45 (alpha 6) expression delineates an extended conduction system in the embryonic and mature rodent heart.

We previously demonstrated that alpha 6 (Cx45), one of the three connexins of the mammalian myocardium, is preferentially expressed in the peripheral portion of the ventricular conduction system in rats and mice. Here we report that alpha 6 is also prominently immunolocalized in the atrioventricular node and His bundle of these species. The distribution of immunolocalized alpha 6 reveals that the node and bundle form part of an extended central conductive network circumscribing the AV and outflow junctional regions of the fetal, and less continuously, the adult heart. Of the three cardiac connexins, alpha 6 is the isoform most continuously expressed by conduction tissues, and may thus account for the recently reported viability of the alpha 5 (Cx40) knockout mouse. It is concluded that alpha 6 expression is a defining feature of the heterogenous tissues comprising the atrioventricular conduction system of the rodent heart.

Metaiodobenzylguanidine as an index of atrioventricular nodal adrenergic activity.

UNLABELLED: Despite its importance, little is known about the uptake, storage and release of catecholamines in the atrioventricular (AV) node and His bundle. Previous in vitro studies have been limited by metabolism of norepinephrine. Metaiodobenzylguanidine (MIBG) shares many transport properties with norepinephrine and is considered a functional marker of adrenergic activity. METHODS: We used [125I]MIBG +/- 99mTc-sestamibi (99mTc-MIBI) and [123I]MIBG +/- 201TI] to evaluate regional differences in adrenergic activity between cardiac conductive and contractile elements in rats. Histological localization of the AV node and His bundle was performed using stains for acetylcholinesterase. RESULTS: Densitometric evaluation of autoradiographs, obtained from 20-mu thick sections of hearts from rats injected with either [125I]MIBG +/- 99mTc-MIBI (n = 4) and [123I]MIBG +/- 201TI (n = 6), revealed that there was approximately 30% more MIBG uptake in the AV node and His bundle compared to atrial or ventricular muscle (p < 0.05). Color-coded functional maps, generated by computer to simultaneously display 123I or [125I]MIBG and perfusion markers, revealed that the heterogeneous distribution of MIBG was independent of myocardial blood flow. CONCLUSION: When used as a selective functional marker of adrenergic activity in the cardiac conduction system, 123I- or [125I]MIBG autoradiography demonstrates increased adrenergic activity in the AV node and His bundle compared with the left ventricle. MIBG imaging provides a new research technique to probe in vivo modulation of AV nodal and His bundle sympathetic activity.

Immunoelectron microscopic localization of HNK-1 in the embryonic rat heart.

To confirm the role of HNK-1 in conduction tissue, the ultrastructural localization of monoclonal antibody HNK-1 was analyzed in developing rat hearts at embryonal day 14.5 by immunoelectron microscopic labeling procedures with post-embedding immunogold staining. Tissue sections in different planes containing the sino-atrial (SA) node, atrio-ventricular (AV) node and His bundle were used to demonstrate HNK-1. Immunogold labeling was detected on the cell surfaces and in the extracellular matrices of cells that had features common to conduction tissue cells. Non-specialized contractile myocytes were not labeled by this antibody. Furthermore, immunogold labeling was more prominent in wide intracellular spaces than in narrow intercellular spaces, and rarely observed in cell-cell contact regions. The cell surfaces and extracellular matrices of mesenchymal cells in the endocardial cushion, which contacts the His bundle, were also positive, suggesting the involvement of tract formation to the AV node. These findings may indicate that HNK-1 plays an important role in cell-cell adhesion processes both temporally and spatially in the developing conduction tissue. It was concluded, therefore, that HNK-1 is a suitable marker of the embryonic heart conduction system and might be useful in analyzing anomalous conduction systems, as in congenital heart disease.

Alpha-B crystallin in the normal human myocardium and cardiac conducting system.

The alpha crystallins are major protein components of the ocular lens and show both structural and functional homology with the family of small heat shock proteins. alpha B crystallin is also present in various extra-lenticular tissues, with a high concentration in cardiac muscle. In this study, the myocardium and conducting system from 15 adult and 25 fetal and infant hearts were examined by immunohistochemistry using a previously characterized antiserum to alpha B crystallin. Contractile myocardium showed moderate staining, with particular localization to Z bands and intercalated discs. Fibres of the sino-atrial and atrio-ventricular nodes and His bundle showed less intense staining than contractile fibres, whereas fibres of the left and right bundle branches showed more intense staining. This distribution is similar to that previously demonstrated for the intermediate filament desmin. This observation, together with currently available evidence, suggests that cardiac alpha B crystallin may play a role in protecting the cytoskeleton during cell stress. For practical purposes, immunostaining with alpha B crystallin greatly facilitates the identification of cardiac conducting fibres.

Beta-adrenoceptor subtypes in the atrioventricular conducting system and myocardium of spontaneously hypertensive rats: effects of angiotensin-converting enzyme inhibition by perindopril.

Quantitative autoradiography was used to determine the density and distribution of beta 1- and beta 2-adrenoceptors in the atrioventricular (AV) conducting system and surrounding myocardium of spontaneously hypertensive rats (SHR) after chronic infusion of perindopril (1 mg/kg/day) for 14 days by osmotic mini-pumps. Systolic blood pressure (SBP) was measured for a period of 4 weeks (2 weeks before and 2 weeks during perindopril infusion) in control and treated animals. Animals infused with vehicle (water) had a mean SBP of 248 mm Hg (measured on day 29); animals treated with perindopril had lower SBP (121 mm Hg, day 29). Perindopril treatment also prevented development of cardiac hypertrophy in SHR. beta-Adrenoceptor densities were measured in the AV node, His bundle, left and right bundle branches (LB, BB), interventricular and interatrial septa (IVS, IAS), mitral valve (MV), right papillary muscle, left and right ventricles (LV, RV), left and right atria (LA, RA), and apex. Perindopril produced no significant change in beta-adrenoceptors in any cardiac region examined. The results suggest that under experimental conditions in which perindopril treatment prevented cardiac hypertrophy and decreased SBP, there was no significant interaction between the renin-angiotensin system (RAS) and beta-adrenoceptor system in rat heart.

Autoradiographic localization and quantitation of beta 1- and beta 2-adrenoceptors in the human atrioventricular conducting system: a comparison of patients with idiopathic dilated cardiomyopathy and ischemic heart disease.

The density and distribution of beta 1- and beta 2-adrenoceptors in the atrioventricular conducting system and interatrial and interventricular septa from human hearts with idiopathic dilated cardiomyopathy and ischemic heart disease was determined by quantitative autoradiography using (-)[125I]cyanopindolol and the selective beta 1-adrenoceptor antagonist CGP 20712A and the selective beta 2-adrenoceptor antagonist ICI 118,551. Both beta 1- and beta 2-adrenoceptors were present in the atrioventricular node, bundle of His, interatrial and interventricular septa. No differences in the density or proportions of beta 1- and beta 2-adrenoceptors in the atrioventricular node, bundle of His, interatrial septum and interventricular septum were observed between hearts with idiopathic dilated cardiomyopathy or ischemic heart disease (P > 0.05) so further analysis did not distinguish between the two aetiologies. The density of beta 1-adrenoceptors was lower in the bundle of His (5.0 +/- 1.7 fmol/mg protein) than in the atrioventricular node (22.2 +/- 5.7 fmol/mg protein, P < 0.05), the interatrial septum (29.6 +/- 4.5 fmol/mg protein, P < 0.001) and interventricular septum (24.9 +/- 5.2 fmol/mg protein, P < 0.005, n = 8 for all values). The atrioventricular node, interatrial and interventricular septa had similar densities of beta 1-adrenoceptors (P = 0.60, ANOVA). The distribution of beta 2-adrenoceptors in the atrioventricular node (21.5 +/- 4.1 fmol/mg protein), bundle of His (12.9 +/- 2.6 fmol/mg protein) and atrial (16.7 +/- 2.3 fmol/mg protein) and septal myocardium (13.8 +/- 2.5 fmol/mg protein, n = 8 for all values) was uniform (P = 0.18, ANOVA). The percentage of beta 1- and beta 2-adrenoceptors in the atrioventricular node, bundle of His, interatrial and interventricular septa was uneven (P < 0.001, ANOVA). There was a higher proportion of beta 2-adrenoceptors in the bundle of His (72 +/- 6%) than in the atrioventricular node (51 +/- 3%, P < 0.01), interatrial septum (36 +/- 1%, P < 0.001) and interventricular septum (36 +/- 1%, P < 0.001).

Characterization and localization of endothelin receptor subtypes in the human atrioventricular conducting system and myocardium.

The characterization and localization of endothelin A (ETA) and endothelin B (ETB) receptors have been determined in tissue sections of the human atrioventricular conducting system, surrounding regions of atrial and ventricular myocardium, and the left ventricular free wall by use of radioligand binding, polymerase chain reaction, and in situ hybridization. Selective ETA (BQ123) and ETB (BQ3020) compounds in conjunction with [125I]endothelin-1 revealed the presence of ETA and ETB receptors in the left ventricular free wall (BQ123: 57 +/- 5% ETA, 43 +/- 2% ETB, n = 3; BQ3020: 67 +/- 3% ETA, 33 +/- 3% ETB, n = 3). Autoradiography using [125I]endothelin-1 in the absence or presence of BQ3020, BQ123, or endothelin-1 showed ETA and ETB receptors localized to atrial and ventricular myocardium, the atrioventricular conducting system, and endocardial cells. There was a higher proportion of ETB receptors in the atrioventricular node and the penetrating and branching bundles of His than in the surrounding interventricular and interatrial septa (p < 0.0001). There was a lower density of ETB receptors in the interventricular septum compared with the interatrial septum and the atrioventricular conducting system (p = 0.009) and a lower density of ETA receptors in the atrioventricular conducting system compared with interatrial and interventricular septa (p = 0.008). Isolated right atrial myocytes showed a higher proportion of ETA receptors (91 +/- 12%, n = 3). Amplification of left ventricular free wall cDNA by polymerase chain reaction revealed the presence of ETA and ETB receptor mRNA. mRNA for both subtypes was detected in isolated atrial myocytes. In situ hybridization showed ETA and ETB receptor mRNA localization to atrial and ventricular myocardium, the atrioventricular conducting system, and endocardial cells. These studies demonstrate the presence of ETA and ETB receptors in human myocardium and the atrioventricular conducting system.

[Findings in the atrioventricular node and the bundle of His in sudden and unexpected death in childhood. Histochemical study]. / Nálezy v atrioventrikulárním uzlu a Hisove svazku u náhlého a necekaného úmrtí v detském veku. Histochemická studie.

In 21 sudden and unexpected deaths of infants and three young children who died unexpectedly with known pathological conditions the authors examined by histochemical methods the atrioventricular node and bundle of His. The activity of "glycogen dependent" phosphorylase and the PAS reaction was in the majority week or negative. The probable cause of this finding is hypoxia, although other influences cannot be ruled out (inflammation, hypotrophy, autolysis). The activities of other enzymes (oxido-reductases ATPase, acetylcholine sterase, non-specific 1-naphtyl acetate esterase and acid phosphatase) were similar as in the normal conduction system.