Differentiation of Purkinje fibres and ordinary ventricular and atrial myocytes in the bovine heart: an immuno-and enzyme histochemical study

Summary The differentiation of Purkinje fibres and ordinary ventricular and atrial myocytes in bovine hearts was studied with specific antibodies against M-line proteins (MM-creatine kinase and myomesin) and with enzyme histochemistry (succinate dehydrogenase and mitochondrial glycerol-3-phosphate dehydrogenase). MM-creatine kinase was detected at an earlier stage in Purkinje fibres and atrial myocytes than in ordinary ventricular myocytes. The findings are in agreement with previous ultrastructural observations that an earlier appearance of a dense M-band occurs in Purkinje fibres than in ordinary ventricular myocytes. Myomesin was detected in all three cell types even at early foetal stages, in accordance with suggestions that it is an integral component of the myofibrillar structure. The activity of succinate dehydrogenase gradually increased in both ordinary ventricular and atrial myocytes, while the activity of mitochondrial glycerol-3-phosphate dehydrogenase was high at different stages of early foetal development in the two tissues, finally becoming low in the adult stage. The activity of succinate dehydrogenase and mitochondrial glycerol-3-phosphate dehydrogenase seemed to remain unchanged in the Purkinje fibres from early to late foetal stages. The present study shows that the Purkinje fibres are already different from ordinary ventricular myocytes at early foetal stages and that the two cell types differentiate in different ways. It is concluded that there are also developmental differences between ordinary ventricular and atrial myocytes.     

The atrioventricular node and bundle in the ferret heart: a light and quantitative electron microscopic study.

The cells of the atrioventricular (AV) junction in the ferret heart were examined using light microscopy, a wax-model reconstruction and quantitative electron microscopy to determine their organization and characteristics. A series of subdivisions of the specialized tissues of the AV junction was apparent at both the light and electron microscopic levels. A transitional zone was observed interposed between the atrial muscle cells and the AV node. The AV node consisted of a coronary sinus portion, a superficial portion and a deep portion. The AV bundle had a segment above the anulus fibrosus, a segment which penetrated the right fibrous trigone, a non-branching segment below the anulus fibrosus and a branched segment. At the ultrastructural level the AV junctional conduction tissues had fewer irregularly oriented myofibrils than did working atrial myocardial cells. T-tubules, present in atrial muscle cells, were not observed in the modified muscle cells of the AV node and bundle. Conventional intercalated discs also were not observed between the cells of the AV node or the AV bundle. Atrial myocardial cells had the highest percentage of the plasma membrane occupied by desmosomes, fasciae adherentes and gap junctions. The AV bundle cells had the highest percentage of appositional surface membrane and a relatively large fraction of plasma membrane occupied by gap junctions. Cells of the superficial portion of the AV node had the smallest percentage of the plasma membrane composed of gap junctions, desmosomes or fasciae adherentes, as well as the smallest fraction of the cell membrane apposed to adjacent cells. The stereological data indicate that the most useful distinguishing characteristic between atrial muscle cells and conduction cells was that a smaller percentage of the conduction cell sarcoplasm was occupied by mitochondria and myofibrils. The most useful characteristics that could be used to differentiate between the regions of the AV junctional conduction tissues were the amounts and types of surface membrane specializations in the respective cell types.     

The development of the conduction system in the mouse embryo heart: IV. Differentiation of the atrioventricular conduction system

The development of the atrioventricular conduction system in the mouse heart has been studied by light and electron microscopy from the time of the completion of ventricular septation to fetal stage II, 13–16 days postcoitum. At the beginning of this period the already established atrioventricular node (AVN) enlarges rapidly into the dorsal AV cushion from the primitive AV tract, reaching almost its full fetal size when septation is complete. The development of the atrionodal interconnections is a slow and complex process. The dorsal atrial myocardium develops on both sides of the node, establishing a muscular overlay over its proximal aspect, and also incorporating the former AV tract. At this time also, the developing muscular interatrial septum grows downward to establish contact with the node, the sinus venosus, and the myocardium of the right and left atrial walls. The distally proceeding differentiation of the ab initio continuous conduction pathway along the AVN, His bundle, and bundle branches demonstrates a progressive and sequential development of high cellular glycogen content. Progressive isolation of the atrioventricular conduction system leading to (still incomplete) insulation by connective tissue, has been observed.     

An immunocytochemical study of the sinuatrial node and atrioventricular conducting system of the rat for atrial natriuretic peptide distribution

Summary Immunocytochemical studies of the adult rat heart show that specific heart granules and atrial natriuretic peptide immunoreactivity are absent from the majority of the myocytes of the specialized nodes, atrioventricular bundle and bundle branches. Immunoreactive granules are present in a small proportion of the transitional sinuatrial and atrioventricular nodal myocytes but, in these regions, they are smaller than their counterparts in the general atrial myocytes. A rarer type of cell profile, identical to general atrial myocytes but lacking immunoreactive granules, is also present at the periphery of the sinuatrial node. A very small proportion of myocytes in the ventricular myocardium, generally in the subendocardial layers subjacent to the terminal ramifications of the bundle branches, contain a few immunoreactive granules.     

The distribution of sympathetic nerve fibres in the AV node and AV bundle of the bovine heart

The sympathetic nervous system has important effects on the properties of the heart, including the conduction of the impulse. However, it is not known how this nervous system is distributed in the atrioventricular (AV) bundle, which together with the AV node constitutes the only conduction pathway between the atria and ventricles in normal hearts. Therefore, in the present study the adrenergic innervation in the bovine AV node/AV bundle was examined by use of the glyoxylic acid induced method for histofluorescence demonstration of catecholamines. Acetylcholinesterase (AChE) histochemistry was also used. It was found that the AChE-positive nerve fascicles in these regions partly contain sympathetic nerve fibres, that sympathetic nerve fibres occur in the proximity of some of the ganglionic cells that occur outside the AV node/AV bundle, that the arteries supplying AV bundle tissue as well as AV nodal tissue have perivascular plexuses of sympathetic nerve fibres, and that there is a substantial number of sympathetic nerve fibres outside Purkinje fibre bundle surfaces. The observations give new insight into the question of the distribution of the sympathetic nerves in the AV bundle in relation to the distribution of these nerves in the AV node. Possible functional implications of the observations are discussed.     

The early development of the atrioventricular node and bundle of His in the embryonic chick heart. An electrophysiological and morphological study

The development of the atrioventricular node and bundle of His of embryonic chick hearts was studied by electrophysiological and morphological techniques. The dorsal wall of the AV canal and the interatrial septum were explored to determine if they contribute to the formation of the AV node and bundle of His. The resting membrane and action potentials of the interatrial septum cells were systematically analyzed and found to undergo progressive differentiation with development. The earliest identification of the AV node and upper bundle of His group of cells was achieved at 5 1/2-6 days of development by the electrical recording of their corresponding characteristic action potentials, from a circumscribed area located in the lowest and dorsal segment of the interatrial septum. The morphological and anatomical characterization of the cells was made following electrical recording and labelling with charcoal particles. The earlier AV node and bundle of His responses had similar characteristics to those of the adult heart. It is concluded that the AV node and upper bundle of His cells derive from the low interatrial septum. The possibility that AV canal cells contribute to this event was discarded. The functional relationship of the Av node and bundle of His with other cardiac tissues during the early development of the heart is discussed.     

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.     

Location and functional characterization of the right atrioventricular pacemaker ring in the adult avian heart

Previous histological studies showed that in addition to a sinus node, an atrioventricular (AV) node, an AV bundle, left and right bundle branches, birds also possess a right AV‐Purkinje ring that is located in the atrial sheet of the right muscular AV‐valve along all its base length. The functionality of the AV‐Purkinje ring is unknown. In this work, we studied the topology of pacemaker myocytes in the atrial side of the isolated chicken spontaneously contracting right muscular AV‐valve using the method of microelectrode mapping of action potentials. We show that AV‐cells having the ability to show pacemaking reside in the right muscular AV‐valve. Pacemaker action potentials were exclusively recorded close to the base of the valve along its whole length from dorsal to the ventral attachment to the interventricular septum. These action potentials have much slower rate of depolarization, lower amplitude, and higher diastolic depolarization than action potentials of Purkinje (conducting) cells. We conclude the right AV‐valve has a ring bundle of pacemaker cells (but not Purkinje cells) in the adult chicken heart. J. Morphol. 277:363–369, 2016. © 2015 Wiley Periodicals, Inc.     

Morphological study of sarcoplasmic reticulum in the atrioventricular node and bundle cells in guinea pig hearts

The osmium-ferrocyanide method for staining of the sarcoplasmic reticulum (SR) was used for a morphological investigation of the various components of the SR in the atrioventricular node and bundle (AVNB) cells of guinea pig hearts. On the basis of light microscopic observations, the AVNB tissue in guinea pig hearts can be divided into five regions: atrionodal junction, midnode, proximal bundle, distal bundle, and bundle branches. Electron microscopic observations revealed two types of junctional SR (j-SR) saccules in the cells from all the regions of AVNB tissue. One is similar to that seen in the working cardiac cells, i.e., flattened saccules with junctional granules. The second type is dilated and contains electron-dense granular material throughout its lumen. The flattened type is seen more often than the dilated type in atrionodal junctional cells and midnode cells, whereas the dilated type occurs more often in distal bundle cells and bundle branch cells. In most cells from the atrionodal junction and midnode regions, the j-SR saccules are apposed more often to sarcolemmal areas associated with nonspecialized regions of intercellular junctions than to other sarcolemmal areas. This distribution was not found in the distal bundle and bundle branch cells. Free SR tubules around the myofilament bundles are poorly developed in the midnode cells, generally in accord with the extent of development of myofibrils. Z-tubules are found in cells from all regions but are poorly developed in midnode cells. Corbular SR vesicles are found in cells from all the regions of AVNB tissues but are rare in midnode cells. Thus, each of the regions in the AVNB tissue has a different, characteristic distribution of SR components. Because of their possible relationship to the regulation of the intracellular concentrations of calcium, these differences in SR morphology may contribute to the diverse physiological properties of the different regions of the AV node and bundle.     

A mathematical model of human atrioventricular nodal function incorporating concealed conduction

This work develops a mathematical model for the atrioventricular (AV) node in the human heart, based on recordings of electrical activity in the atria (the upper chambers of the heart) and the ventricles (the lower chambers of the heart). Intracardiac recordings of the atrial and ventricular activities were recorded from one patient with atrial flutter and one with atrial fibrillation. During these arrhythmias, not all beats in the atria are conducted to the ventricles. Some are blocked (concealed). However, the blocked beats can affect the properties of the AV node. The activation times of the atrial events were regarded as inputs to a mathematical model of conduction in the AV node, including a representation of AV nodal concealment. The model output was compared to the recorded ventricular response to search for and identify the best possible parameter combinations of the model. Good agreement between the distribution of interbeat intervals in the model and data for durations of 5 min was achieved. A model of AV nodal behavior during atrial flutter and atrial fibrillation could potentially help to understand the relative roles of atrial input activity and intrinsic AV nodal properties in determining the ventricular response.     

Probit analysis of the atrioventricular (AV) junctional tissues of the ferret heart

Probit frequency analysis, a graphic method for determining whether a population is normally distributed, skewed, or multinodal, was used to determine whether P cells are present in different regions of the AV junction in the ferret heart. This analysis indicated that at least 95% of the cells of the transitional zone, superficial AV node, deep AV node, and distal AV bundle of the ferret heart are morphologically homogeneous. In the proximal AV bundle a large cell population is found in addition to the AV bundle cells. The probit analysis was also used to characterize the shape of the cells of each region of the AV junction further. AV nodal cells are not as elongated as the atrial muscle cells and AV bundle cells. These nodal cells also do not branch as extensively as the AV bundle cells.     

Inducible recombination in the cardiac conduction system of minK: CreERT² BAC transgenic mice

Inducible Cre recombination is a powerful technology that allows for spatial and temporal modulation of gene expression in vivo. Diseases of the cardiac conduction system (CCS) pose a significant clinical burden but are not currently well understood at the molecular level. To enable inducible recombination in the murine CCS, we created a minK:CreERT(2) bacterial artificial chromosome (BAC) transgenic mouse line. Cre activity is present after tamoxifen administration in the atrioventricular (AV) node, AV bundle, and bundle branches of adult transgenic mice. We anticipate that by enabling inducible recombination specifically in the AV node, bundle, and bundle branches, minK:CreERT(2) BAC transgenic mice will prove useful in advancing our understanding of CCS disease and function.     

Apoptotic myocardial degeneration in thrombotic thrombocytopenic purpura

The objective of this study was to determine whether the known myocardial degeneration in TTP is due to apoptosis. In TTP the heart is often involved, including the cardiac conduction system. Despite many platelet occlusions of small coronary arteries, there is little myocardial necrosis. Why the intermittent clinical episodes begin or end is unknown. Six hearts of patients dying with TTP were examined with routine and immunohistochemical stains. In addition to ventricular and atrial myocardium we examined the cardiac conduction system and coronary chemoreceptor. Numerous small coronary arteries were occluded with platelet thrombi in all these sites, including especially the sinus node, AV node and His bundle. The myocardial degeneration we found was conspicuously devoid of inflammation and the myocytes were relatively intact. These characteristics combined with TUNEL-positivity in the degenerating cells are typical of apoptosis. The focal degeneration in TTP is primarily apoptotic. Because circulating serotonin is carried by platelets and is released during aggregation, and because serotonin can cause a powerful cardiogenic hypertensive chemoreflex, we suggest that such a response may dislodge early platelet aggregations. Lessons from TTP may have special relevance for better understanding of myocardial reperfusion problems associated with angioplasty, thrombolysis and ischemic preconditioning.     

The Spread of Excitation in the Embryonic Chick Heart

The spread of excitation in embryonic chick hearts, ranging in age from 7 to 20 days, was studied with both intracellular and extracellular electrodes. Evidence that the delay in ventricular excitation could be attributed to the cells of the entire atrioventricular (AV) ring was obtained, in part, from sagittal sections of the heart. In the intact preparation, uniform propagation occurred throughout the atrial roof at an apparent conduction velocity of 0.4 to 0.5 meter/sec. Delay of impulse propagation was localized in a very narrow band of tissue which extended across the AV ring. The apparent conduction velocity of this tissue was between 0.003 and 0.005 meter/sec. Both normal and retrograde propagation revealed the spread of conduction across the AV ring to be decremental in nature. This finding was supported by high frequency stimulation experiments which gave rise to AV block localized in the cells of the AV ring. Cardiac rhythmicity and AV transmission were responsive to acetylcholine and norepinephrine in much the same manner as in the adult mammalian heart. The present findings are in support of the hypothesis that the embryonic AV ring is the functional counterpart of the adult AV node.     

Myocardial cell heterogeneity in the human heart with respect to myosin ATPase activity

Summary The pH sensitivity of the Ca²⁺-activated myosin ATPase in atrial, ventricular and conduction tissue of human hearts has been established. Heterogeneity with respect to ATPase activity is shown not only to exist between the atrial, the ventricular myocardium and the conduction system but alsowithin both the ordinary atrial and ventricular myocardium andwithin the conduction system. These observations are related to the polymorphism of the myosin molecule and suggest that fibre types with different contractile properties co-exist in the human heart.     

Functional mathematical model of dual pathway AV nodal conduction

Dual atrioventricular (AV) nodal pathway physiology is described as two different wave fronts that propagate from the atria to the His bundle: one with a longer effective refractory period [fast pathway (FP)] and a second with a shorter effective refractory period [slow pathway (SP)]. By using His electrogram alternance, we have developed a mathematical model of AV conduction that incorporates dual AV nodal pathway physiology. Experiments were performed on five rabbit atrial-AV nodal preparations to develop and test the presented model. His electrogram alternances from the inferior margin of the His bundle were used to identify fast and slow wave front propagations. The ability to predict AV conduction time and the interaction between FP and SP wave fronts have been analyzed during regular and irregular atrial rhythms (e.g., atrial fibrillation). In addition, the role of dual AV nodal pathway wave fronts in the generation of Wenckebach periodicities has been illustrated. Finally, AV node ablative modifications have been evaluated. The model accurately reproduced interactions between FP and SP during regular and irregular atrial pacing protocols. In all experiments, specificity and sensitivity higher than 85% were obtained in the prediction of the pathway responsible for conduction. It has been shown that, during atrial fibrillation, the SP ablation significantly increased the mean HH interval (204 ± 39 vs. 274 ± 50 ms, P < 0.05), whereas FP ablation did not produce significant slowing of ventricular rate. The presented mathematical model can help in understanding some of the intriguing AV node mechanisms and should be considered as a step forward in the studies of AV nodal conduction.     

Atrioventricular conduction with and without AV nodal delay: two pathways to the bundle of His in the rabbit heart

The electrophysiological properties of atrioventricular (AV) nodal dual pathways have traditionally been investigated with premature stimuli delivered with right atrial pacing. However, little is known about the functional characteristics of AV nodal inputs outside of this context. Superfused rabbit triangle of Koch preparations (n = 8) and Langendorff-perfused hearts (n = 10) were paced throughout the triangle of Koch and mapped electrically and optically for activation pattern, electrogram and optical action potential morphologies, stimulation thresholds, and stimulus-His (S-H) intervals. Optical mapping and changes in His electrogram morphology were used to confirm the activation pathway. Pacing stimuli >or=2 mm above the tricuspid valve caused fast-pathway activation of the AV node and His with a threshold of 2.4 +/- 1.6 mA. An area directly below the coronary sinus had high thresholds (8.6 +/- 1.4 mA) that also resulted in fast-pathway excitation (P < 0.001). S-H intervals (81 +/- 19 ms) for fast-pathway activation remained constant throughout the triangle of Koch, reflecting the AV delay. Stimuli applied <2 mm from the tricuspid valve resulted in slow pathway (SP) excitation or direct His excitation (4.4 +/- 2.2 mA threshold; P < 0.001 compared with fast pathway). For SP/His pacing, S-H intervals showed a strong dependence on the distance from the His electrode and were significantly lower than S-H intervals for fast-pathway activation. SP/His pacing also displayed characteristic changes in His electrogram morphology. In conclusion, optical maps and S-H intervals for SP/His activation suggest that AV conduction via SP bypasses the compact AV node via the lower nodal bundle, which may be utilized to achieve long-term ventricular synchronization.     

The effect of propranolol and dimethylpropranolol on cardiac conduction.

Isolated dog hearts perfused with blood from a donor dogand driven at two heart rates were used to compare the effects of propranolol with those of its quaternary ammonium derivative on atrial, atrioventricular (AV) nodal, and His-Purkinje conduction. Propranolol slowed only AV-nodal conduction, increasing the minimal conduction time and the effect of prematurity, without affecting fatigue. Practolol did not have this effect. Dimethylpropranolol had similar but not identical effects on the AV node, but also slowed atrial and ventricular conduction. In contrast with the quaternary derivative of lidocaine, dimethylpropranolol's effect on atrial and ventricular conduction was not dependent on the heart rate. The effect of dimethylpropranolol on ventricular conduction was observed at doses lower than those reported by others to be antiarrhythmic.