Enzyme histochemical studies on the conducting system of the human heart

Summary In this communication, the results of applying various histochemical techniques for the localization of oxidoreductases, transferases, hydrolases and isomerases in the human heart are presented. The Purkinje fibres of the atrioventricular conducting system of the human heart differ from the myocardium proper in containing a slightly higher activity of most of the glycolytic and gluconeogenetic enzymes investigated. The relatively higher activity of 6-phosphofructokinase, the key enzyme in anaerobic carbohydrate metabolism, is especially noteworthy. On the other hand, the activities of some of the enzymes that play a part in the aerobic energy metabolism is slightly less than those in the myocardium fibres.As for the activity of the NADPH regenerating enzymes, the activity of 6-phosphogluconate dehydrogenase and malate dehydrogenase (oxaloacetate-decarboxylating) is somewhat higher, and the activity of glucose-6-phosphate dehydrogenase similar, in the Purkinje fibres compared to that in the myocardial fibres. The activity of myosin ATPase is similar for both types of fibre. Likewise, the fibres of the conducting system and of the myocardium show a similar activity of acid phosphatase, -glucuronidase, non-specific naphthylesterase and peroxidase. The neurogenic function of the conducting system of the human heart was demonstrated by the high activity of acetylcholinesterase in the Purkinje fibres and in the atrioventricular node. All these histochemical findings in Purkinje fibres are similar at widely differing levels of the conducting system.     

Histochemische,fluoreszenzmikroskopische und experimentelle Untersuchungen am Reizleitungssystem von Goldhamster,Maus und Ratte

Zusammenfassung Im Reizleitungssystem (RLS) von Goldhamster, Maus und Ratte sind nicht nur die oxidativen sondern auch die glykolytischen Enzymaktivitäten geringer als in der Arbeitsmuskulatur. Innerhalb der Reizleitungsmuskulatur überwiegt die Glykolyse. Demgegenüber besitzen Glucose-6-phosphat-Dehydrogenase und lysosomale Enzyme im RLS höhere Aktivitäten als im Myokard. Die Lactat-Dehydrogenase-Isoenzyme verhalten sich in beiden Muskelgeweben weitgehend gleich. Entsprechend dem reduzierten glykolytischen und oxidativen Stoffwechsel ist die Kapillardichte im RLS gering. Die Aktivitäten der Enzyme des Glykogenstoffwechsels laufen in RLS und Myokard in etwa dem Glykogengehalt parallel. Klare artspezifische und regionale Stoffwechselunterschiede fehlen innerhalb der Reizleitungsmuskulatur.Durch hohe cholinerge Nervenfaserdichte zeichnen sich alle Teile des RLS von Goldhamster und Maus aus. Eine dichte adrenerge Innervation ist nur beim Goldhamster im gesamten RLS anzutreffen. Im Mäuseherzen bevorzugen die adrenergen Fasern den Sinusknoten.Im AV-System von Ratten nimmt das Glykogen nach Hypoxie, Katecholamingaben und Hunger ab. Beim Goldhamster tritt überzeugender Glykogenverlust im AV-System nur nach Sauerstoffmangel und Katecholaminapplikation ein.

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Histochemical fluorescence microscopical and experimental investigations on the impulse conducting system of golden hamsters, mice and rats

Summary In the impulse conducting system (ICS) of golden hamsters, mice and rats the activities of glycolytic as well as of oxidative enzymes are lower than in the myocardium. However, in the ICS the glycolytic pathway is relatively more active in comparison with the oxidative metabolism. On the contrary, glucose-6-phosphate dehydrogenase and the lysosomal enzymes activities are higher in the ICS than in the ordinary cardiac muscle. The lactate dehydrogenase isoenzyme pattern is nearly the same in both heart muscle tissues. In agreement with the lowered glycolytic and oxidative metabolism a reduced capillarisation is found in the ICS. The activities of enzymes involved in glycogen metabolism are related to the amount of glycogen in the impulse conducting tissue and working muscle. Within the ICS clear speciesdependent and regional metabolic differences have not been observed.A dense cholinergic innervation occurs in all parts of the ICS of golden hamsters and mice. An overall rich adrenergic innervation is typical only for the ICS of golden hamsters. In the mouse heart, however, the adrenergic nerve fibers prefer the sinatrial node.In the AV system of rats glycogen decreases following hypoxia, catecholamine application and hunger. In the same ICS-region of golden hamsters a marked loss of glycogen can only be produced by hypoxia and treatment with catecholamines.

     

Skeletin immunoreactivity in heart Purkinje fibers from several species.

Ever since its discovery, the identification of the specialized conducting system of the heart has been a matter of debate. In some species, a main distinguishing feature under the electron microscope, as compared with ordinary myocytes, is the presence of large pools of juxtanuclear filaments, so called intermediate or skeletin filaments. In the present study, we have adopted the indirect immunofluorescence method and anti-skeletin antisera for the identification of the ventricular conducting system in several species. It was found that anti-skeletin reactivity generally exceeded that of ordinary myocytes. The degree of immunofluorescence could be related to a previous classification model of the differentiation of the conducting cells. It is suggested that skeletin is highly conserved throughout phylogeny and that anti-skeletin may serve as an additional tool for the identification of conducting cells at the light microscopic level.     

Tissue composition of the sinoauricular area of the human heart (a quantitative electron microscopic analysis)

An original technique for fixation, treatment and oriented embedding of the sinoauricular area material of the human heart makes it possible to study ultrastructure of the conducting myocardial in the sinus node (SN) and that of the parasinusoid working myocardium in the right atrium (RA), while the spatial interrelations, between the structures of the area examined are fully kept intact. Quantitative analysis of the SN and RA myocardial tissue composition has been performed. Contents of muscular, connective tissue, vascular and nervous components in the SN and RA have been estimated in hearth of 8 men died from causes not connected with any heart disease. Informativity degree of each component studied is discussed for differentiating the SN conducting myocardium from the RA working myocardium in ultrastructural investigations of the human heart in autopsies.     

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.     

Microquantitative determination of lactate dehydrogenase isoenzymes in the myocardium and the conducting system

Summary A newly developed technique was used for the electrophoretic separation of lactate dehydrogenase (LDH) isoenzymes from lyophilized tissue samples in the nanogram range. In this study portions of 10–200 ng from the myocardium and the conducting system of cattle, sheep, pig and man were microdissected and analysed.In the heart tissues of cattle, sheep and pig, the isoforms LDH1, LDH2 and LDH3 were detected in species-specific varying amounts. In all these animals, the conducting system is marked by high LDH1 activity, which is present at a ratio of about 2:1 compared with the myocardium. The values in man, however, differ from these values, but this might be due to post-mortem changes. The findings are discussed with respect to possible aerobic-anaerobic functions.     

Phosphorylase activity as a histochemical marker of specialized tissue of heart

Synopsis Phosphorylase activity has been investigated histochemically under controlled conditions in the specialized tissue and the general myocardium of goat heart using glucose-1-phosphate as the substrate. It has been observed that the phosphorylase content of the nodal and conducting tissue is very high as compared to the general myocardium. The phosphorylase content of the cardiac neural elements is also high. Since the connective tissue which surrounds the nodal and conducting tissue has almost no phosphorylase activity, the nodal and conducting tissue is sharply demarcated by the histochemical reaction for phosphorylase. This reaction can, therefore, be used as a specific and expedient method for the localization of specialized tissue as an exploratory and confirmatory test.     

In vivo induction of cardiac Purkinje fiber differentiation by coexpression of preproendothelin-1 and endothelin converting enzyme-1

The rhythmic heart beat is coordinated by electrical impulses transmitted from Purkinje fibers of the cardiac conduction system. During embryogenesis, the impulse-conducting cells differentiate from cardiac myocytes in direct association with the developing endocardium and coronary arteries, but not with the venous system. This conversion of myocytes into Purkinje fibers requires a paracrine interaction with blood vessels in vivo, and can be induced in vitro by exposing embryonic myocytes to endothelin-1 (ET-1), an endothelial cell-associated paracrine factor. These results suggest that an endothelial cell-derived signal is capable of inducing juxtaposed myocytes to differentiate into Purkinje fibers. It remains unexplained how Purkinje fiber recruitment is restricted to subendocardial and periarterial sites but not those juxtaposed to veins. Here we show that while the ET-receptor is expressed throughout the embryonic myocardium, introduction of the ET-1 precursor (preproET-1) in the embryonic myocardium is not sufficient to induce myocytes to differentiate into conducting cells. ET converting enzyme-1 (ECE-1), however, is expressed preferentially in endothelial cells of the endocardium and coronary arteries where Purkinje fiber recruitment takes place. Retroviral-mediated coexpression of both preproET-1 and ECE-1 in the embryonic myocardium induces myocytes to express Purkinje fiber markers ectopically and precociously. These results suggest that expression of ECE-1 plays a key role in defining an active site of ET signaling in the heart, thereby determining the timing and location of Purkinje fiber differentiation within the embryonic myocardium.     

The possible role of intracellular Ca2+-stores in the rhythm-inotropic relationship of frog heart muscle (a simulation study)

The hypothesis that intracellular calcium stores play an essential role in determining force-frequency relationships of frog myocardium was tested quantitatively. A simplified mathematical model of excitation-contraction coupling in frog heart muscle was developed and its behaviour under various patterns of stimulation was analysed by means of computer simulation. The model represents a system of ordinary differential equations for individual fluxes within the cell Ca2+-recirculation system and includes a one-compartmental intracellular pool as opposed to the two-compartmental structure of the mammalian sarcoplasmic reticulum (Kaufmann et al. 1974). The behaviour of the model is consistent with available experimental data concerning the basic rhythm-inotropic characteristics of amphibian myocardium and offers some evidence in favour of the basic concept. Within the framework of the proposed model the staircase phenomena in amphibia were accounted for and the impact of different intracellular Ca-movements on the resulting contractile response and rhythm-inotropic phenomena was elucidated.     

Ventricular activation is impaired in aged rat hearts

Ventricular arrhythmias are frequently observed in the elderly population secondary to alterations of electrophysiological properties that occur with the normal aging process of the heart. However, the underlying mechanisms remain poorly understood. The aim of the present study was to determine specific age-related changes in electrophysiological properties and myocardial structure in the ventricles that can be related to a structural-functional arrhythmogenic substrate. Multiple unipolar electrograms were recorded in vivo on the anterior ventricular surface of four control and seven aged rats during normal sinus rhythm and ventricular pacing. Electrical data were related to morphometric and immunohistochemical parameters of the underlying ventricular myocardium. In aged hearts total ventricular activation time was significantly delayed (QRS duration: +69%), while ventricular conduction velocity did not change significantly compared with control hearts. Moreover, ventricular activation patterns displayed variable numbers of epicardial breakthrough points whose appearance could change with time. Morphological analysis in aged rats revealed that heart weight and myocyte transverse diameter increased significantly, scattered microfoci of interstitial fibrosis were mostly present in the ventricular subendocardium, and gap junction connexin expression decreased significantly in ventricular myocardium compared with control rats. Our results show that in aged hearts delayed total ventricular activation time and abnormal activation patterns are not due to delayed myocardial conduction and suggest the occurrence of impaired impulse propagation through the conduction system leading to uncoordinated myocardial excitation. Impaired interaction between the conduction system and ventricular myocardium might create a potential reentry substrate, contributing to a higher incidence of ventricular arrhythmias in the elderly population.     

Developmental patterns of expression and coexpression of myosin heavy chains in atria and ventricles of the avian heart

Monoclonal antibodies (mAbs), electrophoresis, immunoblotting, and immunohistochemistry were used to determine the molecular properties of cardiac myosin heavy chain (MHC) isoforms and the regions of the developing chicken heart in which they were expressed. Adult atria expressed three electrophoretically distinct MHCs that reacted specifically with mAbs F18, F59, or S58. During embryonic Days 2-4, when the atrial and ventricular chambers are forming, MHCs that reacted with mAbs F18, F59, or S58 were expressed in both the atria and ventricles. The atria continued to express MHCs that reacted with mAbs F18, F59, or S58 at all stages of development and in the adult. In the ventricles, expression of the MHCs reacting with these mAbs was found to be developmentally regulated. By embryonic Day 16, MHC(s) reacting with mAb F18 had disappeared from the developing ventricles, whereas MHCs reacting with S58 and F59 continued to be expressed throughout the ventricles. As development continued, MHC(s) reacting with S58 in the ventricle became restricted to expression in only the ventricular conducting system. MHC(s) reacting with F59 were expressed in both the ventricular myocytes and the ventricular conducting system throughout development and in the adult. Thus, in contrast to the embryonic chicken heart where at least three MHC isoforms were expressed in both the atria and ventricles, we found in the adult chicken heart that-at a minimum-three MHC isoforms were expressed in the atria, two MHC isoforms were expressed in the ventricular conducting system, and one MHC isoform in the ventricular myocardium. MHC isoform expression in the developing avian heart appears to be more complex than previously recognized.     

Dual effects of dopamine on the adult heart of the isopod crustacean Ligia exotica

In the adult heart of the isopod crustacean Ligia exotica, the cardiac ganglion acts as the primary pacemaker with the myocardium having a latent pacemaker property. We show several lines of evidence that dopamine modulates the heartbeat of adult L. exotica affecting both pacemaker sites in the heart. Dopamine caused positive chronotropic (frequency increase) and inotropic (amplitude increase) effects on the heartbeat in a concentration dependent manner. The time courses of these effects were considerably different and the inotropic effect appeared later and lasted longer than the chronotropic effect. Dopamine rapidly increased the frequency of the bursting activity in the cardiac ganglion neurons and each impulse burst of the cardiac ganglion was always followed by a heartbeat. Moreover, dopamine slowly increased the amplitude and duration of the action potential plateau (plateau potential) of the myocardium. When the myocardial pacemaker activity was induced by application of tetrodotoxin, which suppresses cardiac ganglion activity, dopamine slowly increased the amplitude and duration of the myocardial plateau potential while decreasing its frequency. These results suggest that dopamine modulates the heartbeat in adult L. exotica producing a dual effect on the two pacemaker sites in the heart, the cardiac ganglion and myocardium.     

Enzyme histochemical studies on the Purkinje fibres of the arrioventricular system of the bovine and porcine hearts

Synopsis In this communication the results of applying various histochemical semipermeable membrane techniques to the localization of several enzymes in bovine and procine heart are presented. The Purkinje fibres of the atrioventricular conducting system of the bovine heart differ from the myocardium proper in containing a greater activity of the glycolytic and gluconeogenetic enzymes—lactate dehydrogenase, glyceraldehyde-phosphate dehydrogenase, hexokinase, glucosephosphate isomerase and phosphoglucomutase, and less activity of the aerobic enzymes-NADH: nitroBT oxidoreductase and isocitrate dehydrogenase (NADP⁺). The metabolic reactions obtained with Purkinje fibres of the porcine heart are less pronounced. These histochemical findings are in accordance with the impression that Purkinje fibres, compared with the common myocardial fibres, have a higher rate of anaerobic metabolism and a lower rate of aerobic metabolism.The activity of the NADPH regenerating enzymes, glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase (decarboxylating), and the activity of acid hydrolases such as non-specific esterase and acid phosphatase is higher in the Purkinje fibres of both the bovine and porcine heart.     

Effects of adaptation to periodic hypoxia on bioelectric activity of cardiomyocytes of isolated heart in ischemia and reperfusion

Experiments on isolated Wistar rat heart perfused according to Langendorff showed that preliminary adaptation of rats to intermittent hypobaric hypoxia limited the fall of values of the resting potential and the amplitude and duration of action potential characteristic for ischemia. Under similar conditions, adaptation considerably reduced the increased time of impulse conduction along the myocardium. In reperfusion, the parameters enumerated restored much more efficiently in hearts from adapted animals than in controls. The role of these changes in the antiarrhythmic effect of adaptation to intermittent hypoxia is under discussion.     

Angiotensin-converting enzyme inhibitor therapy prevents upregulation of endothelin-converting enzyme-1 in failing human myocardium

In this study, we investigated the role of the renin-angiotensin system in expression of the endothelin system in atrial myocardium of patients with congestive heart failure. Atrial myocardium of control patients without angiotensin-converting enzyme (ACE) inhibitor therapy and heart failure patients without or with ACE inhibitor therapy undergoing aorto-coronary bypass surgery was studied. Endothelin-converting enzyme-1 (ECE-1) expression and endothelin-1 peptide level was upregulated in myocardium of heart failure patients without ACE inhibition. ACE inhibitor therapy prevented upregulation of ECE-1 and endothelin-1 in failing myocardium. Prepro-endothelin-1 and endothelin receptor A expression were not affected by heart failure. Endothelin receptor B was downregulated in heart failure patients. Our data demonstrate an upregulation of ECE-1 mRNA expression in failing human myocardium. Inhibition of the renin-angiotensin system by ACE inhibitor treatment prevents upregulation of ECE-1, suggesting that angiotensin II regulates ECE-1 expression in vivo.     

Interaction of gangliosides with serotonin]

It has been shown that ganglioside micellae were able to reversible interaction with serotonin; the interaction is determined by their composition. Ganglioside and ganglioside-serotonin micellae were equal in sizes if pH, the ionic strength and the type of the buffer, the temperature and serotonin concentration were given. When the ganglioside micellae were saturated with serotonin the micallae became able to jumping reconstruction forming the structure able to bind more serotonin than the first one. As the serotonin concentration was increased CCM of mixed serotonin-ganglioside micellae was reached. It has been suggested that the reconstruction of the ganglioside micelle due to its interaction with serotonin can be considered as a model of a cooperative transfer of the postsynaptical membrane when a nervous impulse passes through a synapse.     

Electrical activation of the ventricular myocardium of the crocodile Crocodylus johnstoni: a combined microscopic and electrophysiological study.

We mapped the sequence of ventricular depolarization in the crocodile Crocodylus johnstoni. We also attempted to find specialized conduction tissue within the ventricular myocardium. Electrical recordings with miniature multi-point electrodes revealed two strands of rapidly conducting tissue (channels) within the interventricular septum, suggestive of conductive tissue pathways. From these septal channels, wavefronts of excitation swept around each ventricle. Electrical recordings did not indicate that there was conductive tissue in the wall of either ventricle. Similarly, microscopic studies of the septal channels provided no indication of specialized conductive tissue. We suggest that the channels of early septal depolarization provide the crocodile heart with a high speed depolarization pathway functionally analogous to a rudimentary conductive system.     

Block of Conduction in Partially Depolarized Cardiac Purkinje Fibres induced by an α-Adrenergic Agent

IF discrete areas of the ventricular conducting system are exposed to high concentrations of K⁺, conduction through these areas is slowed so much that the impulse may re-enter the normal part of the conducting system after the end of the refractory period, thereby evoking another excitation of the heart (refs. 1 and 2 and our unpublished work). This slow conduction results from a slow response that resembles the slow component of the cardiac action potential³ and the action potential of normal fibres of the atrioventricular node⁴. Adrenaline enhances the response of Purkinje fibres, depressed by exposure to high K⁺ concentration⁵ and we now report that an α-adrenergic agent, methoxamine, depresses the slow response at concentrations that do not affect the normal action potential. This finding is of interest because very few of the actions of adrenergic agents on the heart can be attributed to the stimulation of α-receptors⁶, because selective depression of the slow response is further evidence that it differs qualitatively from the response of normal fibres (our unpublished work) and because it suggests that α-adrenergic agents may be useful in the prevention of certain cardiac arrhythmias.     

Wnt signaling regulates atrioventricular canal formation upstream of BMP and Tbx2

In the developing heart, the atrioventricular canal (AVC) is essential for separation and alignment of the cardiac chambers, for valve formation, and serves to delay the electrical impulse from the atria to the ventricles. Defects in various aspects of its formation are the most common form of congenital heart defects. Using mutant and transgenic approaches in zebrafish, this study demonstrates that Wnt/β-catenin signaling is both sufficient and required for the induction of BMP4 and Tbx2b expression in the AVC and consequently the proper patterning of the myocardium. Furthermore, genetic analysis shows that Wnt/β-catenin signaling is upstream and in a linear pathway with BMP and Tbx2 during AVC specification.     

Development of the cardiac conduction system

The cardiac conduction system (CCS) is a specialized tissue network that initiates and maintains a rhythmic heartbeat. The CCS consists of several functional subcomponents responsible for producing a pacemaking impulse and distributing action potentials across the heart in a coordinated manner. The formation of the distinct subcomponents of the CCS occurs within a precise temporal and spatial framework; thereby assuring that as the system matures from a tubular to a complex chambered organ, a rhythmic heartbeat is always maintained. Therefore, a defect in differentiation of any CCS component would lead to severe rhythm disturbances. Recent molecular, cell biological and physiological approaches have provided fresh and unexpected perspectives of the relationships between cell fate, gene expression and differentiation of specialized function within the developing myocardium. In particular, biomechanical forces created by the heartbeat itself have important roles in the inductive patterning and functional integration of the developing conduction system. This new understanding of the cellular origin and molecular induction of CCS tissues during embryogenesis may provide the foundation for tissue engineering, replacement and repair of these essential cardiac tissues in the future.