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.     

Quantitative electron microscopic research on the structure of the myocytes in the atrioventricular node and perinodal myocardium of the rat interatrial septum

A quantitative electron microscopical examination of specialized conducting myocytes of types II and III in the atrioventricular node and working myocytes in perinodal region was performed. The volume fractions of myofibrils, mitochondria, nuclei, sarcoplasmic reticulum, glycogen, atrial granules and "clear" cytoplasm were measured, in addition to the diameters of conducting and working myocytes. These data were compared with those for conducting myocytes of types I, II, and III from sinoatrial region and internodal tracts of the rat heart, and with evidence for atrioventricular node of other animals. Principles of an universal classification of conducting myocytes of the heart are discussed.     

Ultrastructure of the DNA-synthesizing cells of the sinoatrial node in mouse embryos according to electron microscopic autoradiographic data

By means of electron microscopic autoradiography with 3H-thymidine a study was made of the differentiation degree of DNA synthesizing muscle cells in the sinoatrial node (SAN) of the heart conductive system of the 18 day old mouse embryos. Clear myocytes (CM), predominating in the SAN at this stage, are irregular in shape, with interdigitating protrusions. Nuclei are clear, spherical or ellipsoidal. One hour following 3H-thymidine injection, about 6% of CM display labeled nuclei; this index is considerably lower than in working ventricular myocardium. Like unlabeled myocytes, CM being in phase S contain sparse, randomly located thin myofibrilles. In some areas of the sarcoplasm, only myofilament bundles and Z-disk material can be seen. The number of CM myofibrilles is always considerably less than in the working ventricular myocytes. Accumulations of intermediate (8--11 nm) filaments are present. Mitochondria with a few cristae are not numerous. The sarcoplasmic reticulum and Golgi apparatus being relatively well developed, multivesicular bodies, centrioles, and occasional cilia are often seen. Near the centrioles (basal bodies), striated filamentous bundles are found sometimes showing periodic dense lines separated by 50--70 nm. Specialized contacts between CM are rare, being presented only by desmosomes and primitive intercalated discs. Besides CM, sparse small dark cells occur filled with myofibrilles and mitochondria. In the peripheral regions of the node "transitional" cells are seen. The SAN of the 18 day old embryo mouse heart grown due to proliferation of CM with a poorly developed myofibrillar apparatus.     

Embryonic histogenesis of the atrioventricular segment of the heart conduction system of swine (Sus porcus L.)]

Histological, histochemical and neurochemical methods were used in order to study the features of the histogenesis of structures of the atrioventricular area of the conducting system of the heart and the formation of their cholinergic innervation in the postnatal period of the hog. Under study were 175 hearts of embryos of the hog at the age of 3, 4, 5, 6, 8, 10, 12, 14 and 15 weeks and 22 hearts of adult hogs. The formation of different areas of this system was shown to be asynchronous. The atrioventricular fascicle is formed during the 4th week, the atrioventricular node -- during the 6th week and the conducting muscle fibres--during the 8th week of embryogenesis. The fascicle and the node have a complicated structure and different cellular composition. In the process of prenatal ontogenesis the increased amount of glycogen and increased activity of phosphorylase were noted in the cytoplasm of myocytes of the atrioventricular area of the conducting system of the heart. The cholinergic nerve fibres grow up to the structures of the atrioventricular area of the conducting system of the heart during the 6th week of embryogenesis, and by the end of the prenatal development they form a thick network in all its structures.     

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.     

Levels of DNA and sex chromatin bodies in the myocyte nuclei of different sections of the human heart

Nuclei of ventricular, atrial and atrioventricular node myocytes of normal and hypertrophied human heart were studied on squash preparations and on 12 micron sections after the Feulgen staining. The cytophotometric DNA measurements have shown a distinction in the degree of polyploidization of nuclei in different heart compartments. In contrast to ventricular and atrial myocardia, in which polyploid nuclei predominate, the conduction system myocytes contain 77-88% of diploid nuclei. A correlation between DNA content and the number of sex chromatin bodies was observed for myocyte nuclei from all the compartments under investigation.     

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.     

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.     

A comparative enzyme histochemical study of glucose metabolism in the conduction system of mammalian hearts.

A histochemical study of some enzymes of glucose metabolism was performed on the heart conduction system of rat, dog, rabbit, pig, calf and lamb. Histochemical activities revealed a higher rate of anaerobic metabolism and a lower rate of aerobic metabolism in the conducting cells in comparison with the working myocardial fibres. An increase of the histochemical activities from the atrioventricular node to the distal portions of bundle branches was noted. The importance of the high glycogen content and the high phosphorylase activity in the heart conduction system was discussed.     

Nerve elements of the heart of the rat after right-sided vagotomy

A quantitative electron microscopical investigation of the sinus node of the atrioventricular His' bundle and of the perinodal working myocardium in intact rats and 7, 15 and 30 days after right-sided vagotomy has revealed variable character of changes in the neural elements of the zones mentioned. Certain ultrastructural rearrangements in the neural apparatus are described; they reflect a combination of destructive and regenerative processes in the heart under vagotomy. Thirty days after the operation, regeneration of the neural elements in the sinoauricular area is of restorative and in the atrioventricular area--of excess character. The data are presented on dynamics of changes in the diameter of the amyelinated neural fibers in the cardiac areas investigated. Participation of both nervi vagi in innervation of the main and additional pacemakers of the organ is discussed.     

Localization of cholinergic innervation and neurturin receptors in adult mouse heart and expression of the neurturin gene

Neurturin (NRTN) is a neurotrophic factor required during development for normal cholinergic innervation of the heart, but whether NRTN continues to function in the adult heart is unknown. We have therefore evaluated NRTN expression in adult mouse heart and the association of NRTN receptors with intracardiac cholinergic neurons and nerve fibers. Mapping the regional distribution and density of cholinergic nerves in mouse heart was an integral part of this goal. Analysis of RNA from adult C57BL/6 mouse hearts demonstrated NRTN expression in atrial and ventricular tissue. Virtually all neurons in the cardiac parasympathetic ganglia exhibited the cholinergic phenotype, and over 90% of these cells contained both components of the NRTN receptor, Ret tyrosine kinase and GDNF family receptor α2 (GFRα2). Cholinergic nerve fibers, identified by labeling for the high affinity choline transporter, were abundant in the sinus and atrioventricular nodes, ventricular conducting system, interatrial septum, and much of the right atrium, but less abundant in the left atrium. The right ventricular myocardium contained a low density of cholinergic nerves, which were sparse in other regions of the working ventricular myocardium. Some cholinergic nerves were also associated with coronary vessels. GFRα2 was present in most cholinergic nerve fibers and in Schwann cells and their processes throughout the heart. Some cholinergic nerve fibers, such as those in the sinus node, also exhibited Ret immunoreactivity. These findings provide the first detailed mapping of cholinergic nerves in mouse heart and suggest that the neurotrophic influence of NRTN on cardiac cholinergic innervation continues in mature animals.This study was supported by the National Heart, Lung, and Blood Institute (grant HL-54633).     

Similarity of the effects of first-generation antiarrhythmia agents and decreased extracellular concentration of sodium ions on action potentials of atrial myocytes]

In rabbit right atria beating in a spontaneous sinus rhythm, myocytes of two types were studied, which differ by the initial form of action potentials. First-class antiarrhythmics and a gradual decrease in extracellular Na+ concentration induced qualitatively similar and unidirectional changes in the form of action potentials of myocytes. In some myocytes of the "conducting system" type, a slow diastolic depolarization was observed after repolarization, and the form of their action potentials became similar to that of the pacemaker cells. An enhancement of the action caused short-time arrhythmias.     

Ultrastructure of the ascospores of some species of the Torulaspora group

Development and germination of the ascospores in species of the Torulaspora group of yeasts have been described. Most species had warty spores which, in sections, showed a dark outer layer consisting of the outer unit membrane of the prospore wall and a layer underneath formed at an early stage of development of the spores. In mature spores the light inner layer of the wall was delimited at the outside by a thin dark layer. The warts often contained dark material. The ascospores of two Pichia and three Debaryomyces species were studied for comparison; they differed in sections from the Torulaspora spores. The taxonomic implications of the ultrastructural observations have been discussed.     

The structure and innervation of the sinu-atrial node of the mole heart

Summary The sinu-atrial node in the heart of the mole, Mogera wogera, contains myocytes which are devoid of atrial specific granules and which may be classified into two types: electron-lucent (majority) and dark (minority). Numerous unmyelinated axon terminals, containing synaptic vesicles, face the nodal myocyte surface with interspaces of less than 300 nm. There are about 6 terminals per myocyte profile and about 10 per profile at nuclear levels. Of a total of 2717 of these terminals 85.2% are 50–200 nm from the nodal myocytes, and 9.6% form closer neuromuscular junctions, with less than 50 nm interspace and some membrane specialization. Such specializations are almost exclusively found in relation to the dark, minority-type of nodal myocyte.Myelinated nerve fibers are also numerous within the mole SA node, and may terminate almost directly on a nodal myocyte. A few nerve fibers contain many mitochondria and may represent afferent endings.Nodal capillaries are smaller and fewer than capillaries in the juxtanodal myocardium, probably indicating a lesser blood supply to the SA node than to the ordinary myocardium.The author wishes to thank Professor A. Yamauchi, Department of Anatomy, for suggesting this investigation and for constant guidance in the course of the work, Professor K. Shiraishi, Department of Biology, for his encouragement and support, and Miss. K. Ito for technical assistance     

Ontogeny of human fetal lymph nodes.

Developing lymph nodes from 30 human embryos and fetuses with crown-rump lengths (CRL) of 18 mm (5.6 wk) to 245 mm (26 wk) were examined by light microscopy. The nodes were embedded in araldite, and the sections examined were approximately 1 mu in thickness. The development of nodes was divided into three stages: 1. the lymphatic plexus and connective tissue invagination (30 mm to 67 mm CRL); 2. the early fetal lymph node (43 mm to ,5 mm CRL); and 3. the late fetal lymph node (CRL greater than 75 mm). The lymphatic plexus was formed by connective tissue invaginations and bridges which divided a lymph sac into a meshwork of channels and spaces. Connective tissue invaginations were endothelially-lined and were surrounded by lymphatic space. Reticular cells, macrophages, and blood vessels were found in these invaginations. Early fetal lymph nodes were formed from invaginations when the cellular density and lymphocyte content increased. The lymphatic space surrounding the early node was the developing subcapsular sinus. With further development the early node became packed with lymphocytes, increasing the cellular density and size of the node. The connective tissue surrounding the subcapsular sinus condensed to form the capsule. Afferent lymphatic vessels pierced the capsule. Capillaries, veins, postcapillary venules, and occasional arteries were found in early and late nodes.     

The caveolae in rabbit sinus node and atrium

Summary Caveolae or membrane vesicles are commonly observed in smooth and skeletal muscle as well as in working heart muscle. Using sections of fixed tissue and replicas of freeze-cleaved material, we show in this study that caveolae are also very numerous in sinus node cells of the rabbit, and to a lesser degree, in the atrial cells.Caveolae increase the plasma membrane surface area by 115% in the leading sinus node, and by 56% in the atrial cells. In these two cell types, the membrane of the caveolae contains four times fewer intramembranous particles than the rest of the plasma membrane, and this difference applies to both PF and EF faces. The role of the caveolae is still unclear, but it does not seem that they have a pinocytotic function.     

Culture and characterization of fetal human atrial and ventricular cardiac muscle cells

Summary Atrial and ventricular cardiac muscle cells isolated from 14- to 18-wk old fetal human hearts were grown in culture and characterized. Once established in culture the flattened cells contracted spontaneously and possessed differentiated ultrastructural characteristics including organized sarcomeres, intercalated discs, and transverse tubules with couplings. Atrial granules were present in the cultured atrial cells. Some cultured ventricular myocytes also contained electron-dense granules associated with Golgi cisternae, which were similar in size and appearance to atrial granules. The cultured ventricular myocytes divided and expressed the genes for thymidine kinase, histone H4, myosin heavy chain, muscle-specific creatine kinase, atrial natriuretic factor, and insulin-like growth factor II. These results establish that differentiated fetal human heart muscle cells can be cultured in sufficient quantities for biochemical, molecular, and morphological analyses. This work was supported by a postdoctoral fellowship from the American Heart Association, Louisiana Affiliate (JBD) and the National Institutes of Health, Bethesda, MD (HL-35632) (WCC).     

Characterization of isolated ventricular myocytes from adult zebrafish (Danio rerio)

The zebrafish is widely used for human related disease studies. Surprisingly, there is no information about the electrical activity of single myocytes freshly isolated from adult zebrafish ventricle. In this study, we present an enzymatic method to isolate ventricular myocytes from zebrafish heart that yield a large number of calcium tolerant cells. Ventricular myocytes from zebrafish were imaged using light and confocal microscopy. Myocytes were mostly rod shaped and responded by vigorous contraction to field electrical stimulation. Whole cell configuration of the patch clamp technique was used to record electrophysiological characteristics of myocytes. Action potentials present a long duration and a plateau phase and action potential duration decreases when increasing stimulation frequency (as observed in larger mammals). Together these results indicate that zebrafish is a species ideally suited for investigation of ion channels related mutation screening of cardiac alteration important in human.     

The human gene coding for HCN2, a pacemaker channel of the heart.

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels, underlying 'pacemaker' currents (I(f)/Ih), are involved in pacemaker activity of cardiac sinoatrial node myocytes and central neurons. Several cDNAs deriving from four different genes were recently identified which code for channels characterized by six transmembrane domains and a cyclic nucleotide binding domain. We report here the identification of the human HCN2 gene and show that its functional expression in a human kidney cell line generates a current with properties similar to the native pacemaker f-channel of the heart. The hHCN2 gene maps to the telomeric region of chromosome 19, band p13.3. This is the first identification of a genetic locus coding for an HCN channel.