Morphological study of the atrioventricular conduction system and Purkinje fibers in yak.

We studied the morphology of the atrioventricular conduction system (AVCS) and Purkinje fibers of the yak. Light and transmission electron microscopy were used to study the histological features of AVCS. The distributional characteristics of the His-bundle, the left bundle branch (LBB), right bundle branch (RBB), and Purkinje fiber network of yak hearts were examined using gross dissection, ink injection, and ABS casting. The results showed that the atrioventricular node (AVN) of yak located in the right side of interatrial septum and had a flattened ovoid shape. The AVN of yak is composed of the slender, interweaving cells formed almost entirely of the transitional cells (T-cells). The His-bundle extended from the AVN, and split into left LBB and RBB at the crest of the interventricular septum. The LBB descended along the left side of interventricular septum. At approximately the upper 1/3 of the interventricular septum, the LBB typically divided into three branches. The RBB ran under the endocardium of the right side of interventricular septum, and extended to the base of septal papillary muscle, passed into the moderator band, crossed the right ventricular cavity to reach the base of anterior papillary muscle, and divided into four fascicles under the subendocardial layer. The Purkinje fibers in the ventricle formed a complex spatial network. The distributional and cellular component characteristics of the AVCS and Purkinje fibers ensured normal cardiac function.

Immuno-histochemistry and three-dimensional architecture of the intermediate filaments in Purkinje cells in mammalian hearts.

In mammalian hearts, Purkinje cells varied greatly in morphological appearance in different species, and were divided into three groups. Bovine Purkinje cells corresponding to group I were a large size, and had a few myofibrils and abundant intermediate filaments throughout the cytoplasm. The aim of the present study was to clarify the more detailed distribution and three-dimensional architecture of intermediate filaments in Purkinje cells. The hearts in various mammals including humans were investigated by both immuno-histochemistry and scanning electron microscopy (SEM).Immuno-histochemical studies demonstrated that sheep Purkinje cells in group I had a great number of intermediate filaments of 10 nm positive for desmin antibody. Purkinje cells in group II (humans, monkeys and dogs) and group III (mice) were somewhat larger or smaller in size than myocardial cells, but also showed a strong positive reaction for desmin antibody. The saponin or NaOH treatment of cardiac tissues in sheep and humans enabled us to view intermediate filaments by SEM three-dimensionally. Intermediate filaments in sheep Purkinje cells formed a considerably delicate network, and were distributed throughout the cytoplasm. In contrast, those in human Purkinje cells were lower in density, and were present around the nucleus and between myofibrils. It was concluded that a delicate network of intermediate filaments in Purkinje cells of mammalian hearts acted as the cytoskeleton to maintain intercellular stability.

Cocaine causes atrial Purkinje fiber damage.

Comparisons of atrial tissues from Syrian hamster offspring born from cocaine-treated mothers during the last days of pregnancy with sham-treated ones demonstrate irreversible focal ischemic damage in the Purkinje myofibers and minor endocardial damages as well as minute cardiomyocyte vacuolization. These defects are consistent with the pharmacotoxicity of cocaine or its metabolites. The damaged Purkinje myocytes apparently remain in contact with adjacent cardiomyocytes but undergo autolytic process similar to that found in autoschizic cell death. Adjacent cell type(s) appear to segregate or engulf the injured cells. Data collected in this report demonstrate why clinical bradyarrhythmias, arrhythmias, or sudden death as cardiac arrest can be found in pre- and postnatal cocaine-abused babies as well as those found in young individuals caused by acute or chronic cocaine abuse.

Morphological varieties of the Purkinje fiber network in mammalian hearts, as revealed by light and electron microscopy.

Purkinje fibers in mammalian hearts are known to comprise the following three groups depending on their structure: group I found commonly in ungulates, group II in humans, monkeys and dogs, and group III in rodents. The aim of the present study was to document precisely the cytoarchitecture of a network of Purkinje fibers in different species by light and electron microscopy. Light microscopy of silver impregnated tissues revealed the reticular fibers ensheathing individual Purkinje strands consisting of 2-8 cells in both the ungulates (i.e., sheep and goats) and cetaceans (whales and dolphins) while they encircled each Purkinje cell in the primates (humans and monkeys), carnivores (dogs and seals), and rodents (rats). Scanning electron microscopy of NaOH digested tissues showed the ungrates (group I) to have a Purkinje fiber network composed of Purkinje strands; the cells in the strands were oval and made side-to-side and/or end-to-end connections. The Purkinje fiber network in the primates and carnivores (group II) was delicate and complicated; the Purkinje cells were usually cylindrical and connected end-to-end, the exception being their polygonal or stellate shapes at the bifurcations. Purkinje cells in the rodents (group III) resembled ventricular cardiac myocytes in cytoarchitecture. Morphologically, whales and seals respectively belonged to Purkinje cells of group I and group II. These findings indicate that the structural variety of the Purkinje fiber network may reflect the conducting function and be related to the phylogeny of the mammalian species.

Selective vacuolar degeneration in dystrophin-deficient canine Purkinje fibers despite preservation of dystrophin-associated proteins with overexpression of Dp71.

BACKGROUND: Respiratory support therapy significantly improves life span in patients with Duchenne muscular dystrophy; cardiac-related fatalities, including lethal arrhythmias, then become a crucial issue. It is therefore important to more thoroughly understand cardiac involvement, especially pathology of the conduction system, in the larger Duchenne muscular dystrophy animal models such as dystrophic dogs. METHODS AND RESULTS: When 10 dogs with canine X-linked muscular dystrophy in Japan (CXMD(J)) were examined at the age of 1 to 13 months, dystrophic changes of the ventricular myocardium were not evident; however, Purkinje fibers showed remarkable vacuolar degeneration as early as 4 months of age. The degeneration of CXMD(J) Purkinje fibers was coincident with overexpression of Dp71 at the sarcolemma and translocation of mu-calpain to the cell periphery near the sarcolemma or in the vacuoles. Immunoblotting of the microdissected fraction showed that mu-calpain-sensitive proteins such as desmin and cardiac troponin-I or -T were selectively degraded in the CXMD(J) Purkinje fibers. Utrophin was highly upregulated in the earlier stage of CXMD(J) Purkinje fibers, but the expression was dislocated when vacuolar degeneration was recognized at 4 months of age. Nevertheless, the expression of dystrophin-associated proteins alpha-, beta-, gamma-, and delta-sarcoglycans and beta-dystroglycan was well maintained at the sarcolemma of Purkinje fibers. CONCLUSIONS: Selective vacuolar degeneration of Purkinje fibers was found in the early stages of dystrophin deficiency. Dislocation of utrophin besides upregulation of Dp71 can be involved with this pathology. The degeneration of Purkinje fibers can be associated with the distinct deep Q waves in ECG and fatal arrhythmia seen in dystrophin deficiency.

T-tubule profiles in Purkinje fibres of mammalian myocardium.

Purkinje (P)-fibres are cardiac myocytes that are specialized for fast conduction of the electrical signal. P-fibres are usually defined as having the following identifying features: lack of T tubules; frequent lateral cell junctions; deep indentations at the intercalated discs level; the CX40 isoforms of gap junction proteins and, in large mammals, paucity of myofibrils and abundance of glycogen. We have examined the ultrastructure of P-fibres in free running P-strands from right and left ventricles of small (mouse and rat) intermediate (rabbit) and large (dog) size mammals focusing on presence and distribution of the T tubules. In contrast with previous studies, we find that P-fibres do have T tubules which form normal dyadic associations with the sarcoplasmic reticulum and that the frequency of tubules varies with the size of the animal. Profiles of T tubules and dyads are present over short segments of individual P-cells flanked by totally T tubule-free segments. It is thought that lack of T tubules in P-cells is necessary to reduce capacitance and thus accelerate action potential spread. This may not be as important in a small heart.

Cytoarchitecture and intercalated disks of the working myocardium and the conduction system in the mammalian heart.

Working and specialized cardiac myocytes and their intercalated disks (ID) in the mammalian heart were examined by transmission and scanning electron microscopy. The NaOH/ultrasonication treatment of cardiac tissues resulted in the digestion of collagen fibers and separation of intercellular junctions. Auricular and ventricular myocytes were cylindrical in shape, bifurcated, and connected end-to-end at the ID. The ID in the working myocardium showed a stair-like profile, consisting of steps (plicate segments) and corresponding risers (interplicate segments). The ventricular myocytes had many steps and risers. The steps were filled with numerous finger-like microprojections, including desmosomes, fasciae adherentes, and small gap junctions. The risers showed the smooth surface, including desmosomes and large gap junctions. The cell strands of the sinoatrial node were oriented linearly, while those of the atrioventricular node formed a reticular network. The ID in both nodal cells was underdeveloped, having few microprojections. Myocytes in the His bundle and its branches were arranged in parallel, and Purkinje cell strands formed reticular networks. The ID in the His-Purkinje system was irregular in appearance, and the microprojections were larger in size and smaller in number than those of working myocytes. There were few microprojections in the sheep Purkinje cells. The gap junctions in the conduction system were few or small in size in the nodal tissue, but large in the His-Purkinje system.

Expression of brain natriuretic peptide in the rat heart studies during heart growth and in relation to sympathectomy.

Brain natriuretic peptide (BNP) might be of importance during heart development and is described to be increasingly expressed in congestive heart failure and to affect the progress of this condition. However, details in the normal expression of BNP are still unclear in various parts of the adult and growing heart, including the conduction system. In this study, we investigated the expression of BNP in relation to that of atrial natriuretic peptide (ANP) in the growing as well as in the adult rat heart. The effects of chemical sympathectomy in adult rats were also examined. Contrary to previous BNP immunohistochemical studies, the BNP antiserum was preabsorbed with an excess of ANP before staining to abolish the crossreactivity with ANP. There was a pronounced BNP immunoreaction in the auricles, the trabeculated ventricular walls, and the peripheral parts of the conduction system at 0-1 days postnatally. The degree of immunoreaction gradually decreased with increasing age. A similar developmental pattern was seen concerning ANP expression, but the magnitude of the latter clearly exceeded that for BNP. Immunoreaction for BNP was never detected in the atrioventricular (AV) node and AV bundle at any stage. In contrast to the situation for ANP previously observed, no obvious changes in BNP immunoreaction patterns were observed in response to sympathectomy. This is the first study to thoroughly demonstrate the expression of BNP in the various regions of the rat heart during growth and in the normal and sympathectomized adult stage. The observations are related to possible functions of natriuretic peptides in the growing and adult heart.

Significant damage of the conduction system during cardioplegic arrest is due to necrosis not apoptosis.

OBJECTIVES: Ventricular conduction disturbances following cardioplegic arrest remains a serious, yet unsolved problem. In the present study we examined whether myocardial conduction cells (MCC, Purkinje fibers) are more vulnerable to ischemia/reperfusion injury than working myocardial cells and whether the damage is due to necrosis or apoptosis. METHODS: Mini-pigs were subjected to 60 min of crystalloid (St Thomas; n = 15 group I) or blood (Buckberg; n = 6 group II) cardioplegic arrest followed by 3 h of reperfusion. Animals not subjected to either procedures served as controls (n = 5). Ventricular myocardial specimens were investigated by hematoxylin and eosin (HE) and periodic acid Schiff (PAS) staining and immunohistochemical labeling of apoptosis-associated proteins (Bax, Bcl-2, Caspase-3). DNA-breaks were visualized by in situ end labeling (terminal deoxynucleotidyl transferase dUTP-biotin nick-end labeling, TUNEL). Electron microscopy confirmed apoptosis or necrosis. RESULTS: MCC of control hearts intrinsically expressed Bax, Bcl-2, and Caspase-3 without signs of either apoptotic or necrotic damage. Subendocardial Purkinje fibers of groups I and II hearts exhibited focal damage, with reduced labeling of apoptosis-associated proteins, glycogen loss, karyopycnosis and increased eosinophilia (15/21 hearts). The majority of damaged MCC displayed nuclear TUNEL-positivity (2.8+/-2.5% of MCC), whereas the average TUNEL-rate of the adjacent working myocardium was low (<0.1%). Electron microscopy demonstrated ischemic changes in MCC consistent with cellular necrosis. CONCLUSIONS: Ischemia/reperfusion injury due to cardioplegic arrest inflicts significant damage on subendocardial MCC, but not on working myocardium. Ultrastructural and light-microscopic findings are consistent with coagulation necrosis, rather than apoptosis.

[Changes of the heart conduction system after exposure to fluorouracil]. / Izmeneniia provodiashchei sistemy serdtsa pod vliianiem ftoruratsila.

Using 20 male rats the electron microscopic study of conducting system of the heart was performed to assess the effect of fluorouracil--an antineoplastic drug belonging to antimetabolite group. A daily dose of 15 mg/kg of fluorouracil was injected intraperitoneally for 5 days; three courses of injections with the intervals of three weeks were performed. Toxic effect of fluorouracil is demonstrated after the first course of injections and is revealed as a predominant injury of pacemaker cells in sinoatrial and atrioventricular nodes. Latent pacemaker myocytes and Purkinje fibers undergo "calcium injury" during the second and the third courses of fluorouracil injections. The irreversible damage of contractile and specialized myocytes results in fibrosis which develops predominantly in the areas of atrioventricular bundle and its branches.

Histochemical and ultrastructural characterisation of an arrhythmogenic substrate in ischemic pig heart.

The aim of the present study was to reveal by enzyme histochemistry and ultrastructural examination the possible anatomic substrate that may be the cause of high susceptibility of the pig heart to ischemia and/or reperfusion-induced severe arrhythmias. The heart of landrace pigs was subjected to 90 min of left coronary occlusion followed by 30 min reperfusion, whereby both conditions elicited arrhythmias and often even ventricular fibrillation. We found for the first time, besides common contractile cardiomyocytes, Purkinje fibers, and "transitional cells" in mid-myocardium. Transitional cells likely correspond to the recently described M cells. Importantly, these cells and Purkinje fibers exhibited reversible ischemia-related subcellular alterations, whereas the majority of contractile cardiomyocytes were irreversibly injured in the area of infarction. In correlation with these findings, glycogen-dependent phosphorylase activity was abolished, whereas it was still persistent in Purkinje fibers and small islands of contractile cardiomyocytes. Moreover, a distinct heterogeneity in the activity of all enzymes selected and subcellular alterations within a border zone were observed. These results suggest that particularly the preserved viability of specialized conducting cells spanning the ventricular wall may account for electrical disturbances that consequently contribute to increased susceptibility of the pig heart to ischemia- and reperfusion-induced severe arrhythmias.

Comparison of responses of spontaneously active cells in the cerebellar Purkinje layer to parallel fibre stimulation in slice preparations and urethane-anaesthetised rats: effects of benzodiazepine receptor ligands.

1. GABA-mediated inhibitory responses were induced in spontaneously active Purkinje cells by parallel fibre stimulation in cerebellar slices or in urethane-anaesthetised rats. Effects of agonist and inverse agonist benzodiazepine (BDZ) receptor ligands were compared in the preparations. 2. Purkinje cells fired simple spikes at higher rates in slice preparations while complex spikes were seldom (in vivo) or never observed (slice). Cells fired more regularly in vivo resulting in the occurrence of rhythmic postinhibitory responses in the PSTH analysis in some preparations. 3. Single pulse stimulation of parallel fibres at just suprathreshold intensity induced inhibition of Purkinje cell activity in both preparations. At lower firing rates there was a marked increase in the duration of this response, which was more evident in vivo where more slowly firing cells were encountered. 4. BDZ receptor ligands modified inhibitory responses in slice preparations with only weak effects on the firing rates of the cells. These compounds predominately induced changes in firing rate in the anaesthetised rat with little evidence of direct modification of GABA-mediated synaptic transmission. 5. In a few experiments, following injection of the partial inverse agonists beta-CCE and beta-CCM, block of the inhibitory response was observed independent of changes in firing rate. Bidirectional efficacy of BDZ receptor ligand (agonists decrease firing and increase inhibitory response, inverse agonists increase firing and decrease inhibitory response) was demonstrated for modulation of inhibitory responses in slices and for changes in firing rate in vivo. The increased firing rate response in vivo was biphasic the magnitude of the later phase being correlated with efficacy of inverse agonists. 6. It is concluded that cerebellar slice preparations are more appropriate for studying direct effects of BDZ receptor ligands on GABA-mediated synaptic inhibition than in vivo preparations.

Restrictive cardiomyopathy due to desmin accumulation in a family with evidence of autosomal dominant inheritance.

OBJECTIVE: A familial case of restrictive cardiomyopathy due to desmin accumulation characterized by severe disturbances of cardiac conduction is described. BACKGROUND: Desmin is an intermediate filament normally present in the myocardium, particularly in the Purkinje fibres, in the skeletal and in the smooth muscle. METHODS: Resting electrocardiogram, 2-dimensional and Doppler echocardiogram, cardiac catheterization, electrophysiological study have been performed in all siblings. Informed consent for endomyocardial biopsy was obtained only in one patient. RESULTS: The mother showed bilateral pes cavus and complained of episodes of vertigo at the age of 36 years. At that time she was submitted to electrophysiological study and to permanent pacing. After 15 years of good health conditions, she developed heart failure and underwent cardiac transplantation. A 21 year old son had a syncope; his ECG was similar to that of his mother; a permanent pacemaker was implanted and a diagnosis of restrictive cardiomyopathy with desmin accumulation was confirmed at histopathology study. Afterwards, another 24 year old sib had a syncope with head trauma: ECG showed right atrial enlargement, left bundle branch block. After electrophysiological study, he started antiarrhythmic therapy. This patient showed bilateral pes cavus. CONCLUSIONS: The early manifestation of desmin accumulation may be intraventricular conduction disorders that can be often controlled by pacemaker implantation. Clinical symptoms of heart failure may be absent for a long period of time. Pedigree analysis is most consistent of autosomal dominant inheritance.

The intercalated disc of monkey myocardial cells and Purkinje fibers as revealed by scanning electron microscopy.

The intercalated discs of working myocardium and Purkinje fibers of the monkey heart were examined by scanning and transmission electron microscopy. The NaOH/ultrasonication technique resulted in the digestion of connective tissue and a separation of the intercellular junctions of intercalated discs, such that these could be visualized three-dimensionally. The intercalated discs of ventricular myocytes, atrial myocytes and Purkinje fibers vary considerably in number and configuration, as do the intercalated discs of the three different layers of the ventricular myocardium. Myocytes in the subepicardial, middle and subendocardial layers of the ventricle have 1-3, 4-5 and 5-6 intercalated discs at the end of these cells, respectively. Those in the endocardial layer are characterized by the presence of small laterally-placed intercalated discs. Atrial myocytes and Purkinje fibers usually only have 1-2 intercalated discs. Individual intercalated discs in ventricular myocytes have complicated stairs with 10-30 steps and corresponding risers, while those of atrial myocytes and Purkinje fibers have simple stairs with 1-3 steps and risers. Steps equivalent to the plicate segments are characterized by densely-packed microplicae and finger-like microprojections which greatly increase surface area in ventricular myocytes. Microprojections in atrial myocytes and Purkinje fibers are sparse by comparison. Risers equivalent to the interplicate segments containing large gap junctional areas are most numerous in left ventricular myocytes, followed by right ventricular myocytes, Purkinje fibers and atrial myocytes in decreasing order. The geometric arrangement of the various types of myocytes may be related with impulse propagation. Large intercalated discs of cell trunks and series branches may participate in longitudinal propagation, while small laterally-placed ones may be the site of transverse propagation.

[Quantitative electron microscopic analysis of tissue components of the subendocardial region of the dog heart]. / Kolichestvennyi élektronno-mikroskopicheskii analiz tkanevykh komponentov subéndokardial'noi oblasti serdtsa sobak.

Quantity and distribution of Purkinje conducting cells, contractile cardiomyocytes, connective tissue, capillaries and nervous structures in the conducting and working myocardium (CM and WM) of the subendocardial area of the anterior papillary muscles of the dog heart were studied. Predominance of the contractile cardiomyocytes volume density over that of Purkinje cells and a higher level of the volume density of capillaries and nervous structures were characteristic of WM comparing to CM while the connective tissue volume density was higher in CM than in WM. These variations in the distribution and quantitative content of tissue structures may determine the primary damage of the subendocardium as well as non-similar response of different animals to the same impact.

Ultrastructural and morphometric features of nodal and impulse-conducting cardiac myocytes of the bat Pipistrellus pipistrellus.

Cells of the impulse-generating and conducting tissues of the insect-eating bat Pipistrellus pipistrellus were studied and evaluated using ultrastructural morphometry. Sinoatrial node cells are smaller than working atrial cells and measure about 6.5 microm in diameter. Their mitochondira and myofibril content constitute 23% and 19% of cytoplasmic volume, respectively. Corresponding values for working atrial cells are 23% and 52%. Atrioventricular node cells are 4.2 microm in diameter and contain abundant glycogen in the cytoplasm. The fractional volume of mitochondria in about 24% while that of myofibrils is 7%. Cells of the bundle of His are larger (6-8 microm diameter) and contain more cellular organelles than do nodal cells. Their mitochondria and myofibril contents are 25% and 25%, respectively. Cells in the proximal part of the right bundle branch are slender with diameters averaging 3.4 microm. Mitochondrial content is 23% while myofibrils occupy 20% of the cytoplasmic volume of these cells. Distally located bundle branch cells measure 7-10 microm in diameter with mitochondria and myofibril volumes of 30% and 33%. Subendocardial cells in the ventricular free wall are large reaching 28 microm in diameter (cf. 14-18 microm in working ventricular cells) and have mitochondira and myofibril volume fractions of 32% and 29%, respectively (35% & 40% for working ventricular cells).

Immunohistochemical delineation of the conduction system. II: The atrioventricular node and Purkinje fibers.

Using an antibody that reacts specifically with the myocytes of the conduction system of the bovine heart, we have studied the atrioventricular node and the spatial distribution of the Purkinje fibers in the bovine heart. This study was complemented by studying the distribution of the gap junction protein connexin43 in these areas in the bovine heart and in the human heart. The large Purkinje fibers in the bovine heart are arranged in a two-dimensional network underneath the endocardium. At discrete sites, these fibers branch to the Purkinje fibers situated between the muscle bundles of the ventricular mass. These intramural Purkinje fibers are arranged in sheets that form a complex three-dimensional network of lamellas. Contacts with the ventricular myocytes are found throughout the myocardial wall, with the exception of a subepicardial layer of 2-mm thickness, ie, 10% to 15% of the wall thickness. The spatial arrangement of the Purkinje fibers correlates well with data on electrophysiology. Connexin43 was not detected in the myocytes of the atrioventricular node, whereas in the Purkinje fibers of the atrioventricular bundle and of the bundle branches, abundant expression of connexin43 was found in both humans and cows. In the bovine Purkinje fibers, a remarkable subcellular distribution of connexin43 is found: it occupies the entire plasma membrane facing other Purkinje cells but not that facing the surrounding connective tissue. The structural differences in architecture of the ventricular conduction system in humans and cows seems not to result in substantial differences in conduction velocities. However, the Purkinje fiber network in the bovine heart may explain the efficient ventricular excitation, as reflected by the relatively short QRS complex compared with that in the human heart, where intramural Purkinje fibers are not found.

Demonstration of connective tissue sheaths surrounding working myocardial cells and Purkinje cells of the sheep moderator band.

Morphological studies were carried out to delineate the characteristics of connective tissue sheaths surrounding working myocardial cells and Purkinje cells in the moderator band of adult sheep hearts, using a series of techniques including silver staining, immunohistochemistry, transmission electron microscopy (TEM) and scanning electron microscopy (SEM). For SEM, tissue blocks were treated with 2N NaOH at room temperature to digest cellular elements. Individual working myocardial cells were ensheathed by thin argyrophil fibers (reticular fibers), while fascicles of 4-8 Purkinje cells (Purkinje strands) were encircled by rather thick reticular fibers. Some collagen fibers were located between masses of myocardial cells as well as between the Purkinje strands. Immunohistochemical analyses indicated that reticular fibers directly surrounding both myocardial cells and Purkinje strands showed moderately positive reactions for anti-type I collagen and intensely positive reactions for anti-type III collagen. Deserves particular note is that the three-dimensional architecture of the reticular sheaths varied widely at different places. The thin reticular sheaths surrounding each myocardial cell consisted of fibrils which were arranged in a coarse network and directed circularly along the long axis of cells. By contrast, the sheaths enclosing Purkinje strands were thicker, and their reticular fibrils were woven into a compact "felt-like" texture. The functional significance of these connective tissue sheaths is also discussed.