Differentiation of the atrioventricular node, the atrioventricular bundle and the bundle branches in the bovine heart: an immunohistochemical and enzyme histochemical study

The previous observations of differences between different cardiac regions (ventricular myocardium, atrial myocardium, Purkinje fibre system) with respect to the maturation of the M-line region and the establishment of mature metabolic characteristics, have been extended. It was found that M-line maturation proceeds differently also between different regions of the conduction system. The M-line proteins, myomesin and MM-creatine kinase, were detected earlier, by means of immunohistochemistry, in the AV bundle and bundle branch cells than in the AV node cells. Also, a difference was observed in large foetuses. Striations in the AV node were less evident than in the AV bundle and the bundle branches in sections incubated with antibodies against myomesin as well as against MM-creatine kinase. Using enzyme histochemistry it was observed that the differences in metabolic properties between the AV node, the AV bundle and the bundle branches on the one hand, and the ordinary myocardium on the other, of adult hearts, are not established at the early stages. No clear difference in activity of succinate dehydrogenase was seen between the conduction tissues and the ordinary myocardium in the foetal hearts, while the conduction tissues showed a lower activity in the adult hearts. Furthermore, the pattern of activity of mitochondrial glycerol-3-phosphate dehydrogenase between the conduction tissues and the atrial and ventricular myocardium was quite different in early foetal stages compared with the adult stage.     

Gangliosides of the bovine heart impulse conducting system

The ganglioside analysis of the heart impulse conducting system was carried out, comparing it with that of ordinary myocardium. The heart impulse conducting system contained about 3-times more gangliosides than ordinary myocardium and showed a distinctly different ganglioside composition. These observations seemed to indicate that the differentiation between myoblasts and each type of cardiac muscle cell, impulse conducting system and ordinary myocardium cells, resulted in their characteristic ganglioside compositions.     

初生与成年牦牛心肌的组织学结构比较

为探究牦牛心肌发育过程中组织学结构的变化,采用组织学、组织化学和免疫组织化学方法对初生和成年牦牛心肌的组织学结构、心钠素（ANP）和缝隙连接蛋白43（Cx43）的表达情况进行了观察。结果显示,初生牦牛心肌细胞数量少且细短,闰盘为直线型或V型,心肌细胞间以Ⅲ型胶原纤维为主,Ⅰ型胶原纤维较少;成年牦牛心肌细胞粗长,其数量是初生牦牛的2倍,闰盘呈竹节样吻合或阶梯状,心肌细胞间以Ⅰ型胶原纤维为主。初生和成年牦牛,ANP在心房肌细胞中均表达强烈,且右心房表达量显著高于左心房,心室肌细胞中不表达。ANP在普肯耶纤维中有少量表达。初生牦牛心房肌细胞ANP表达量高于成年牦牛。在心肌细胞膜和胞质内,初生牦牛Cx43表达强于成年牦牛,但在闰盘内,成年牦牛Cx43表达强于初生牦牛。Cx43在初生和成年牦牛的普肯耶纤维中均呈膜阳性。结果提示,初生与成年牦牛相比,成年牦牛的心肌细胞变粗长且数量增多,ANP在心房肌表达减弱,Cx43在闰盘表达更显著,说明成年牦牛较初生牦牛适应循环变化的代偿能力呈进行性减弱;细胞间牢固性增加,有助于实现心肌收缩信号的迅速传导。     

Developmental changes in heart photosensitivity of the isopod crustacean Ligia exotica

During juvenile development, the cardiac pacemaker of the isopod crustacean Ligia exotica is transferred from the myocardium to the cardiac ganglion of the neurogenic heart. In adult, light stimulus decreases the beat frequency of the heart. To elucidate developmental changes in the photosensitivity of the juvenile Ligia heart, we examined the effect of a light stimulus on the semi-isolated heart of juveniles at various developmental stages by the recording membrane potential of the myocardium. We also examined the effect of hyperpolarizing current injection into the myocardium, because this causes different effects on the beat frequency between myogenic and neurogenic hearts. In newly hatched juveniles, beat frequency decreased upon current injection but exhibited no response to white light. In contrast, 10 days after hatching, beat frequency did not change upon current injection, but decreased in response to white light. The heart photoresponse of juveniles was reversibly eliminated by application of tetrodotoxin, which changes the heartbeat from neurogenic to myogenic by suppressing cardiac ganglion activity. The proportion of juveniles exhibiting a heart photoresponse increased gradually up to 100% during the period between 3 and 10 days after hatching. The results suggest that the heart photoresponse of L. exotica appears in association with transfer of the cardiac pacemaker from the myocardium to the cardiac ganglion during juvenile development.     

乙酰胆碱对豚鼠心房肌和心室肌的动作电位和收缩力的不同作用

实验采用标准玻璃微电极细胞内记录技术记录心肌细胞动作电位（action potential,AP）、肌力换能器记录心肌收缩力（force contraction,Fc）,研究乙酰胆碱（acetylcholine,ACh）对离体豚鼠心房肌、心室肌的作用。结果表明,10μmol/L ACh可缩短心房肌、心室肌动作电位的时程（action potential duration,APD）。心房肌APD在给药前后分别为208.57±36.05ms及101.78±14.41ms（n=6,P<0.01）,心室肌APD在给药前后分别为286.73±36.11ms及265.16±30.06 ms（n=6,P<0.01）。心房肌动作电位的幅度（action potential amplitude,APA）也降低,给药前后分别为88.00±9.35 mV及62.62±20.50 mV（n=6,P<0.01）,而心室肌APA无明显变化。ACh还降低心房肌、心室肌的收缩力,心房肌、心室肌Fc的抑制率分别为100％（n=6,P<0.01）和37.57±2.58％（n=6,P<0.01）。ACh对心房肌、心室肌APD和Fc的抑制作用在一定范围内（1nmol/L～100μmol/L）随ACh浓度的增高而增强。用Scott法求出ACh对心房肌、心室肌APD缩短作用的KD值,分别为0.275和0.575μmol/L,对Fc抑制作用的KD值分别为0.135和0.676μmol/L。各浓度下ACh对心房肌效应与心室肌效应作组间t检验,从10nmol/L到0.1mmol/L均有显著的统计学差异。此外,10μmol/L阿托品及20mmol/L     

Myosin heavy chain isoforms in the myocardium of the atrioventricular junction of Scyliorhinus canicula (Chondrichthyes,Carcharhiniformes)

The atrioventricular junction of the fish heart, namely the segment interposed between the single atrium and the single ventricle, has been studied anatomically and histologically in several chondrichthyan and teleost species. Nonetheless, knowledge about myosin heavy chain (MyHC) in the atrioventricular myocardium remains scarce. The present report is the first one to provide data on the MyHC isoform distribution in the myocardium of the atrioventricular junction in chondrichthyans, specifically in the lesser spotted dogfish, Scyliorhinus canicula, a shark species whose heart reflects the primitive cardiac anatomical design in gnathostomes. Hearts from five dogfish were examined using histochemical and immunohistochemical techniques. The anti-MyHC A4.1025 antibody was used to detect differences in the occurrence of MyHC isoforms in the dogfish, as the fast-twitch isoforms MYH2 and MYH6 have a higher affinity for this antibody than the slow-twitch isoforms MYH7 and MYH7B. The histochemical findings show that myocardium of the atrioventricular junction connects the trabeculated myocardium of the atrium with the trabeculated layer of the ventricular myocardium. The immunohistochemical results indicate that the distribution of MyHC isoforms in the atrioventricular junction is not homogeneous. The atrial portion of the atrioventricular myocardium shows a positive reactivity against the A4.1025 antibody similar to that of the atrial myocardium. In contrast, the ventricular portion of the atrioventricular junction is not labelled, as is the case with the ventricular myocardium. This dual condition suggests that the myocardium of the atrioventricular junction has two contraction patterns: the myocardium of the atrial portion contracts in line with the atrial myocardium, whereas that of the ventricular portion follows the contraction pattern of the ventricular myocardium. Thus, the transition of the contraction wave from the atrium to the ventricle may be established in the atrioventricular segment because of its heterogeneous MyHC isoform distribution. The findings support the hypothesis that a distinct MyHC isoform distribution in the atrioventricular myocardium enables a synchronous contraction of inflow and outflow cardiac segments in vertebrates lacking a specialized cardiac conduction system.     

氦氖激光对大鼠心肌超微结构、SOD和MDA影响的观察

为了解低强度氦氖激光对心肌细胞超微结构、超氧化物歧化酶（SOD）和丙二醛（MDA）的影响,采用2．87J／cm^2,7.17J/cm^2和28.66J/cm^2三种剂量氦氖激光照射大鼠心前区,电镜观察心肌超微结构,测定心肌组织SOD和MDA的含量。结果发现心肌线粒体有轻微损伤,心肌组织SOD含量在7．17J／cm^2剂量显著下降（P＜0．05）,而在28．66J／cm^2剂量显著升高（P＜0．05）,心肌组织MDA含量在7．17J／cm^2和28．66J／cm^2两种剂量下降有显著升高（P＜0．01）,在2．87J／cm^2剂量,心肌组织SOD和MDA含量均无显著变化（P＜0．05）。结果提示心肌SOD含量和脂质过氧化随不同的激光剂量而变化。     

Correlation of myofibrillar ATPase activity and myosin heavy chain content in ventricular and atrial myocardium of fish heart

In the fish heart, ventricular and atrial muscles contain different isoforms of native myosin and myosin heavy chain (MyHC) but the significance of this diversity is still not known. We have analysed ventricular and atrial myocardium of six freshwater fish species (goldfish, roach, bream, rudd, perch and pike-perch) using histochemical staining for myofibrillar ATPase activity as well as non-denaturing and SDS gel electrophoreses for native myosin and MyHC content. In the range of fish species studied, the intensity of ATPase reaction was higher in the atrial myocardium than in the ventricular myocardium and the composition of native myosin isoforms differed between these two muscles. The MyHC content in the cardiac muscle showed some species-related differences. In the goldfish, both atrial and ventricular cardiac muscle contained electrophoretically similar MyHC. In the other fish species, however, the ventricular myocardium showed electrophoretically faster MyHC than that present in the atrial myocardium. These results indicate that there are consistent and characteristic species-related differences between the ventricular and atrial muscles at the level of ATPase staining and the type of MyHC expressed. The findings suggest that fish ventricular and atrial muscles may differ in their contractile properties.     

Force-frequency-relation in human atrial and ventricular myocardium

In human heart failure, an increase in frequency of stimulation is followed by a reduced force of contractionin vivo andin vitro. The present study aimed to investigate whether a different origin of the myocardial sample or pretreatment with the cardioprotective agent 2,3-butanedione-monoxime (BDM) influences the force-frequency-relationship in electrically driven muscle strips taken from failing and nonfailing human myocardium. With as well as without pretreatment with BDM, the altered force-frequency-relationship in failing compared to nonfailing human ventricular myocardium can be observed. The effectiveness and the potency to increase force of contraction following an increase in frequency of stimulation was significantly higher in atrial than in ventricular myocardium in nonfailing and failing tissue. The different observations in atrial and ventricular myocardium provide evidence for functionally relevant differences in the electromechanical coupling between the human atrial and ventricular myocardium.     

Ultrastructure of the myocardial junctions of the chick embryo at an early stage in the organogenesis of the heart

The ultrastructural features of the myocardiocyte junctional systems have been studied in the heart right myocardium of 6 day chick embryos in order to analyze the relationships between the spatial arrangement of the myocardial fibres and the formation of the myocardiocyte junctions. The myocardiocytes are remarkably branched, joined in small fibres to form a loose network with large intercellular spaces and appear well differentiated, rich in organelles, myofibrils and glycogen. Adherent-type junctions and well differentiated desmosomes are detectable at the level of the termino-terminal contacts between myocardiocytes, where the plasma membranes appear more or less complicately interconnected; nexus-type junctions are seen in the latero-lateral contact regions. The results indicate that already at an early stage of the heart organogenesis the myocardium intercellular junctions are similar, on the whole, to those described in the adult, so that they would not seem to be transitory or modifiable structures. The changes in the spatial orientation of the myocardial fibres, which take place after the 6th incubation day, could be allowed by the network-like arrangement of the primitive myocardium and presumably conditioned by the blood flow in the developing heart.     

Physiological Evaluation of the Results of Myocardial Revascularization in Patients with Ischemic Heart Disease

Intraoperative assessment of the myocardial blood flow was performed in different heart areas in 94 patients with ischemic heart disease prior to and after myocardial revascularization. An ALF-21 laser–Doppler flowmeter (Transonic Systems Inc.) was used to measure the myocardial blood flow near the anterior and posterior surfaces of the left ventricle (LV_asand LV_ps, respectively) and the anterior surface of the right ventricle (RV_as). According to the results of revascularization, the patients were divided into four groups: group I with increased myocardial blood flow in all areas tested, group II and group III with blood flow redistributed along LV and RV, and group IV with moderately decreased myocardial blood flow in all test areas. All groups showed a postoperative decrease in blood flow differences between different myocardium areas and the leveling of blood flow in the whole myocardium. The efficiency of revascularization depended on a number of factors, including the initial level of myocardial blood flow, completeness of revascularization of ischemic areas, the number and quality of bypass grafts, and the patients' age. The most pronounced positive results were observed in patients under the age of 60 years.     

Experimental-theoretical study of the force-interval relationship in the developing chicken myocardium]

The force-interval relationship was studied on myocardium preparations from chick embryos and hatched chickens. It is shown that the force-interval relationships of myocardium change during ontogenesis. A negative staircase (a decrease in the isometric force with increasing stimulation rate) in the chick embryo myocardium and a positive steady-state relationship in hatched stage myocardium were revealed. Changes in the force after switching from one stimulation frequency to another, the effects of poststimulation potentiation, as well as responses to the introduction of pauses and extrasystols at a constant stimulation rate were recorded. All the effects observed in the transient processes in preparations from hatched stage myocardium were more pronounced than in embryo myocardium. Our previous mathematical model of calcium recirculation in cardiomyocytes was adapted for simulating the main features of force-interval relationships in embryonal and relatively developed myocardium. The main source of regulatory calcium in the model of hatched stage myocardium is sarcoplasmic reticulum. In the model of embryo myocardium, it was postulated, based on data available in literature, that the main regulator of contractile response of the muscle is calcium that enters cardiomyocytes from extracellular medium. To describe force-interval relationships, by this model, the decreasing dependence of the entry of extracellular calcium on the intervals between stimuli was introduced.     

Growth allometry of the myocardium in human embryos (from stages 15 to 23).

The relative growth of the myocardium was studied in 27 staged human embryos (Carnegie stages). The volume of the myocardium was determined for each embryo according to Cavalieri's principle (by using point-counting planimetry to determine the area of the profiles of the myocardium). The volume of the myocardium (variable Y) was correlated to embryonic crown-rump length (variable X in millimeters) and age (in days). The bivariate allometric equation was used as Y = aXb. The scatterplot was discontinuous, presenting two trends during the postsomitic period. The first part was composed of embryos staged from stages 15 to 20, and the second part by embryos staged from stages 21 to 23. The breakpoint between these different trends was found at the level of stage 20 (embryo of 22 mm in crown-rump length and age nearly of 52 days). From stages 15 to 20, the growth rate of the myocardium was allometrically negative. On the other hand, from stages 21 to 23 this growth rate was moderately allometrically positive. These differences in growth of the myocardium were analyzed and, at least partially, might be due to the functional circulatory increase in the peripheral vascular bed in correlation to the cardiac hemodynamic demand required at the end of the embryonic period proper.     

Quick adaptation of the myocardium and autonomic nervous regulation of the cardiac rhythm in 10- to 11-year-old children operating a computer

A study of quick adaptation of the myocardium of subjects operating a computer, depending on the adaptive capacities of the body, was conducted in 100 children with ages varying between 10 and 11 years using the electrocardiography method for the analysis of heart rate variability. Significant differences in the bioelectrical processes in the myocardium and autonomic nervous regulation of the cardiac rhythm (CR) were found in children with different adaptive capacities of the body. Quick adaptation to the operator activity in children with a good adaptive capacity is characterized by intensification of atrial activity and metabolic processes in the myocardium, as well as by a shortened duration of the cardiac cycle due to a shorter diastolic time, determined by increased sympathetic influences on the CR. In children with a decreased adaptive capacity, a decrease in the atrial excitability and myocardial metabolism, an increase in the systolic time, a decrease in the diastolic time, and an increase in parasympathetic influences on the CR are observed.     

Demonstration of myoglobin and CK-M in myocardium. Comparison of five fixation methods and three immunohistochemical techniques

The results of immunohistochemical staining vary depending on the tissue, fixative, antigen-antibody system, and immunohistochemical staining methods used. The purpose of this study was to evaluate the effect of different methods of fixation, different antigen-antibody systems, and different immunohistochemical methods on immunohistochemical staining of myocardium. Samples of normal fresh canine myocardium from six dogs were fresh frozen and fixed in 10% neutral buffered formalin, Bouin's, Bayley's and Carnoy's fixatives. Immunohistochemical staining for myoglobin and creatine kinase M was performed using the ABC (avidin-biotin complex) and indirect peroxidase-antiperoxidase (PAP) techniques. Tissues fixed in formalin showed the most intense specific staining for both antigens with the least background and nonspecific staining. All other fixation methods and frozen section techniques gave a more variable degree of specific positive staining and substantial background staining and/or nonspecific staining. ABC and PAP techniques gave similar results with both antigen-antibody systems and with each fixation method. Thus, no differences in specificity or sensitivity were observed between ABC and PAP techniques. Differences in staining intensity and pattern were related primarily to differences in fixation methods.     

Effect of chronic stress on the ultrastructure of the myocardium and hypothalamus of "emotional" and "nonemotional" rats

The influence of chronic stress on the ultrastructure of the myocardium and hypothalamus was studied in experiments on male rats with different levels of emotional-behavioral reactivity. "Emotional" rats manifested a pronounced increase in glycogen granules in myocytes and intercellular space, appearance of the areas of overcontraction of myofibrils, conglomerates of aggregated platelets in myocardial capillaries, and red cell egress from myocardial and hypothalamic capillaries. Alterations in the ultrastructure of the myocardium and hypothalamus in "nonemotional" rats were less marked and consisted in the appearance of the areas of overcontraction of myofibrils, enlargement of sarcoplasmic reticulum caverns, and in an increase in the lipid content in cardiomyocytes. The ultrastructural changes in the myocardium and hypothalamus of "emotional" and "nonemotional" rats indicate different reactivity of the animals and are likely to be accounted for by different levels of activation of their adrenergic systems.     

Expression of major gap junction connexin types in the working myocardium of eight chordates.

The alpha1 connexin (connexin43) is regarded as the major gap junction protein of the myocardium because it predominates there in mammals. Here, we show that it is not the major connexin of the working myocardium in non-mammalian vertebrates, which instead express beta1-like connexins homologous to mammalian connexin32. A phylogenetic series of hearts was immunostained with seven antibodies raised against peptide sequences specific for three distinct members of the gap junction connexin family: alpha1, beta1 and alpha5 (mammalian connexin40/avian connexin42). Working myocardium from two ascidian chordates (Ciona and Mogula), a teleost (Carassius), a frog (Xenopus) and two reptiles (Anolis and Alligator) was found to express a beta1-like connexin, rather than an alpha1-like connexin. An alpha1-like connexin was nevertheless often detected in other cardiac tissues. In the chicken (by ancestry a reptile), the developing myocardium expressed a beta1-like connexin strongly on embryonic day 6 but less strongly at hatching, and minimally in the adult. Myocardial expression of alpha5 connexin increased during development, but remained strongest in the coronary vascular endothelial and cardiac conduction tissues. The arteriolar smooth muscle of the chicken expressed alpha1 connexin throughout development, but its myocardium did not. In contrast, the working myocardium of a marsupial mammal (the opossum Trichosurus) strongly expressed an alpha1 connexin just like placental mammals. These results imply that a shift from beta1 to alpha1 connexin expression in the heart occurred prior to the evolution of the opossums. The beta and alpha connexin subfamilies have different permeabilities and gating properties, and we discuss factors that might have made this shift beneficial.     

Morphological analysis of the adaptive changes in the myocardium under individually measured physical load]

Studying morphological changes of the myocardium in the organic, cellular and subcellular levels of experimental animals (dogs) has shown that standard physical load on tradmill band graded only by external parameters of work (time of running, its speed etc.) causes different changes in different animals. In contrast to it, the method of individually graded physical load proposed by the author (1970, 1972) considers the depth of myocardium responses, the degree of its morphofunctional changes corresponding to certain stages of changes in the parameters of cardio-vascular activity and respiratory system. Using the relationship of pulse rate and frequency of respiration as a diagnostic test made it possible to establish 6 stages of myocardium responses to physical load. Each of these stages has a complex of morpho-functional changes in the myocardium and corresponds to certain adaptive shifts on the organism level. On these foundations one can prognoze the depth of morphological changes in the myocardium basing upon diagnostic tests. So, the proposed experimental model of individual selection of the value of physical load according to responses of the organism rather than to external parameters of work as well as the established principle of the stage character of adaptive responses may be used in the practice of investigators for directed influence on the myocardium to form its certain qualities.     

Cardiac telocytes exist in the adult Xenopus tropicalis heart

Recent research has revealed that cardiac telocytes (CTs) play an important role in cardiac physiopathology and the regeneration of injured myocardium. Recently, we reported that the adult Xenopus tropicalis heart can regenerate perfectly in a nearly scar‐free manner after injury via apical resection. However, whether telocytes exist in the X tropicalis heart and are affected in the regeneration of injured X tropicalis myocardium is still unknown. The present ultrastructural and immunofluorescent double staining results clearly showed that CTs exist in the X tropicalis myocardium. CTs in the X tropicalis myocardium were mainly twined around the surface of cardiomyocyte trabeculae and linked via nanocontacts between the ends of the telopodes, forming a three‐dimensional network. CTs might play a role in the regeneration of injured myocardium.