Development changes in regulation of embryonic chick heart gap junctions

Summary Embryonic chick myocyte pairs were isolated from ventricular tissue of 4-day, 14-day, and 18-day heart for the purpose of examining the relationship between macroscopic junctional conductance and transjunctional voltage during cardiac development. The double whole-cell patch-clamp technique was employed to directly measure junctional conductance over a transjunctional voltage range of ±100 mV. At all ages, the instantaneous junctional current (or conductance=current/voltage) varied linearly with respect to transjunctional voltage. This initial response was followed by a time- and voltage-dependent decline in junctional current to new steady-state values. For every experiment, the steady-state junctional conductance was normalized to the instantaneous value obtained at each potential and the data was pooled according to developmental age. The mean steadystate junctional conductance-voltage relationship for each age group was fit using a two-state Boltzmann distribution described previously for other voltage-dependent gap junctions. From this model, it was revealed that half-inactivation voltage for the transjunctional voltage-sensitive conductance shifted towards larger potentials by 10 mV, the equivalent gating charge increased by approximately 1 electron, and the minimal voltage-insensitive conductance exactly doubled (increased from 18 to 36%) between 4 and 18 days of development. Decay time constants were similar at all ages examined as rate increased with increasing transjunctional potential. This data provides the first direct experimental evidence for developmental changes in the regulation of intercellular communication within a given tissue. This information is consistent with the hypothesis that developmental expression of multiple gap junction proteins (connexins) may confer different regulatory mechanisms on intercellular communication pathways within a given cell or tissue.     

Fine structural changes in embryonic chick heart ventricle induced by lead poisoning.

The embryonic chick heart ventricle of day 11 was studied electron microscopically to learn the structural changes that develop in lead poisoning. The chick embryos were administered with 0.015 mg/egg of lead acetate at day 2. The most pronounced changes observed in the ventricle were: malformed mitochondria, disorganized, short and scanty myofibrils and abundance of swollen vacuoles. The ultrastructure of the ventricle from the control chick embryos was normal. The most frequent change noted in the ventricular tissue was an alteration in the myofibrils. This study indicates that electron microscopic changes can be induced in the embryonic chick heart ventricle by lead poisoning.     

Different effect of propranolol and verapamil on isoprenaline-induced changes in the chick embryonic heart

Isoprenaline (IPRO) has been reported to cause pathological lesions of the embryonic heart. The purpose of the present study was to ascertain whether the development of IPRO-induced changes can be reduced--similarly as in adults--by beta blockade or calcium antagonists. IPRO was administered to 10-day-old chick embryos intraamnially (i.a.) in a dose of 2 X 10 mg.kg-1 per 48 h; propranolol (Inderal) and verapamil (Isoptin) were injected i.a. in a dose of 1.0 or 10.0 mg.kg-1 before each injection of IPRO. It was found that propranolol completely blocked the cardiac IPRO-induced changes, i.e. cardiomegaly, avascular areas and elevation of cAMP. On the other hand, verapamil was found to have no protective effect in any dose used. Furthermore, it increased the mortality of experimental embryos. This fact support the hypothesis that cardiac sensitivity to calcium antagonists may differ during prenatal development.     

The effect of oxidized isoprenaline on the chick embryonic heart

Intra-amnial administration of isoprenaline (IPRO) to chick embryos induces a number of myocardial lesions. The purpose of the present study was to investigate whether similar changes may also be induced after injection of spontaneously oxidized isoprenaline and commercially obtained adrenochrome. Cardiotoxicity of these substances has been demonstrated in adult animals. IPRO, oxidized IPRO, or adrenochrome were administered intra-amnially to 10-day-old chick embryos at doses of 0.1, 1.0, 10.0, and 100.0 mg X kg-1. Parallel experimental groups received propranolol at a dose of 1 mg X kg-1, 15 s before injection of IPRO or oxidized IPRO. The cAMP level in the heart was determined by radioimmunoassay 2 and 30 min after administration of IPRO, oxidized IPRO, or adrenochrome at a single dose of 10.0 mg X kg-1. It has been found that in embryos the effect of IPRO and oxidized IPRO is dose dependent. The rise in mortality and development of cardiomegaly together with increased hydration and disturbances of the development of coronary vascularization were highly significant starting from the dose of 10 mg X kg-1. Furthermore, both drugs significantly increased cAMP levels in the embryonic heart. On the other hand, the administration of adrenochrome was without any effect. The changes induced by IPRO were prevented by the administration of the beta-blocking agent propranolol; the lesions induced by spontaneously oxidized IPRO were, however, prevented only partially.     

The effect of graded hypoxia on the embryonic chick heart

Embryonic ventricular function in the chick was measured in response to graded levels of hypoxia. Myocardial contractility, as measured by cinephotoanalysis and expressed as shortening fraction, was significantly depressed after 1 hour of moderate hypoxia (6% O2) and after 5 hours of milder (16% O2 and 11% O2) levels of hypoxia (P less than .05). Microscopy confirmed associated myocyte damage with cell death noted after 5 hours of moderate hypoxic stress. Heart rate change was not related to the severity of hypoxia. The greatest level of tachycardia was noted with conditions of mildest hypoxia (16% O2). The data confirm that cardiac contractility, as measured by shortening fraction, is depressed on exposure to hypoxia, with impairment of function related to the severity of the hypoxic conditions.     

Morphological and biochemical changes of LLC-PK1 cells during adaptation to glucose-free culture conditions.

The established renal epithelial cell line LLC-PK1 retained in tissue culture several differentiated properties of renal proximal tubular cells. By adapting LLC-PK1 cells to glucose-free culture conditions, we recently succeeded in isolating a gluconeogenic strain of LLC-PK1 cells capable of growing in the absence of hexoses. In contrast to the parental wild type, the isolated strain expressed fructose-1,6-bisphosphatase activity and was, therefore, designated LLC-PK1-FBPase+. Besides the differences in glucose metabolism, the isolated gluconeogenic substrain differs form the parental wild type with respect to morphological appearance and the expression of apical membrane marker enzymes. LLC-PK1-FBPase+ cells display a drastic accumulation of autophagic vacuoles, disappearance of apical membrane alkaline phosphatase activity, and increased gamma-glutamyltranspeptidase activity. In order to find out whether or not a low alkaline phosphatase activity in combination with the enhanced formation of autophagic vacuoles is related to a change in apical membrane surface, we utilized a combined light and electron microscopic morphometric procedure to determine the absolute amount of organelle volumes and membrane surface areas. This stereologic approach shows that LLC-PK1-FBPase+ cells display a tenfold increase in the volume of autophagic vacuoles and the lysosomal compartment. Analysis of lysosomal enzyme activities, however, revealed no changes as compared to wild-type cells. The apical membrane surface of gluconeogenic cells was found to be increased by 80%. Karyotype analysis revealed that LLC-PK1 wild-type cells were diploid, whereas FBPase+ cells exhibited polyploidy with a high percentage of tetraploid nuclei. Culturing LLC-PK1-FBPase+ cells in the presence of 5 mM glucose does not abolish the morphological and biochemical changes described, indicating the stability of the FBPase+ strain.     

Effect of ouabain on the innervated and noninnervated embryonic chick heart.

Attempts to assess the importance of the role played by endogenous catecholamines in the positive inotropic response to ouabain have produced contradictory results. The sympathetic nervous system is not present in the 4-day-old chicken embryo heart but is fully developed after 7 days of embryonic life. This was confirmed by the fact 4-day-old hearts do not respond to tyramine and cocaine while the usual positive inotropic and chronotropic responses were observed when these drugs were administered to 7-day-old hearts. In spite of this difference the positive inotropic response to ouabain was virtually identical at these two stages of development.     

Changes in the nuclear polyamine content of chick erythrocytes during embryonic development.

The polyamine content of the circulating erythrocyte population in the embryonic chick was studied during its development. Total cellular polyamine content fell dramatically between 5 and 7 days of development, paralleling the decrease in metabolic activity exhibited by these cells. Nuclei were isolated from the erythrocytes by a non-aqueous technique, which not only eliminated the polyamine loss that occurred with aqueous isolation, but also prevented redistribution of the polyamines from the cytoplasm. Nuclear spermidine and spermine contents decreased markedly between 5 and 6 days of development from 31 to 10 pmol/microgram of DNA and from 33 to 18 pmol/microgram of DNA respectively. Thereafter the spermine content remained constant, but the spermidine content continued to decline. Good correlations between spermidine and RNA contents were observed in both cells and nuclei, and similarly between spermine and RNA contents in cells, but no such correlation was observed between spermine and RNA in nuclei.     

Hyaluronidase activity and hyaluronate content of the developing chick embryo heart.

Hyaluronidase activity and hyaluronate content were measured in the developing chick heart from embryonic day 3 through posthatching stages. High levels of both enzyme and substrate were found during the earliest stages examined. Hyaluronidase activity gradually declined to 63% of the initial (day 3) level by embryonic day 16. Enzyme activity decreased more sharply during the next 4 days to 30% of the initial level and remained constant through 2 weeks after hatching. Low levels of enzyme activity (about 10% initial levels) were still detectable in 10-week-old chicken hearts. The heart hyaluronidase is an endoglycosidase with an estimated molecular weight of 62,000, which degrades hyaluronate and, to a lesser extent, chondroitin sulfate at an acid pH optimum. Hyaluronate constituted approximately 50% of the total glycosaminoglycan content at embryonic day 5. Between embryonic days 5 and 12, the concentration of hyaluronate decreased to 25–30% of the initial level and remained constant thereafter. The level of other glycosaminoglycans decreased more gradually than hyaluronate and did not reach a constant level until hatching. This pattern of hyaluronidase activity and hyaluronate concentration presumably reflects the extensive tissue remodeling which transforms the developing heart from a thin-walled tube containing extensive regions of extracellular matrix to a compact, thick-walled myocardium having a limited extracellular compartment.     

Fatty acid synthesis in chick embryonic heart and liver during development.

Fatty acid synthesis by subcellular fractions of heart and liver of chick embryos at varying stages of development has been studied. Fatty acid synthetase activity is associated with the embryonic heart at early stages of development, as suggested by substrate requirement, Schmidt decarboxylation of synthesized fatty acids and gas liquid chromatographic identification of the products as palmitic and stearic acids. The fatty acid synthetase activity decreases in heart cytosol with age of the embryo and is absent in the newly hatched chick and in older chicken. The acetyl CoA carboxylase activity is negligible in embryonic and adult chicken heart. The fatty acid synthetase activity in liver is low, but measurable during the entire embryonic development. The activity increases by about three-fold on hatching and thereafter in fed, newly hatched chicks by about 35-fold, over the basal embryonic activity. The acetyl and malonyl transacylase activities in the heart and liver cytosols during development followed closely the fatty acid synthetase activities in heart and liver, respectively. A non-coordinate induction of fatty acid synthetase and acetyl CoA carboxylase activities in liver was observed during development. The microsomal chain elongation in liver and heart followed the pattern of fatty acid synthetase activity in liver and heart, respectively. The mitochondrial chain elongation in embryonic heart is initially low and increases with age; while this activity in liver is higher in early stages of embryonic development than in the older embryos and the chicks. Measurement of lipogenesis from acetate-1-¹⁴C by liver and heart slices from chick embryos and newly hatched chicks support the conclusions reached in the studies with the subcellular fractions. The results obtained indicate that the major system of fatty acid synthesis in embryonic and adult heart is the mitochondrial chain elongation. In embryonic liver, fatty acid synthesis proceeds by chain elongation, while the de novo system is the major contributor to the lipogenic capacity of the liver after hatching.     

Phase resetting of embryonic chick atrial heart cell aggregates. Experiment and theory.

The influence of brief duration current pulses on the spontaneous electrical activity of embryonic chick atrial heart cell aggregates was investigated experimentally and theoretically. A pulse could either delay or advance the time of the action potential subsequent to the pulse depending upon the time in the control cycle at which it was applied. The perturbed cycle length throughout the transition from delay to advance was a continuous function of the time of the pulse for small pulse amplitudes, but was discontinuous for larger pulse amplitudes. Similar results were obtained using a model of the ionic currents which underlie spontaneous activity in these preparations. The primary ion current components which contribute to phase resetting are the fast inward sodium ion current, INa, and the primary, potassium ion repolarization current, IX1. The origin of the discontinuity in phase resetting of the model can be elucidated by a detailed examination of the current-voltage trajectories in the region of the phase response curve where the discontinuity occurs.     

85Sr uptake by the chick embryonic heart: effect of high doses of isoproterenol.

The aim of the present study was to establish whether intraamnial administration of toxic doses of isoproterenol to chick embryos increases cardiac accumulation of strontium, the homologue element of calcium. It has been shown that the ability of embryonic tissues (blood, heart, and liver) to accumulate 85Sr decreases significantly during ontogeny. Administration of isoproterenol to chick embryos did not elevate the concentration of 85Sr in the heart. It seems, therefore, that isoproterenol-induced developmental changes in the chick embryonic myocardium are not necessarily due to intracellular calcium (as measured by 85Sr) overload.     

Morphologic study of ventricular trabeculation in the embryonic chick heart

This paper presents a morphologic study of ventricular trabeculation in chick embryo hearts between days 2 and 5 of incubation. Trabeculation appears to be the expression of three closely interrelated events: the formation of endocardial outgrowths that eventually invade the myocardium; the development of large intercellular spaces between the myocytes, and the decrease in thickness of the cardiac jelly. Endocardial cells present morphologic differences between trabeculated and nontrabeculated areas of the ventricular region. The elongation of the endocardial cells in the endocardial outgrowths and the presence of mitoses suggest that the endocardium grows out by means of an increase in cell number and by redistribution and elongation of the preexisting endocardial cells. The intercellular spaces of the myocardium appear filled with abundant extracellular material. It is suggested that the continuous synthesis of extracellular material by the myocytes may increase the hydrostatic pressure within the myocardium, inducing the formation and the enlargement of these intercellular spaces. The development and later rupture of endocardium-covered cords is described here. These cords are made up of a core of cardiac jelly material revested by endocardium. The cords may be engaged in the removal of substantial amounts of cardiac jelly during the formation of the trabeculae.     

中华蟾蜍糖原含量的季节变化

于2005年11月至2006年10月,用硫酸-蒽酮比色法和比重法测定了中华蟾蜍各月份的肝糖原和肌糖原含量及肝比重。结果显示:冬眠期间(11月至次年的2月),糖原含量逐月下降;2月份时出现临时回升,然后继续下降;4月份时肝糖原含量最低;5月份起,肝糖原含量逐渐上升;5月份时肌糖原含量为最低;6月份起,肌糖原含量逐渐上升。虽然在7-8月间出现过下降,两种糖原的含量在10月份时达到一年中的最高值。这些结果表明,蟾蜍糖原含量在一年中呈现显著的季节性波动。越冬前所储备糖原的一部分可能用于越冬期间维持高水平的血糖,一部分用于基础代谢。2月份时糖原含量的临时上升,可能是血液中作为防冻保护剂的葡萄糖运回肝脏和肌肉中再合成糖原的结果。7-8月间糖原含量降低可能与蟾蜍夏蛰有关。雌性5月至10月期间的肝糖原总体水平显著低于雄性,可能与依赖可得到葡萄糖的卵母细胞中的糖原合成有关。糖原含量的季节变化与蟾蜍的生活状态(越冬、繁殖等)有关,并与血糖含量有联动关系。