Avian Heart Research Articles

Onset of expression and regional deposition of alpha-smooth and sarcomeric actin during avian heart development.

The sequential appearance of mRNAs for smooth, cardiac, and skeletal alpha-actin has been described during development of the chicken heart (Ruzicka, D.L., and R.J. Schwartz 1988 J. Cell Biol., 107:2575-2586). To assess whether this reflects the deposition of corresponding isoproteins, we have immunocytochemically localized smooth and sarcomeric (cardiac and skeletal) alpha-actin in Hamburger-Hamilton (H-H) stage 7-18 embryos using monoclonal antibodies. Within the developing embryo at stage 9-, smooth muscle alpha-actin was exclusively detected in the developing heart, upon fusion of the endocardial tubes; sarcomeric alpha-actin was observed later (stage 9). By the onset of contraction at stage 10+, intense immunostaining of both smooth and sarcomeric isoproteins was observed in the ventricle; at this time smooth muscle alpha-actin was also detected in splanchnic mesoderm of the pre-vitelline area, in a cellular layer adjacent to the only embryonic cells that exhibited factor VIII (von Willebrand factor) antigens. Double immunostaining of the myocardium at stage 11, at which time striations were first detected, revealed the co-existence of smooth and sarcomeric actin in developing sarcomeres. Intense expression of sarcomeric actin continued in the heart after stage 11, whereas smooth muscle alpha-actin was down-regulated in the ventricle and became regionalized to the inflow and outflow tracts. As expected, smooth muscle alpha-actin was detected around intra- and extra-embryonic vascular structures at later developmental stages, while sarcomeric actin was observed in somites.

Urokinase activity in the developing avian heart: a spatial and temporal analysis.

Early events in cardiac morphogenesis are characterized by cell migrations and extensive tissue remodeling. This study was undertaken to determine the levels of urokinase in specific regions of the avian heart during early stages of development. Urokinase has previously been shown to be involved in both cell migration and matrix turnover. Elevated urokinase activity and mRNA levels were associated with the onset of ventricular trabeculation and mesenchymal cell migration in the endocardial cushion tissues. Urokinase was localized by immunostaining to the endocardial and mesenchymal cells of the developing atrioventricular canal (AVC) and outflow tract (OFT) as well as with evaginating ventricular endocardium. No immunoreactivity was seen associated directly with the matrix, suggesting that the enzyme remained mostly cell associated, a finding which was confirmed in isolated endocardial cells. Results from this study suggest a role for urokinase in the tissue remodeling and cell migration that occurs during the early stages of cardiac morphogenesis.

Changes in force and calcium sensitivity in the developing avian heart

The aim of this study was to characterize the development of the contractile properties of intact and chemically skinned muscle from chicken heart and to compare these characteristics with those of developing mammalian heart reported by others. Small trabeculae were dissected from left ventricles of Arbor Acre chickens between embryonic day 7 and young adulthood (7 weeks post-hatching). At all ages, increasing extracellular calcium (0.45-3.6 mM) progressively increased twitch force of electrically stimulated trabeculae. Twitch force at 1.8 mM extracellular calcium, normalized to cross-sectional area, increased to a maximum at 1 day post-hatching, remained constant through 3 weeks post-hatching, but then decreased at 7 weeks post-hatching. The maximal calcium-activated force of trabeculae chemically skinned with Triton X-100 detergent increased to a maximum 2 days before the time of hatching and was not significantly changed up to 7 weeks post-hatching. Over the ages studied, average twitch force in 1.8 mM calcium was between 26 and 66% of maximal calcium-activated force after skinning, suggesting that the contractile apparatus is not fully activated during the twitch in normal Ringer. In skinned trabeculae, the calcium sensitivity of the contractile apparatus was higher in the embryo than in the young adult. These age-dependent changes in calcium sensitivity are correlated with isoform switching in troponin T. A decrease in pH from 7.0 to 6.5 decreased the calcium sensitivity of the contractile apparatus to a greater degree in skinned trabeculae from young adult hearts than in those from embryonic hearts. This change in susceptibility to acidosis is temporally associated with isoform switching in troponin I.(ABSTRACT TRUNCATED AT 250 WORDS)

MC29-immortalized clonal avian heart cell lines can partially differentiate in vitro

We established quail clonal heart muscle cell lines from cardiac rhabdomyosarcomas developed in embryos injected in ovo with the MC29 virus containing the v- myc oncogene. These clones were characterized by means of antibodies detecting markers of striated muscle cells. Two clones were selected for further characterization on the basis of a distribution of myogenic markers similar to that in normal early embryonic cardiac muscle cells. However, these muscle markers progressively disappeared with time in culture. Cardiomyocytic differentiation could be reinduced in culture, by associating the avian cardiac cells with 3T3 cells in a defined synthetic medium. Muscle markers were then reexpressed in all cardiac cells as soon as Day 1 after coculture. Multiplication of cardiac cells continued at the same time. This is characteristic of cardiac clones since MC29-infected quail myoblasts and MC29-infected quail fibroblasts exhibited a split response to 3T3 association, i.e., decreased growth and enhanced differentiation. The cardiac clones were maintained in vitro for more than 60 generations (6 months) without morphological changes. To our knowledge, this is the first description of clonal embryonic avian heart cell lines.

Regulation of proliferation of embryonic heart mesenchyme: Role of transforming growth factor-β1 and the interstitial matrix

Proliferation of atrioventricular cushion mesenchyme of the embryonic avian heart maintained in three-dimensional aggregate culture is stimulated by interaction with the interstitial matrix. Chicken serum or transforming growth factor-β1, which stimulates proliferation, induces matrix deposition in regions of the aggregate showing high labeling indices with tritiated thymidine. Dispersed heart mesenchyme interstitial matrix introduced into serum-free culture is incorporated into the aggregate and stimulates cellular proliferation similar to serum or transforming growth factor-β1. Proliferation is reversibly inhibited by the peptide Gly-Arg-Gly-Asp-Ser-Pro. It is suggested that transforming growth factor-β1 stimulates the production of interstitial matrix and that a sufficient stimulus for proliferation in this system is the presence of the matrix, which acts as the adhesive support for cellular anchorage.

Couplings, EJSR, Gap junctions, ECC, in Ratite Heart Conduction Fibers

The ultrastructure of avian hearts has provided seminal information regarding striated muscle function. Extended junctional SR (EJSR) in avian hearts suggests that excitation-contraction coupling (ECC) is accomplished through a diffusible transmitter substance, but raises questions regarding the function of “couplings” in general. Other information gleaned from small avian hearts has elucidated adaptations of the individual and appositional geometry of the conduction fibers (CF) to functional demands imposed by different heart sizes and rates, as well as the relationship between the size of gap junctions (GJ), cell input resistance and coupling resistance between cells. The hearts of ratites (ostrich, emu, rhea) are very large (> 1 kg) and beat at about 50/min. Their working myocytes are almost identical to those found in chickens.We investigated the following questions: 1. Do ratite CF contain “couplings”, EJSR and corbular SR? 2. Are there differences in geometry between the CF of small, fast beating, and large, slow beating hearts as is the case in mammals?

Development of Cholinergic Neuroeffector Transmission in the Avian Heart

A brief outline of the ontogenesis of cholinergic neuroeffector transmission in the embryonic chick heart is given. It is important to define the development of neuroeffector transmission to evaluate the possible effects on postjunctional membrane properties. The membrane response of the aneural chick heart undergoes a qualitative change to muscarinic agonist at a time well before cholinergic innervation. This suppressed response can be revealed later in life, even in adults, when provision is made to exclude Gi/Go and or K channel activity.

An instructive role for the interstitial matrix in tissue patterning: tissue segregation and intercellular invasion.

Intercellular invasion is the intrusion of the cells of one tissue into space occupied by a second tissue. The alternative situation to invasion, one characteristic of most coherent tissues, is segregation, with identifiable boundaries existing between contiguous tissues. The interfaces between mesenchymal and myocardial tissues in the developing avian heart show a profoundly different character in different regions of the heart: the interface between epicardial mesenchyme and heart wall myocardium is planar, without intermingling of the two cell types, whereas the interface between endocardial cushion mesenchyme and myocardium is diffuse, with extensive invasion of both tissue types across the border to produce intermingling of the two tissues. Thus, invasion and tissue segregation coexist in different regions of the mesenchyme-myocardium contact zone. Investigation of the involvement of the interstitial matrix in invasion and segregation has been conducted by maintaining the two tissues in mutual contact in organ culture. Investigation of the mechanisms by which the two cell types sort out in randomized chimeric tissue reaggregates has provided insight into the conditions for tissue segregation. We have modeled invasion in organ culture by fusing aggregates of myocardial cells with aggregates of cardiac mesenchymal cells. Cells of both tissues invaded the partner aggregate during a period of 1-3 d of coculture. Both invasion and segregation in the aggregates appear to depend on the presence or absence of a fibronectin-rich interstitial matrix elaborated by the cardiac mesenchyme. During sorting, the matrix appears selectively in regions occupied by the mesenchyme. Under conditions of culture that are nonpermissive for matrix deposition, sorting fails to occur. Stimulation of matrix deposition by addition of serum, transforming growth factor beta, or isolated matrix itself is accompanied by sorting out of the two tissues. Sorting out is blocked reversibly by inclusion of the fibronectin adhesion site peptide, GRGDSP. Invasion of fused aggregates is preceded by a redistribution of the fibronectin-containing matrix of the mesenchymal aggregate such that matrix-poor regions come to occupy the interface with the myocardial partner aggregate. The invasion that ensues involves mesenchymal cells emigrating from, and myocardial cells intruding into, matrix-poor regions of the mesenchymal aggregate.(ABSTRACT TRUNCATED AT 400 WORDS)

Relationship between intracellular pH (pH i) and calcium (Ca i2+) in avian heart fibroblasts

Measurements of pH i and Ca i 2+ were made in single isolated avian heart fibroblasts using the fluorescent dyes 2,3-dicyanohydroquinone (DCH) and Indo-1. The resting level of Ca i 2+ is in part maintained by an influx of Ca 2+ from the external medium. This flux was reduced in the absence of Ca 0 2+ or by adding 2 m M LaCl 3 or CoCl 2 to the bathing medium; however, it was insensitive to calcium channel blockers nifedipine and verapamil. BAPTA (25 μ M), a calcium chelator, also reduced Ca i 2+. Changes in Ca i 2+ brought about by any of these methods were found to be accompanied by an intracellular acidification. Experiments were carried out altering pH i using trimethylamine, propionate, and ammonium chloride to determine whether pH i could influence Ca i 2+. It was found that an intracellular acidification induced a fall in Ca i 2+ and any rise in pH i induced a rise in Ca i 2+. These results suggest a direct interaction between Ca i 2+ and pH i. Various models are described which may account for the experimental observations. The findings are discussed in terms of the possible roles for pH i and Ca i 2+ and their interactions to influence cell motility and adhesion.

Reactivity of rat and rabbit intrafusal fibers with monoclonal antibodies directed against myosin heavy chains.

Serial cross and longitudinal sections from the intracapsular portions of intrafusal fibers of rat and rabbit tibialis anterior muscles were examined by fluorescence microscopy with a library of monoclonal antibodies directed against different epitopes on myosin heavy chains. Intrafusal fiber types were identified with the histochemical reactions for acid-stable and alkali-stable actomyosin ATPase. Three antibodies, known to react with avian heart and slow-tonic myosins, produced fluorescent staining in intrafusal fibers. Nuclear bag2 fibers reacted with all three antibodies, chain fibers with two, and nuclear bag1 fibers with only one. These results indicate that in rat and rabbit tibialis anterior muscle spindles nuclear bag2 fibers and chain fibers contain more than one myosin isoform. They also demonstrate that, in addition to the histochemical actomysin ATPase reaction, nuclear chain fibers and the two types of nuclear bag fibers can be identified by the selective reactivities of their myosin heavy chains.

Noncontact measurements of avian embryo heart rate by means of the laser speckle: comparison with contact measurements

A new, noncontact system for measuring the heart rate of avian embryos in the egg has been developed using a laser speckle phenomenon. The system was based upon detection of egg movements attributable to cardiac contractions of the embryo by measuring the intensity fluctuation of speckle produced in the proximity of the egg under laser light illumination (i.e. noncontact measurement). The applicability of the noncontact system to determine the heart rate of developing chick embryos was examined simultaneously with a contact system employing an audio cartridge. Both systems were found to be feasible in determining the heart rate of embryos during the late prenatal and paranatal stages. With the aid of adeques signal processing, the measurement of cardiogenic movements of the egg can be used to count the heart rate during the middle stages of incubation.

Characterization of avian angiotensin II cardiac receptors: Coupling to mechanical activity and phosphoinositide metabolism

We have characterized the avian angiotensin II (AII) cardiac receptor and provide data that this receptor couples to both mechanical activity and phospholipid metabolism in the avian heart. In 10-day-old chicks, 125I-AII bound to a high affinity site ( K d = 10 nmol ) and a low affinity site ( K d = 79.4 nmol ) with estimated binding capacities of 531 and 1330 fmol/mg protein, respectively. The 125I-AII binding was rapid, saturable, reversible and modulated by divalent cations and guanine nucleotides. The potency order for the competitive binding of angiotensin I and II paralleled that observed for in vitro contractile force development in bioassays utilizing left atrial tissue. In avian heart, AII also produced a dose-dependent accumulation of inositol-1-phosphate which was inhibited by the angiotensin antagonist [Sar 1, Ile 8]AII. The data demonstrate specific avian AII cardiac receptors and provide the first evidence that these receptors couple to both mechanical activity and phosphoinositide metabolism in avian heart.

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

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

Evidence for distinct phosphorylatable myosin light chains in avian heart and slow skeletal muscle

In mammalian organisms the regulatory or phosphorylatable myosin light chains in heart and slow skeletal muscle have been shown to be identical and presumably constitute the product of a single gene. We analyzed the expression of the avian cardiac myosin light chain (MLC) 2-A in heart and slow skeletal muscle by a combination of experimental approaches, e.g., two-dimensional gel electrophoresis of the protein and hybridization of mRNA to specific MLC 2-A sequences cloned from chicken. The investigations have indicated that, unlike in mammals, in avian organisms the phosphorylatable myosin light chains from heart and slow skeletal muscle are distinct proteins and therefore products of different genes. The expression of MLC 2-A is restricted to the myocardium and no evidence was found that it is shared with slow skeletal muscle.

Development of physiological responsiveness to glucagon during embryogenesis of avian heart

Glucagon increases contractility of the heart muscle by stimulation of adenylate cyclase activity and elevation of cAMP. We have investigated the specific time of onset of glucagon sensitivity of heart muscle during development of the chick embryo. Using both isolated heart preparation and cultured cardiac cells, we have found that the contractile response to glucagon cannot be detected prior to Day 4 of development. Binding studies, carried out with heart cells prepared from 3-, 5-, 7-, and 11-day chick embryos, showed a significant increase in the number of glucagon binding sites between Days 3 and 5. Scatchard analysis showed that for Day 5 cells maximum binding capacity was 0.56 pmole/mg of protein with K d of 16.0 n M, while for Day 3, maximal binding was only 0.16 pmole/mg with K d of 15.1 n M. Therefore in this 2-day interval there was a marked increase in the receptor number, without changes in the receptor affinity. Since hormonal stimulation of adenylate cyclase depends on the presence of the regulatory component ( N s), we have used cholera toxin-induced chronotropic effect as an assay for functional N s. No response to cholera toxin could be detected prior to Day 4 of chick heart development. Therefore, emergence of the cholera toxin sensitivity correlates well with the onset of responsiveness to glucagon. We conclude that as the heart develops it acquires a physiological responsiveness to glucagon. The acquisition of the hormonal sensitivity correlates with the increase in the receptor number and the functional levels of regulatory component.

Anatomy of the crista supraventricularis: Its importance for understanding right ventricular function, right ventricular infarction and related conditions

During careful studies of the human cardiac conduction system the anatomy of the crista supraventricularis is an inescapable concomitant demonstration. For the purpose of this report the observations from about 1,000 human hearts were combined with special additional studies of 75 human hearts (50 adults, 25 infants), 30 dogs and 5 chickens. The crista supraventricularis is similar in human and canine hearts. Avian hearts differ from mammal hearts in that they contain only a muscular right atrioventricular valve which replaces the crista supraventricularis. In addition to dividing the inflow and outflow pathways of the right ventricle, the crista supraventricularis is crucially located to join the interventricular septum and left ventricle to much of the right ventricular free wall, thereby playing an important role in emptying the right ventricle and closing the tricuspid valve. On the basis of these observations, the function of the crista supraventricularis is examined relative to right ventricular systole, right ventricular infarction, various electrophysiologic problems, the performance of cardiac surgery and new questions in cardiac imaging.

The atrioventricular conducting system of the avian heart.

The atrioventricular (AV) conducting system of the heart was histologically investigated in 14 species in 9 orders of aves. The main part of the AV conducting system in aves was fundamentally similar to that in mammals, but the avian AV bundle was longer than the mammalian one and its left and right limbs had no branches crossing the ventricular cavities. In the avian heart, the accessory part of the AV conducting system was well developed. It consisted of the right AV Purkinje ring, which originated from the AV node, and the recurrent branch, which emanated from thd AV bundle. In general, the former encircled the right AV orifice and the latter encircled the aortic root, together forming an 8-shaped loop. However, the degree of development of the accessory part varied among avian species, hence allowing six types of the accessory part to be distinguished.

Block of avian cardiac fast sodium channels by tetrodotoxin is enhanced by repetitive depolarization but not by steady depolarization

The blockade of Na+ channels by tetrodotoxin (TTX) was studied in the avian heart with the maximum rate of rise (Vmax) of phase 0 of the action potential used as an indicator of Na+ conductance (gNa). Inhibition by TTX of Vmax occurred at lower concentrations (IC50 congruent to 20 nM) than those reported in mammalian hearts (IC50, 1 to 10 microM). The IC50 was not affected by K+-induced membrane depolarization. Inhibition of closed Na+ channels by TTX was demonstrated and the degree of inhibition was increased by repetitive excitation. The time constant for recovery (tau Rec) from inactivation of Vmax was increased by TTX, a result consistent with the ability of the toxin to trap Na+ channels in the inactivated state. Reduction of the external Na+ concentration [( Na+]0 by 50% reduced the IC50 5.3-fold. This shift can largely be accounted for by the non-linear relationship between Vmax and gNa, that is, there need not be an important effect of [Na+]0 on toxin binding to its receptor. The interaction between TTX and its receptor in the avian heart is about as sensitive as that observed in peripheral nerve. However, like its less-sensitive mammalian heart counterpart, the TTX-Na+ channel interaction is frequency-dependent and apparently little influenced by membrane voltage or [Na+]0.

The valva atrioventricularis dextra of the avian heart.

AbstractIn this descriptive acount the author corrects an error in the literature about the structure of the right atroventricular valve in the avian heart.

Carbonic anhydrase in developing avian heart, blood and chorioallantoic membrane

1. 1. Carbonic anhydrase has been detected in cardiac tissue, blood, and chorioallantoic membrane of embryonic and hatching chicks by biochemical assay and autoradiography. 2. 2. Enzyme activity in heart was low (0.14 to 0.29 units/mg protein) during embryonic development and peaked (0.85 units/mg protein) near the time of hatching. The role of carbonic anhydrase in chick heart is postulated to be facilitation of diffusion of metabolic CO 2 out of the tissue with consequent prevention of acidosis. 3. 3. Carbonic anhydrase was detectable in blood at around day 10 of embryonic development and increased rapidly from day 14 until hatching, an event which parallels rising plasma pCO 2. At hatching, blood levels were about half the adult level of 6.8 units/mg protein. 4. 4. In chorioallantoic membrane, carbonic anhydrase also appeared around the 10th day of development, increasing to 1.1 units/mg protein by day 20. Carbonic anhydrase in this tissue is thought to be associated with respiration and calcium release from the shell. 5. 5. Comparison of the activity profiles suggest different signals for enzyme synthesis in the different tissues.