Embryonic Chick Heart Research Articles

Visualization of sulfated glycoconjugates in the chicken embryonic heart by whole-mount alcian blue staining.

In the atrioventricular canal (AVC) and outflow tract (OT) of the developing heart, endothelial cells transform specifically to mesenchymal cells. The mesenchymal cells migrate into the underlying acellular matrix termed cardiac jelly and form endocardial cushion tissue. It is believed the that the highly hydrated nature of cardiac jelly is ascribed to sulfated glycoconjugates in the components of jelly matrix. In the present study, we have visualized the distribution and its temporal changes of sulfated glycoconjugates in the embryonic heart from stage 12 to 26 using whole mount alcian blue (AB) histochemistry. Atrial matrix was AB-negative in all the stages examined. Cardiac jelly in the AVC and OT were positive and the staining intensity increased as heart development proceeded, while AB-positive staining in the matrix of the ventricle became negative by stage 19. At stages later than 19, AB-positive matrix was localized in only the AVC and OT where endothelially-derived mesenchymal cells populated.

P-607 - Effects of Ca channel Blockers Under the Different Light Condition in Chick-embryonic Heart

P-607 - Effects of Ca channel Blockers Under the Different Light Condition in Chick-embryonic Heart

Mitochondrial gene expression is impaired by ethanol exposure in cultured chick cardiac myocytes.

A depression in cytochrome c oxidase (COX) activity occurs following chronic embryonic ethanol exposure in vivo. The aim of this study was to examine the effect of chronic ethanol exposure on COX activity in isolated cardiac cells maintained in vitro. Additionally, the mechanism by which ethanol produces an impairment in COX activity was evaluated by examining mitochondrial gene expression. Spontaneously beating cardiac myocyte cultures were established from 10-day embryonic chick hearts. Various concentrations of ethanol (0-250 mM) were introduced at the time of plating and cells were harvested over 7 days. COX activity was determined in myocyte homogenates. The levels of nuclear-encoded (COXIV) and mitochondrial-encoded (COXII) subunit proteins were measured by Western blotting. Relative levels of mitochondrial DNA and the mitochondrially-encoded COXIII mRNA were determined by Southern and Northern blotting. A consistent decrease in COX activity in ethanol-exposed cardiac myocytes of approximately 30% was observed with an ethanol concentration of 25 mM. Increasing the ethanol concentration to 250 mM produced only a minor enhancement of this effect, while severely decreasing cellular viability. The content of the mitochondrially-encoded COXII subunit was reduced by ethanol exposure, while that of the nuclear-encoded COXIV subunit was unchanged. The content of the mitochondrially-encoded COXIII mRNA was unchanged by ethanol exposure. However, prolonged ethanol exposure produced an increase in mitochondrial DNA levels in cardiac myocytes. Ethanol exposure of cardiac myocytes produces deficits in COX activity in the absence of systemic variables, indicating that ethanol has a direct effect on cardiac mitochondria. The ethanol-induced decrease in COX activity is correlated with a specific decrease in at least one mitochondrially-encoded gene product, COXII. No changes were observed in the level of the nuclear-encoded COXIV subunit, indicating that expression of this nuclear-encoded gene is not impaired by ethanol exposure.

Taurine indirectly increases [Ca] i by inducing Ca 2+ influx through the Na + -Ca 2+ exchanger

Recent studies in heart cells have shown taurine to induce a sustained increase of both intracellular Ca2+ and Na+. These results led us to believe that the increase in Na+ by taurine could be due to Na+ entry through the taurine-Na+ cotransporter which in turn favours transarcolemmal Ca2+ influx through Na+-Ca2+ exchange. Therefore, we investigated the effect of β-alanine, a blocker of the taurine-Na+ cotransporter and low concentrations of CBDMB (a pyrazine derivative, 5-(N-4chlorobenzyl)-2′,4′-dimethylbenzamil), a Na+-Ca2+ exchanger blocker on taurine-induced [Ca]i increase in embryonic chick heart cells. Using Fura-2 Ca2+ imaging and Fluo-3 Ca2+ confocal microscopy techniques, taurine (20 mM) as expected, induced a sustained increase in [Ca]i at both the cytosolic and the nuclear levels. Preexposure to 500 μM of the blocker of the taurine-Na+ cotransporter, β-alanine, prevented the amino acid-induced increase of total [Ca]i. On the other hand, application of β-alanine did not reverse the action of taurine on total [Ca]i. However, low concentrations of the Na+-Ca2+ exchanger blocker, CBDMB, reversed the taurine-induced sustained increase of cytosolic and nuclear free calcium (in presence or absence of β-alanine). Thus, the effect of taurine on [Ca]i in heart cells appears to be due to Na+ entry through the taurine-Na+ cotransporter which in turn favours transarcolemmal Ca2+ influx through the Na+-Ca2+ exchanger.

Induction of the cholesterol metabolic pathway regulates the farnesylation of RAS in embryonic chick heart cells: a new role for ras in regulating the expression of muscarinic receptors and G proteins.

We propose a novel mechanism for the regulation of the processing of Ras and demonstrate a new function for Ras in regulating the expression of cardiac autonomic receptors and their associated G proteins. We have demonstrated previously that induction of endogenous cholesterol synthesis in cultured cardiac myocytes resulted in a coordinated increase in expression of muscarinic receptors, the G protein alpha-subunit, G-alphai2, and the inward rectifying K+ channel, GIRK1. These changes in gene expression were associated with a marked increase in the response of heart cells to parasympathetic stimulation. In this study, we demonstrate that the induction of the cholesterol metabolic pathway regulates Ras processing and that Ras regulates expression of G-alphai2. We show that in primary cultured myocytes most of the RAS is localized to the cytoplasm in an unfarnesylated form. Induction of the cholesterol metabolic pathway results in increased farnesylation and membrane association of RAS. Studies of Ras mutants expressed in cultured heart cells demonstrate that activation of Ras by induction of the cholesterol metabolic pathway results in increased expression of G-alphai2 mRNA. Hence farnesylation of Ras is a regulatable process that plays a novel role in the control of second messenger pathways.

Excitation-contraction coupling in the day 15 embryonic chick heart with persistent truncus arteriosus.

Ca2+ transients were examined in embryonic chick hearts with an experimentally induced cardiac neural crest-related outflow tract defect known as persistent truncus arteriosus (PTA). In all of the animal models of neural crest-related heart defects, prenatal mortality is too high to be attributed to structural defects of the heart alone, suggesting that there is altered development of the myocardium. Earlier reports indicating reduced L-type Ca2+ current in hearts with PTA suggest that poor viability may be related to impairment of cardiac excitation-contraction coupling. To test this hypothesis, direct measurements of the systolic Ca2+ transient in fura-2-loaded myocytes from normal hearts and hearts with PTA were carried out. We found that Ca2+ transients were severely depressed in hearts with PTA and difficult to measure above background noise unless signal averaged or treated with isoproterenol (ISO). We confirmed that the reduced Ca2+ transients were due, at least partly, to a reduction in L-type Ca2+ current. In addition we found that although ISO could raise the L-type current in hearts with PTA to the level found in normal hearts in the absence of ISO, it could not fully restore the Ca2+ transient. Furthermore, caffeine-stimulated Ca2+ transients were diminished in size and the time-to-peak and the decaying phase were significantly slowed. Interestingly, these observations were not accompanied by a reduction in the number of Ca2+ release channels. These results indicated an impairment of SR function in addition to the reduction in L-type Ca2+ current. These results strongly support our hypothesis that the poor viability of embryos with PTA is due to impaired cardiac excitation-contraction coupling.

Production of a monoclonal antibody by in vitro immunization that recognizes a native chondroitin sulfate epitope in the embryonic chick limb and heart.

We report the production of a monoclonal antibody (d1C4) by in vitro immunization that has immunoreactivity with a native chondroitin sulfate epitope in embryonic chick limb and heart. Murine lymphocytes were stimulated by direct exposure to unfixed, unsolubilized precartilage mesenchymal aggregates in high-density micromass culture derived from Stage 22-23 chick limb buds. Specificity of d1C4 reactivity was demonstrated by sensitivity of immunohistochemical staining to pretreatment with chondroitinase ABC or AC, preferential immunoreactivity with chondroitin-6-sulfate glycosaminoglycan (CS-C GAG) in ELISA, and competition of immunohistochemical staining with CS-C GAG. Immunohistochemical analysis of the expression of the d1C4 epitope revealed a striking localization of immunoreactivity in the extracellular matrix (ECM) of precartilage aggregates of chick limb mesenchyme in high-density micromass culture by 16 hr and the prechondrogenic limb core at Stage 23 in vivo. Immunoreactivity in both cultured limb mesenchyme and the embryonic limb continued through differentiation of prechondrogenic condensations into cartilage tissue. In the developing chick heart, d1C4 staining was found throughout the ECM of atrioventricular cushion tissue by Stage 25, but was localized to mesenchyme adjacent to the myocardium in the outflow tract cushions. There was an abrupt demarcation between d1C4-reactive intracardiac mesenchyme and unreactive extracardiac mesenchyme of the dorsal mesocardium in the Stage 22 embryo. This study demonstrates the efficacy of in vitro immunization of lymphocytes for the production of MAbs to native ECM constituents, such as CS-GAGs. Immunohistochemical data utilizing d1C4 suggest that CS-GAGs bearing this epitope may be important in early morphogenetic events leading to cartilage differentiation in the limb and valvuloseptal morphogenesis in the heart.

Angiotensin II-Induced Inositol Phosphate Generation is Mediated Through Tyrosine Kinase Pathways in Cardiomyocytes

The objective of this study was to determine whether the G-protein-linked angiotensin II receptor mediated inositol phosphate production involves a tyrosine phosphorylation (tyr phos) dependent pathway in the heart. Cardiomyocytes, in culture, from 7-day-old chick embryonic hearts were incubated with myo [ 3H] inositol for 18–24 h. Cells were incubated with LiCl to inhibit inositol 1-phosphate phosphatase and allow accumulation of inositol phosphates with angiotensin II (ang II) treatment. Inositol fractions were separated on column chromatography. Ang II produced significant ( p < 0.01) increases of InsP 1, InsP 2, and InsP 3, within 1 min of treatment of cardiomyocytes. Tyrosine kinase inhibition with genistein significantly ( p < 0.05) reduced ang II induced inositol phosphate production. This did not occur with the analogue diazdien that is a very weak inhibitor of tyrosine kinase. The ability of ang II to induce tyr phos was demonstrated in whole cell lysates of cardiomyocytes immunoprecipitation with anti-P-Tyr antibodies. Genistein blunted this action of ang II. The rapid activation of a tyr phos dependent pathway by ang II was demonstrated by the similar time course of tyr phos of two different cardiac proteins, 70 and 195 kDa, and peak inositol phosphate production. Tyr phos of these cardiac proteins was mediated predominantly but not exclusively through the AT 1 ang II receptor subtype as it was completely blocked by the AT 1 antagonist losartan, while the AT 2 receptor antagonist PD123319 blunted ang II-induced tyr phos. These results demonstrate a novel role for a tyr phos dependent pathway in the heart for ang II-induced inositol phosphate production and strengthens the concept of the interaction of G-protein coupled receptors with tyrosine kinases.

Passive stress-strain measurements in the stage-16 and stage-18 embryonic chick heart.

The first stress-strain measurements on embryonic cardiovascular tissue are described here, obtained from cyclic uniaxial loading of the primitive ventricle. An excised ventricular segment from Hamburger/Hamilton stage-16 or stage-18 chicks (2-1/2 and 3 days of a 21-day incubation period) was mounted longitudinally between two small wires in oxygenated Krebs-Henseleit cardioplegia solution. One wire was attached to an ultrasensitive force transducer and the other to a Huxley micromanipulator controlled by remote motor drive. A real-time video tracking system calculated three myocardial surface strains based on the positions of three surface markers while the heart was deformed in a triangular wave pattern. Force transducer output was filtered, digitally sampled, and stored with strains and time. Results were plotted as strain (longitudinal, circumferential, shear, and principal) versus time, stress versus time, and stress versus longitudinal strain. The stress-strain curves were nonlinear, even at low strain levels. The hysteresis loops were large; mean hysteresis energy as a proportion of total cycle stored strain energy was 36 percent (stage 16) and 41 percent (stage 18). We created a finite element model of the ventricle and fit the model behavior to the experimental behavior to determine parameters for a stage-18 pseudoelastic strain-energy function of exponential form. The calculated exponential parameter is significantly lower than that found in corresponding uniaxial studies of mature myocardium, possibly indicating the lower fiber content of the immature tissue. The results of this study are the first step in characterizing material properties for comparisons with later developmental stages and with impaired and altered myocardium. The long-term goal is to aid in identifying the biomechanical factors regulating growth and morphogenesis.

Changing activation sequence in the embryonic chick heart. Implications for the development of the His-Purkinje system.

In the mature heart, impulse propagation through the His-Purkinje system (HPS) is required for efficient ventricular contraction in an apex-to-base direction. However, the embryonic heart begins to contract as a myocardial tube without a specialized conduction system. To identify the developmental stage when the HPS begins to function, we mapped the ventricular depolarization sequence from microvolt-level electrograms recorded from embryonic myocardium using 50-micron extracellular electrodes, high-gain amplification, and signal-processing techniques. Analysis of left ventricular activation in 99 embryonic hearts revealed a transition in the activation sequence that was dependent on developmental stage. As the heart develops, a transition in the activation sequence occurred from the primitive base-to-apex pattern (in 20 of 33 hearts) at early stages (Hamburger-Hamilton stages 25 to 28) to the HPS-like apex-to-base pattern (12 of 17 hearts) late in development (stages 33 to 36). Immunohistological experiments (n = 10) also confirm that the expression pattern of two biochemical HPS markers changes in parallel with the change to the mature ventricular activation pattern. These data indicate that the ventricular activation sequence in the chick heart develops to a mature pattern at stages 29 to 31, suggesting that preferential conduction through the HPS begins shortly after ventricular septation is complete.

Effects of Heparin on the Electrophysiological and Mechanical Properties of Early Embryonic Chick Hearts

The effect of heparin (1–200μg/ml) on several functional properties of 4-day-old embryonic hearts was studied. Intracellular recordings either from cells of the atrium, the atrio–ventricular canal or the ventricle were made on the isolated and perfused hearts. In all impaled myocardial cells, heparin produced an increment in the maximal rate of rise and amplitude of the action potentials and the duration was slightly diminished. In addition, heparin provoked a reduction in the spontaneous beating frequency, while the electrical excitability and the conduction velocity of the atrium and the ventricle were increased. The measurements of atrio–ventricular latency revealed that it was diminished, i.e. the propagation of impulses through the atrio–ventricular canal was improved. Simultaneously with these changes, heparin incremented the amplitude of contraction of either the atrium or the ventricle importantly.

Angiotensin II induces activation of phosphatidylinositol 3-kinase in cardiomyocytes.

Phosphatidylinositol 3-kinase phosphorylates membrane lipids at the third position of the inositol ring producing phosphoinositides, not on the pathway for production of 1,4,5-triphosphate. To test the hypotheses that angiotensin II (Ang II) activates phosphatidylinositol 3-kinase in cardiomyocytes and that this pathway is involved in Ang II-induced protein synthesis. Cardiomyocytes, in culture, from 7-day-old chick embryonic hearts were treated with Ang II and the activation of phosphatidylinositol 3-kinase was assessed after immunoprecipitation with antibodies to the p85 subunit of phosphatidylinositol 3-kinase by the conversion of PI (phosphatidylinositol) to phosphatidylinositol 3-monophosphate (PIP) in the presence of gamma-[32P]-ATP and analyzed by thin-layer chromatography. Western blotting was performed after antiphosphotyrosine immunoprecipitation with antibodies to the p85 subunit of phosphatidylinositol 3-kinase. Protein synthesis was assessed by [35S]-methionine incorporation and polyacrylamide gel electrophoresis. Ang II stimulated phosphatidylinositol 3-kinase activity dramatically, with 4.5- and 3.5-fold increases in PIP formation after 1 and 5 min, respectively. The involvement of tyrosine kinases was demonstrated by Western blotting in which Ang II increased tyrosine phosphorylation of a protein recognized by antibodies to the 85 kDa subunit of phosphatidylinositol 3-kinase. Furthermore, the tyrosine kinase inhibitor lavendustin A blocked Ang II-stimulated phosphatidylinositol 3-kinase activity and conversion of phosphatidylinositol to PIP. Ang II increased new protein synthesis as reflected by the significantly (P < 0.05) greater incorporation of [35S]-methionine into cardiomyocytes treated with Ang II. The link between Ang II and protein synthesis was mediated in part through phosphatidylinositol 3-kinase because the phosphatidylinositol 3-kinase inhibitor wortmannin blocked the effect of Ang II on protein synthesis. Increased production both of nuclear and of cytosolic proteins was demonstrated by agarose gel electrophoresis of these cellular components of Ang II-treated cardiomyocytes. Wortmannin produced a general inhibition of the synthesis of nuclear and cytosolic proteins, with a greater effect on nuclear proteins. The action of wortmannin on nuclear protein synthesis was confirmed by similar findings with another phosphatidylinositol 3-kinase inhibitor, LY294002. Phosphatidylinositol 3-kinase activation by Ang II occurs through a pathway utilizing tyrosine phosphorylation. Furthermore, this pathway is involved in cardiomyocyte protein synthesis and the possibility that it is operative in Ang II-mediated cardiac hypertrophy arises.

Regulation of expression and function of m2 and m4 muscarinic receptors in cultured embryonic chick heart cells by transforming growth factor-β 1

Incubation of cultured embryonic chicken heart cells with transforming growth factor β 1 (TGF-β 1) resulted in a concentration- and time-dependent decrease in the number of muscarinic acetylcholine receptors (mAChR), which reached a maximum by 24 hr. Twenty-four hours following TGF-β 1 treatment, cm2 and cm4 mAChR protein levels were decreased 59 and 41%, respectively, and cm2 mRNA and cm4 mRNA levels were both decreased by approximately 40%. Chick heart cells treated wich TGF-β 1 exhibited a decreased sensitivity for carbachol-mediated inhibition of adenylyl cyclase activity compared with control cells. Thus, TGF-β 1 stimulation of chick heart cells results in decreases in the expression of both cm2 and cm4 mAChR and in muscarinic responsiveness.

Anti-invasive activity of alkaloids and polyphenolics in vitro

Invasiveness, the ability of certain tumour cells to migrate beyond their natural tissue boundaries, often leads to metastasis, and usually determines the fatal outcome of cancer. The need for anti-invasive agents has led us to search for possibly active compounds among alkaloids and polyphenolics. One hundred compounds were screened in an assay based on the confrontation of invasive human MCF-7/6 mammary carcinoma cells with fragments of normal embryonic chick heart in vitro. Anti-invasive activity was frequently found among chalcones having a prenyl group. Six compounds were found to inhibit invasion when added to the culture medium at concentrations as low as 1 μM. For at least three of them the anti-invasive effect could be associated with a cytotoxic effect on the MCF-7/6 cells, but not on the heart tissue. This selective cytotoxicity was substantiated by different methods, such as histology and growth assays (volume measurements, cell counts, MTT and sulforhodamine B assays). The anti-invasive effects of the compounds could neither be ascribed to induction of apoptosis nor to the promotion of cell-cell adhesion. Our data indicate that among the alkaloids and polyphenolics a number of molecules can inhibit growth and invasion of human mammary cancer cells via selective cytotoxicity.

Bursting behavior during fixed-delay stimulation of spontaneously beating chick heart cell aggregates.

Spontaneously beating embryonic chick atrial heart cell aggregates were stimulated with depolarizing current pulses delivered at a fixed delay after each action potential. This preparation is an experimental model of a reentrant tachycardia. During fixed-delay stimulation, bursting behavior was typically observed for a wide range of delays. Episodes of bursting at a rate faster (slower) than control were followed by overdrive suppression (underdrive acceleration). We use a simple nonlinear model, based on the interaction between excitability and overdrive suppression, to describe these dynamics. A modified version of the Shrier-Clay ionic model of electrical activity of the embryonic chick heart cell aggregates that includes a simplified Na+ pump term is also presented. We show that the complex patterns during fixed-delay stimulation arise as a result of delicate interactions between overdrive suppression and phase resetting, which can be described in terms of the underlying ionic mechanisms. This study may provide a basis for understanding incessant tachycardias in the intact heart, as well as an alternative mechanism for the emergence of bursting activity in other biologic tissue.

Temporal and spatial patterns of phosphotyrosine immunolocalization during cardiac myofibrillogenesis of the chicken embryo.

To investigate the possible role of phosphorylation of protein tyrosine during myofibrillogenesis (6- to 13-somite stages) of the chicken embryonic heart tube, immunolocalization of phosphotyrosine (P-Tyr) and the relationship between P-Tyr and developing myofibrils were studied by means of confocal scanning laser microscopy and immuno-electron microscopy. The staining pattern of P-Tyr varied in different sites of myocytes at different stages of embryonic development: At the cell-cell boundaries, P-Tyr was localized at the adhesion belt of outer myocardial layer cells (6- to 13-somite stages), non-junctional cell-cell contacts (6- to 13-somite stages) and early intercalated disks of both the outer and inner myocardial layer cells (8- to 13-somite stages). At the cell-extracellular matrix boundaries of inner layer cells, the first stages of myofibril formation appeared as serially aligned areas of P-Tyr localization closely associated with circumferentially aligned thick actin bundles (8- to 9-somite stages). This P-Tyr immunostaining decreased when the thick actin bundles developed into mature striated myofibrils at the 10- to 13-somite stages. These findings suggest that the phosphorylation of protein tyrosine residues is primarily concentrated at the modulating cell-cell and cell-matrix adhesion sites of developing myocytes and myofibrils.

Expression of the cholinergic signal-transduction pathway components during embryonic rat heart development.

Previous studies showed that acetylcholinesterase (AChE) activity is present in the downstream (arterial) part of the embryonic chick and rat heart, but its functional significance was unclear. To establish whether other components of a cholinergic signal-transduction pathway are present in the embryonic heart, we localised the mRNAs encoding choline acetyltransferase (ChAT), acetylcholinesterase (AChE), and the muscarinic receptor isoforms (mAChRs; m1-m5). Messenger RNA detection and localisation by in situ hybridisation and reverse transcriptase-polymerase chain reaction were employed. Expression of ChAT and AChE mRNAs was observed from 15 embryonic days onward in the neural tissue covering the dorsocranial wall of the atria. Muscarinic receptors (m1, m2, m4) were observed at the same localisation as AChE and ChAT mRNAs, both during embryogenesis and after birth. In addition, m1 and m4 mAChRs showed a low level of expression in the atrial myocardium during the fetal period. No expression of the m3 or the m5 mAChRs was observed in or near the embryonic hearts. ChAT, AChE, and mAChRs (m1, m2, m4) mRNAs always colocalised in the cardiac ganglia. However, none of these mRNAs was found at a detectable level in the outflow tract and/or the ventricular trabeculations. The AChE activity in the arterial part of the embryonic heart is probably synthesised elsewhere and subserves a function different from the hydrolysis of locally produced acetylcholine.

Premyofibrils in spreading adult cardiomyocytes in tissue culture: evidence for reexpression of the embryonic program for myofibrillogenesis in adult cells.

Do adult cardiomyocytes use the same pathways hypothesized for the formation of myofibrils in embryonic cardiomyocytes in tissue culture. [Rhee, et al., Cell Motil. Cytoskeleton 28:1-24, 1994]? Premyofibrils in embryonic cardiomyocytes are composed of short sarcomeric units of alpha-actinin (Z-bodies) and actin filaments held together by short nonmuscle myosin IIB filaments. Premyofibrils are believed to be transformed into nascent myofibrils by their capture of muscle-specific myosin II filaments aligned in aperiodic arrays. Nascent myofibrils are thought to transform into mature myofibrils by the loss of nonmuscle myosin IIB, the fusion of the Z-bodies into Z-bands, and the periodic alignment of muscle myosin II filaments into A-bands. Freshly isolated cat and rat adult cardiomyocytes placed in tissue culture lack premyofibrils and nascent myofibrils. Adult cardiomyocytes spreading in culture reinitiate the synthesis of nonmuscle myosin IIB. Moreover, patterns similar to the proposed embryonic myofibrillar program first detected in spreading chick embryonic hearts were also detected in these spreading adult mammalian cardiomyocytes. The isolated adult cardiomyocytes begin to spread after 1 day in culture by sending out lamellipodia. When these cells are injected with fluorescently labeled alpha-actinin, linear arrays of short spacings of beaded alpha-actinin bodies are detected in the spreading edges of the adult cardiomyocytes. These dense bodies (Z-bodies) stain positively for the same sarcomeric-specific isoform of alpha-actinin that is in the Z-bands of mature sarcomeres. These linear arrays of alpha-actinin-containing Z-bodies have other characteristics of premyofibrils and are detected only in the spreading regions of the cells. Thus, these premyofibrils at the edges of the spreading adult cardiomyocytes stain positively for nonmuscle myosin IIB but negatively for muscle-specific myosin II. Initially, no vinculin is associated with any parts of the premyofibrils in the spreading regions of the early spreading cardiomyocytes. However, later, vinculin is found to be associated with the ends of the premyofibrils. Fibers that stain solidly for muscle-specific myosin II (i.e., nascent myofibrils) are localized between the peripheral premyofibrils and the centrally positioned, mature myofibrils. It is suggested that the puzzling ability of cardiomyocytes in hypertrophic hearts to reinitiate the synthesis of fetal sarcomeric proteins may be related to the reinitiation of the embryonic premyofibril program for myofibrillogenesis.

The Challenge of Complex Dynamics for Theoretical Models of Cardiac Activity

Electrical stimulation of excitable cardiac tissue results in a variety of rhythms similar to those clinically recorded. Simplified momlinear equations and ionic models give complementary descriptions of the dynamics in cardiac preparations. Nonlinear midels often capture the essential dynamical features of the experimental observations but do not properly describe the underlying ionic mechanisms. Ionic midels are descriptive, complex, and despite their general sucess in reproducing the cardiac action potential, seldom undergo extensive testing against experimentally determined rhythms. We review work from our group devoted to the modeling of cardiac rhythms, including nonlinear models and the Shrier‐Clay ionic model of electrical activity in embryonic chick heart cell aggregates. Since sustained stimulation may have long lasting consequences on the intrinsic excitability of the tissue, we also discuss recent advances in modeling the stimulation history‐dependent aspects of pacemaking.

Evidence that fibroblast growth factors 1 and 4 participate in regulation of cardiogenesis.

Previous studies in this laboratory have indicated that the early embryonic chick heart depends on fibroblast growth factor-2 (FGF-2; bFGF), sequentially utilized in paracrine and autocrine fashion, for its growth and development (Sugi and Lough, [1995] Dev. Biol. 168-567-574). This view emanated from immunohistochemical detection of FGF-like antigens in endoderm cells at stage 6, and later in the early myocardium at stage 9+ (Parlow et al. [1991] Dev. Biol. 146:139-147). To identify other members of the FGF family that are expressed by these cells, we have used peptide-generated antisera that specifically recognize FGFs 1 and 4. Like FGF-2, FGFs 1 and 4 were exclusively detected in the endoderm at stage 5+ and later in the myocardium, appearing as punctate cytoplasmic deposits. However, whereas FGF-2 is first detected at stage 9+, FGFs 1 and 4 did not appear until stages 11 and 15, respectively. Expression of all FGFs peaked at stages 18-24, decreasing thereafter in parallel with reduced myocardial cell proliferation. To determine these isoproteins' ability to facilitate the completion of terminal cardiac myocyte differentiation, stage 5+ precardiac mesoderm was cultured in defined medium with purified FGFs. Like FGF-2, as little as 5-10 ng/ml FGF-1 or FGF-4 supported the proliferation and differentiation of precardiac myoblasts, resulting in the formation of a vesicle containing an adherent multilayer of synchronously contractile cells. Evidence that this represented FGF receptor-mediated signaling rather than a nonspecific effect of exogenous FGF was indicated by the ability of sodium chlorate to inhibit FGF-mediated cardiogenesis. These findings are consistent with the hypothesis that, like FGF-2, FGFs 1 and 4 participate in the regulation of early heart development via paracrine and autocrine mechanisms.