Immunolocalization of basic fibroblast growth factor during chicken cardiac development

Basic fibroblast growth factor (bFGF) has been identified in cultured cardiac myocytes as well as in myocardial tissue of both embryonic and adult organisms; bFGF has also been demonstrated to regulate proliferation and differentiation of these cells in culture. Such studies suggest a possible role for bFGF in cardiac myogenesis. In vitro studies using cultured endothelial and neuronal cells also suggest that myocyte-derived bFGF may be involved in the regulation of vascularization and/or innervation of the developing heart. We have generated a spatial and temporal map for bFGF in the developing chick heart using immunohistochemical techniques and our monospecific polyclonal rabbit antihuman bFGF IgG. A progressive decrease in bFGF expression was seen in the highly trabeculated region of the ventricular myocardium, relative to the myocardium directly underlying the epicardial tissue, with increasing developmental age. bFGF expression was limited to the cytoplasm of cardiac myocytes; neither vascular endothelium nor smooth muscle contained anti-bFGF immunoreactive material. A correlation between the temporal and spatial pattern of bFGF expression seen here, with the pattern of myocyte proliferation and differentiation reported by others, suggests a role for bFGF in the autocrine regulation of myocyte proliferation and differentiation.     

Loading effect of fibroblast-myocyte coupling on resting potential, impulse propagation, and repolarization: insights from a microstructure model

The numerous nonmyocytes present within the myocardium may establish electrical connections with myocytes through gap junctions, formed naturally or as a result of a cell therapy. The strength of the coupling and its potential impact on action potential characteristics and conduction are not well understood. This study used computer simulation to investigate the load-induced electrophysiological consequences of the coupling of myocytes with fibroblasts, where the fibroblast resting potential, density, distribution, and coupling strength were varied. Conduction velocity (CV), upstroke velocity, and action potential duration (APD) were analyzed for longitudinal and transverse impulse propagation in a two-dimensional microstructure tissue model, developed to represent a monolayer culture of cardiac cells covered by a layer of fibroblasts. The results show that 1) at weak coupling (<0.25 nS), the myocyte resting potential was elevated, leading to CV up to 5% faster than control; 2) at intermediate coupling, the myocyte resting potential elevation saturated, whereas the current flowing from the myocyte to the fibroblast progressively slowed down both CV and upstroke velocity; 3) at strong couplings (>8 nS), all of the effects saturated; and 4) APD at 90% repolarization was usually prolonged by 0-20 ms (up to 60-80 ms for high fibroblast density and coupling) by the coupling to fibroblasts. The changes in APD depended on the fibroblast resting potential. This complex, coupling-dependent interaction of fibroblast and myocytes also has relevance to the integration of other nonmyocytes in the heart, such as those used in cellular therapies.     

SARCOPLASMIC RETICULUM AND T TUBULES IN DIFFERENTIATING RAT SKELETAL MUSCLE

An electron microscope study has been made of the distribution of membrane couplings between the sarcoplasmic reticulum (SR) and either the plasmalemma or the T tubules in fetal and neonatal rat intercostal muscle. Within primitive muscle cells at 12 days of gestation, the SR forms both simple and specialized membrane junctions with the plasmalemma; caveolae are very few, and T tubules are not detected. Undifferentiated cells neighbor muscle cells. Occasionally these cells contain subsurface couplings between the endoplasmic reticulum and plasmalemmae. Possible relationships between these couplings and the peripheral couplings of muscle cells are discussed. By 15–18 days of gestation, caveolae and beaded T tubules, comparable to those of cultured muscle, develop; T tubules lie along-side myofibrils and are rarely transverse. SR couples both to T tubules and to plasmalemmae during this period. T tubules with lineal profiles appear after further development and their orientation transverse to A–I junctions becomes increasingly evident. Membrane couplings between SR and T tubules also increase in number, whereas the incidence of peripheral coupling declines rapidly Evidence suggests that peripheral couplings are swept into myotubes as caveolae proliferate and T tubules form. SR thus appears to initially couple with the plasmalemma and then to await T tubular growth. This contrasts with the developmental pattern described in cultured chick muscle in which peripheral couplings are not reported and T tubules with diads and triads occur at very primitive stages of muscle differentiation.     

Lymph heart in chick--somitic origin, development and embryonic oedema

The lymph heart is a sac-like structure on either side of avian tail. In some adult birds, it empties the lymph from the copulatory organ; however, during embryonic development, it is thought to circulate extra-embryonic lymph. Very little is known about the origin, innervation and the cellular changes it undergoes during development. Using immunohistochemistry and gene expression profiling we show that the musculature of the lymph heart is initially composed solely of striated skeletal muscle but later develops an additional layer composed of smooth myofibroblasts. Chick-quail fate-mapping demonstrates that the lymph heart originates from the hypaxial compartments of somites 34-41. The embryonic lymph heart is transiently innervated by somatic motoneurons with no autonomic input. In comparison to body muscles, the lymph heart has different sensitivity to neuromuscular junction blockers (sensitive only to decamethonium). Furthermore, its abundant bungarotoxin-positive acetylcholinesterase receptors are unique as they completely lack specific acetylcholinesterase activity. Several lines of evidence suggest that the lymph heart may possess an intrinsic pacing mechanism. Finally, we assessed the function of the lymph heart during embryogenesis and demonstrate that it is responsible for preventing embryonic oedema in birds, a role previously thought to be played by body skeletal muscle contractions.     

Sequential growth of fetal sheep cardiac myocytes in response to simultaneous arterial and venous hypertension

While the fetal heart grows by myocyte enlargement and proliferation, myocytes lose their capacity for proliferation in the perinatal period after terminal differentiation. The relationship between myocyte enlargement, proliferation, and terminal differentiation has not been studied under conditions of combined arterial and venous hypertension, as occurs in some clinical conditions. We hypothesize that fetal arterial and venous hypertension initially leads to cardiomyocyte proliferation, followed by myocyte enlargement. Two groups of fetal sheep received intravascular plasma infusions for 4 or 8 days (from 130 days gestation) to increase vascular pressures. Fetal hearts were arrested in diastole and dissociated. Myocyte size, terminal differentiation (%binucleation), and cell cycle activity (Ki-67[+] cells as a % of mononucleated myocytes) were measured. We found that chronic plasma infusion greatly increased venous and arterial pressures. Heart (but not body) weights were approximately 30% greater in hypertensive fetuses than controls. The incidence of cell cycle activity doubled in hypertensive fetuses compared with controls. After 4 days of hypertension, myocytes were (approximately 11%) longer, but only after 8 days were they wider (approximately 12%). After 8 days, %binucleation was approximately 50% greater in hypertensive fetuses. We observed two phases of cardiomyocyte growth and maturation in response to fetal arterial and venous hypertension. In the early phase, the incidence of cell cycle activity increased and myocytes elongated. In the later phase, the incidence of cell cycle activity remained elevated, %binucleation increased, and cross sections were greater. This study highlights unique fetal adaptations of the myocardium and the importance of experimental duration when interpreting fetal cardiac growth data.     

Enhancement by growth factors of cardiac myocyte differentiation from embryonic stem cells: a promising foundation for cardiac regeneration

Cell transplantation is a promising, still novel, potentially therapeutic approach for the treatment of heart diseases. Clinical applications require generation of large number of donor cells. Embryonic stem (ES) cells are capable of self-renewal apparently in an unlimited fashion, in vitro. Theoretically, they can differentiate into any cell type required for cell transplantation, including cardiac myocytes. Diverse growth factors have been implicated in programming diverse cellular processes, including development of the embryonic heart, ES cell self-renewal, and cardiac myocyte differentiation from ES cells. This review addresses the current understanding of the role of growth factors in the differentiation of cardiac myocytes from ES-embryoid body cell systems in vitro as well as cardiac regeneration in vivo.     

Adrenergic neuromuscular junctions in the cerebral arteries of birds

By means of the histochemical and electron microscopical methods, adrenergic innervation of the middle cerebral artery and its branches have been investigated in three sparrows (Passer montanus), blue rock pigeons (Columba livia), hens (Gallus domestica), hazel-grouses (Tetrastes bonasia), large billed crows (Corvus levaillantii), ducks (Anas domestica) and slaty backed gulls (Larus schistisagus). Neuromuscular connections in all the birds investigated have similar organizational features. Some adrenergic effectors are situated 700 nm from the external layer of myocytes in the vascular middle tunic, and they perform the distant transmission. Other effectors are at the distance of 40-80 nm from myocytes, and they are built according to the synaptic type. They produce certain action upon single muscle cells, thus performing local transmission. For the neuromuscular connections of the cerebral vessels in the birds, presence of multiaxonal complexes is specific. Axons of the complex innervate groups of muscle cells and they are at the distance of 50-270 nm from their plasmic membranes. Perhaps, they combine the local and distant means of transmission.     

T-tubes in cultured mammalian myocardial cells

Summary T-tubes are among the last structural elements of the mammalian myocyte to develop in vivo. We were able to identify T-tubes in early cultures of neonatal rat myocytes. Ventricles were excised from 3- to 4-day-old neonatal rats, incubated overnight in cold trypsin, and treated with sequential changes of collagenase-hyaluronidase. Fractions of cells isolated in this manner were pooled and cultured in plastic petri dishes. In cells prepared for transmission electron microscopy, T-tubes were observed at the cell periphery of cultured myocytes, but were more difficult to identify as the cultures aged and became overgrown by fibroblasts. T-tubes were identified by virtue of their continuity with the sarcolemma, their relatively large diameter, and their regular entry at the level of the Z line. Even at optimal culture ages, T-tubes were not present in every myocyte. At the times T-tubes could be located, myocytes were beating and had begun to establish intercalated discs and gap junctions. The de novo formation of T-tubes in cultured myocytes of neonatal rat heart reflects a duplication of in vivo differentiation by the cultured myocyte. The appropriateness of cultured myocytes in the study of the development and physiology of the heart is emphasized by the in vitro formation of T-tubes.Supported by research grants from the Muscular Dystrophy Association, Inc., The Schlieder Foundation, and USPH-Training Grant HL 07098-04. The authors are indebted to Philip Constantin for assistance in dissociating and culturing heart tissue.     

Ultrastructure of the chronically vagotomised atrial myocardium in the monkey (Macaca fascicularis)

The ultrastructure of the atrial myocardium in the monkey (Macaca fascicularis) was studied after bilateral cervical vagotomy and survival times of 100, 175 and 367 days. Changes were observed in the nucleus and the cytoplasm of the myocyte. Sequestered within the nuclei of the affected myocytes were cytoplasmic organelles and inclusions. In the late stages, there was a tendency towards condensation and margination of the heterochromatin. Changes in the cytoplasm included increased glycogen, mitochondrial degeneration and myofibrillar disorganisation and degeneration. There was increased collagen and mononuclear cell infiltration in the extracellular space in the later stages. This study has shown that the long term structural integrity of the atrial myocyte depends on an intact vagal innervation. The survival of the monkey after chronic bilateral vagotomy suggests that this nonhuman primate is a suitable model for functional studies of the parasympathetically denervated heart.     

Modulation of cell junctions during differentiation of the chicken otocyst sensory epithelium.

The differentiation of sensory and support cells within the embryonic chick otocyst is accompanied by alterations in the distribution of preexisting intercellular junctions. Prior to innervation of this epithelium, tight, gap and adhering junctions exist between all cells. Upon differentiation of the epithelium, apical bands of tight and adhering junctions are maintained throughout, while gap junctions and desmosomes are found only between support cells. Thus, some of the gap junctions that join homogeneous epithelial cells prior to innervation are removed as sensory cells differentiate, and a separate population of very large gap junctions is formed between differentiating support cells. Morphological evidence suggests two possible mechanisms which may be responsible for the observed changes in gap junctional distribution: removal of gap junctions by internalization, and formation of gap junctions by aggregation of precursor particles. The temporal correlation between junctional modulation, cytological differentiation of sensory and support cells, and ingrowth of nerve fibers makes the latter event a likely developmental cue for differentiation of this epithelium.     

Fine structure and differentiation of Ascidian muscle. I. Differentiated caudal musculature of Distaplia occidentalis tadpoles

The structure of the caudal muscle in the tadpole larva of the compound ascidian Distaplia occidentalis has been investigated with light and electron microscopy. The two muscle bands are composed of about 1500 flattened cells arranged in longitudinal rows between the epidermis and the notochord. The muscle cells are mononucleate and contain numerous mitochondria, a small Golgi apparatus, lysosomes, proteid-yolk inclusions, and large amounts of glycogen. The myofibrils and sarcoplasmic reticulum are confined to the peripheral sarcoplasm. Myofibrils are discrete along most of their length but branch near the tapered ends of the muscle cell, producing a Felderstruktur. The myofibrils originate and terminate at specialized intercellular junctional complexes. These myomuscular junctions are normal to the primary axes of the myofibrils and resemble the intercalated disks of vertebrate cardiac muscle. The myofibrils insert at the myomuscular junction near the level of a Z-line. Thin filaments (presumably actin) extend from the terminal Z-line and make contact with the sarcolemma. These thin filaments frequently appear to be continuous with filaments in the extracellular junctional space, but other evidence suggests that the extracellular filaments are not myofilaments. A T-system is absent, but numerous peripheral couplings between the sarcolemma and cisternae of the sarcoplasmic reticulum (SR) are present on all cell surfaces. Cisternae coupled to the sarcolemma are continuous with transverse components of SR which encircle the myofibrils at each I-band and H-band. The transverse component over the I-band consists of anastomosing tubules applied as a single layer to the surface of the myofibril. The transverse component over the H-band is also composed of anastomosing tubules, but the myofibrils are invested by a double or triple layer. Two or three tubules of sarcoplasmic reticulum interconnect consecutive transverse components. Each muscle band is surrounded by a thin external lamina. The external lamina does not parallel the irregular cell contours nor does it penetrate the extracellular space between cells. In contracted muscle, the sarcolemmata at the epidermal and notochordal boundaries indent to the level of each Z-line, and peripheral couplings are located at the base of the indentations. The external lamina and basal lamina of the epidermis are displaced toward the indentations. The location, function, and neuromuscular junctions of larval ascidian caudal muscle are similar to vertebrate somatic striated muscle. Other attributes, including the mononucleate condition, transverse myomuscular junctions, prolific gap junctions, active Golgi apparatus, and incomplete nervous innervation are characteristic of vertebrate cardiac muscle cells.     

CARDIAC MUSCLE : Its Ultrastructure in the Finch and Hummingbird with Special Reference to the Sarcoplasmic Reticulum

Cardiac muscle fibers of the hummingbird and finch have no transverse tubules and are smaller in diameter than those of mammalian hearts. The fibers are connected by intercalated discs which are composed of desmosomes and f. adherentes; small nexuses are often interspersed. As in cardiac muscle of several other animals, the junctional SR of the couplings is highly structured in these two birds but, in addition, and after having lost sarcolemmal contact, the junctional SR continues beyond the coupling to extend deep into the interior of the cells and to form belts around the Z-I regions of the sarcomeres. This portion of the sarcoplasmic reticulum, which we have named "extended junctional SR," and which is so prominent and invariant a feature of cardiac cells of hummingbirds and finches, has not been observed in chicken cardiac cells. The morphological differences between these species of birds may be related to respective differences in heart rates characteristic for these birds.     

Myocyte enlargement, differentiation, and proliferation kinetics in the fetal sheep heart.

The generation of new myocytes is an essential process of in utero heart growth. Most, or all, cardiac myocytes lose their capacity for proliferation during the perinatal period through the process of terminal differentiation. An increasing number of studies focus on how experimental interventions affect cardiac myocyte growth in the fetal sheep. Nevertheless, fundamental questions about normal growth of the fetal heart remain unanswered. In this study, we determined that during the last third of gestation the hearts of fetal sheep grew primarily by four processes. 1) Myocyte proliferation contributed substantially to daily cardiac mass gain, and the number of cardiac myocytes continued to increase to term. 2) The (hitherto unrecognized) contribution to cardiac growth by the increase in myocyte size associated with the transition from mononucleation to binucleation (terminal differentiation) became considerable from approximately 115 days of gestational age (dGA) until term (145dGA). Because binucleation became the more frequent outcome of myocyte cell cycle activity after approximately 115dGA, the number of binucleated myocytes increased at the expense of the number of mononucleated myocytes. Both the interval between nuclear divisions and the duration of cell cycle activity in myocytes decreased substantially during this same period. Finally, cardiac growth was in part due to enlargement of 3) mononucleated and 4) binucleated myocytes, which grew in cross-sectional diameter but not length during the last third of gestation. These data on normal cardiac growth may enable a more detailed understanding of the consequences of experimental and pathological interventions in prenatal life.     

Distribution of isomyosin in cultured cardiac myocytes as determined by monoclonal antibodies and adenosine triphosphatase activity

The distribution of isomyosin in cardiac muscle cells in culture has been investigated with monoclonal antibodies and Ca2+-activated myosin ATPase cytochemical staining. With immunofluorescent studies using monoclonal antibodies to isomyosins V1 and V3, the cardiac myocytes grown in a serum-free and thyroxine (T4)-free medium for 7 days contained a predominant population of cells which were strongly reactive to anti-V3 antibody. A small population of myocytes in this culture exhibited weak or no reaction to anti-V3 antibody. When cultures were exposed to anti-V1 antibody, the predominant cardiac myocyte population showed little or no reactivity to this antibody, whereas a small population of the myocytes were strongly reactive. The myosin ATPase staining reaction of the positive myocyte population was significantly less pronounced than that of the V3-negative population which showed a strong reaction. The staining pattern changed dramatically after exposure of cultured myocytes to thyroid hormone for 7 days. Most of the cells were found to react strongly with anti-V1 antibody, while some cells showed little reactivity and some were not stained at all. A small number of cardiac myocytes in this culture showed little or no reactivity to anti-V1 antibody but were strongly reactive to anti-V3 antibody. The predominant anti-V1-positive myocyte population exhibited strong myosin ATPase staining as compared to a smaller V3-positive myocyte population which showed very weak staining. The cytochemical results of ATPase staining in cardiac myocytes agreed well with ATPase activity as determined on pyrophosphate gels containing isomyosin derived from cultured cardiac myocytes with or without T4. This study has demonstrated that cultured myocytes contain a small population of muscle cells which is not responsive to thyroid hormone or to the lack of it.     

Effects of subcultivation and culture medium on differentiation of human fetal cardiac myocytes

Summary Previous work has suggested that subcultivated human fetal heart muscle cell cultures contain immature cardiac muscle cells capable only of limited differentiation after mitogen withdrawal. We studied several human fetal heart cultures (14–15 wk gestation) at several passage levels using immunocytochemistry, autoradiography, and Northern blot analysis. Characteristics in high-mitogen (growth) medium were compared with those after serum withdrawal. Cultured cells from one heart, expanded through 2 passages in growth medium, did not beat; however, 75% of cells did beat after subsequent culture for 24 days in low-serum (differentiation) medium containing insulin. In confluent cultures after 1 passage, there was no detectable difference in the number of cardiac myocytes present in growth medium compared with that 7 days after serum withdrawal. After 4 passages, however, serum withdrawal increased the number of cells expressing immunoreactive sarcomeric myosin heavy chain by 100-fold; expression of immunoreactive sarcomeric actin andα-cardiac actin mRNA also increased in the same cultures. Similar results were obtained in cultures kept in differentiation medium for 20 days before passage and expansion in growth medium. Using isopycinc centrifugation, a high-density cell fraction was isolated which contained no immunostained myocytes in growth medium but numerous myocytes after serum withdrawal. Combined immunocytochemistry/autoradiography showed that myocytes synthesize DNA in growth medium and in serum-free medium containing fibroblast growth factor, but not in serum-free medium alone. The results indicate that a) human fetal cardiac muscle cells proliferate in vitro and can maintain a phenotype characteristic of fetal myocytes after multiple subcultivations followed by serum withdrawal; b) after subcultivation in growth medium, some myocytes modulate their phenotype into one in which detectable levels of cardiac contractile proteins are expressed only after mitogen withdrawal, and c) the phenotype attained after serum withdrawal is in part dependent on passage level. Cultured human fetal myocardial cells my provide a useful experimental system for the study of human cardiac muscle cell biology.     

Contribution of mesenterial muscle dedifferentiation to intestine regeneration in the sea cucumber Holothuria glaberrima

Holothurians (sea cucumbers) have been known from ancient times to have the capacity to regenerate their internal organs. In the species Holothuria glaberrima, intestinal regeneration involves the formation of thickenings along the free mesentery edge; these thickenings will later give rise to the regenerated organ. We have previously documented that a remodeling of the extracellular matrix and changes in the muscle layer occur during the formation of the intestinal primordium. In order to analyze these changes in depth, we have now used immunocytochemical techniques and transmission electron microscopy. Our results show a striking disorganization of the muscle layer together with myocyte dedifferentiation. This dedifferentiation involves nucleic activation, disruptions of intercellular junctions, and the disappearance of cell projections, but more prominently, the loss of the contractile apparatus by the formation and elimination of spindle-like structures. Muscle dedifferentiation can be seen as early as 2 days following evisceration and continues during the next 2 weeks of the regeneration process. Dedifferentiation of myocytes might result in cells that proliferate and give rise to new myocytes. Alternatively, dedifferentiating myocytes could give rise to cells with high nuclear-to-cytoplasmic ratios, with some being eliminated by apoptosis. Our results, together with those in other regeneration models, show that myocyte dedifferentiation is a common event in regeneration processes and that the dedifferentiated cells might play an important role in the formation of the new tissues or organs. This work was supported by NSF (IBN-0110692) and NIH-MBRS (S06GM08102). We also acknowledge partial support from RCMI (RRO-3641-01), the Department of Biology, and the University of Puerto Rico.     

Cell types required to efficiently innervate human muscle cells in vitro

Previous studies carried out in our laboratory have shown that myofibers formed by fusion of muscle satellite cells from donors with spinal muscular atrophy (SMA) type I or II undergo a characteristic degeneration 1.5-3 weeks after innervation with rat embryonic spinal cord explants. The only cells responsible for degeneration of innervated cocultures are SMA muscle satellite cells. In order to study the kinetics of nerve and muscle cell degeneration in nerve-muscle cocultures implicating SMA muscle cells, we attempted to simplify the nervous component of the coculture and identify the nerve cell types necessary for a successful innervation. We demonstrate here that motoneurons alone were unable to innervate myotubes. However, when three cell types (motoneurons, sensory neurons, and Schwann cells) were added onto a reconstituted muscular component consisting of cloned muscle satellite cells and cloned muscular fibroblasts, myotubes contracted, indicating that functional neuromuscular junctions were formed. We concluded that the three cell types were required for a successful innervation. Moreover, we studied the effects of culture medium conditioned by different combinations of nerve cells on innervation; we observed that physical contacts among sensory neurons, motoneurons, and myotubes are required for a successful innervation; in contrast Schwann cells can be replaced by a Schwann-cell-conditioned medium, indicating that these cells produce a putative soluble "innervation-promoting factor." Obviously such a reconstituted system does not reflect the in vivo situation but it allows the formation of functional motor synapses and could therefore allow us to elucidate neuromuscular disease pathogenesis, especially that of spinal muscular atrophy.     

The mammalian myotome: a muscle with no innervation

SUMMARY The segmented muscular myotome is the first muscle to form in all vertebrates. In fish and amphibian embryos, the myotome becomes innervated very early and is essential for larval swimming. Its role in birds and mammals, however, is not clear. Using immunohistochemistry on sections and whole mounts of rat embryos, we demonstrate that the mammalian myotome differentiates and develops over a period of 3 days without being invaded by the outgrowing spinal nerves. In contrast, the limb muscle masses become filled with fine nerve branches from the first time that myocyte differentiation can be detected. Additionally, we show that the mammalian myotome does not express clustered acetylcholine receptors until after embryonic day 13.5, which corresponds to the beginning of its transformation into the adult epaxial muscles, showing that there are no functional myotomal neuromuscular junctions before this age. We suggest that the mammalian myotome has entirely lost the function of neurally controlled muscular contraction: its remaining functions are likely to be as a signaling tissue, as a structural scaffold, and as an incubator for myogenic precursors of the deep back, abdominal, and intercostal muscles.