Effect of bolus injection of 20 ml saline with arm elevation on the onset time of vecuronium administered via a peripheral vein: a randomised controlled trial.

We investigated whether a bolus injection of 20 ml saline with arm elevation might shorten the onset time of vecuronium administered via a dorsal hand vein. Thirty patients were randomly allocated to the bolus saline group or control group. General anaesthesia was induced and maintained with remifentanil and propofol. Vecuronium 0.1 mg.kg(-1) was administered to all patients, followed in the treatment group by bolus injection of 20 ml saline and arm elevation. Response to train-of-four stimulation was measured by acceleromyography at the adductor pollicis muscle. The mean (SD) lag time was 47.2 (14.5) s in the bolus saline group and 67.9 (12.2) s in the control group (p = 0.0002). The time to 95% block of T1 was 104.6 (29.9) s in the bolus saline group and 128.3 (15.8) s in the control group (p = 0.011). Bolus saline injection results in shortened lag time and onset time of neuromuscular block with vecuronium.

Neuromuscular blockade by vecuronium during induction with 5% sevoflurane or propofol.

This randomized trial investigated whether 5% sevoflurane potentiated neuromuscular blockade by vecuronium. General anaesthesia was induced with 5% sevoflurane in oxygen in 16 patients or with propofol in 16 patients. After loss of consciousness, vecuronium was administered to all participants at randomly assigned doses of 25, 30, 35 or 40 µg/kg. Neuromuscular blockade was assessed by use of acceleromyography to measure responses to train-of-four stimuli in the adductor pollicis and corrugator supercilii muscles. Maximum blockade was significantly more intense in the adductor pollicis among patients in the sevoflurane group than in the propofol group, whereas there was no significant between-group difference at the corrugator supercilii muscles. In both groups, maximum blockade at the corrugator supercilii was significantly less intense than that achieved at the adductor pollicis. In the dose-response analysis, the 50% and 95% effective doses were lower for sevoflurane than for propofol in both muscles, although this did not reach statistical significance. It is concluded that induction of general anaesthesia with sevoflurane might provide improved conditions for intubation and reduce airway problems.

Neuromuscular effects of sevoflurane in myasthenia gravis patients.

BACKGROUND: Little information is available regarding the neuromuscular effects of sevoflurane in patients with myasthenia gravis (MG). We evaluated the neuromuscular effects of sevoflurane alone in patients with MG and in those with normal neuromuscular transmission. METHODS: Sixteen patients with generalized type MG (MG group) and 12 otherwise healthy patients (control group) entered into this study. Anaesthesia was induced with propofol, fentanyl, and midazolam followed by nitrous oxide in oxygen. Neuromuscular monitoring was recorded from the adductor pollicis muscle using electromyography with train-of-four stimulation of the ulnar nerve. After a stabilization period, and before sevoflurane administration, baseline T4/T1 was obtained and MG patients were classified as non-fade MG group (baseline T4/T1 > or = 0.90) (n = 10) and fade MG group (baseline T4/T1 < 0.90) (n = 6). End-tidal sevoflurane concentration was kept constant at 1.7% for 30 min and doubled thereafter to 3.4% and maintained for a further 30 min. RESULTS: Sevoflurane produced a concentration-dependent decrease in T1 and T4/T1 values. At 3.4% sevoflurane, T1 and T4/T1 decreased significantly from baseline values in all three groups. From baseline until the patient woke up from anaesthesia, the T4/T1 of the fade MG group was significantly lower than the other groups. At the end of anaesthesia, T4/T1 returned to values similar to the baseline in all three groups. CONCLUSIONS: During sevoflurane anaesthesia, concentration-dependent inhibition of neuromuscular transmission was observed in MG and control patients. The inhibitory effects of sevoflurane were more prominent in MG patients with baseline T4/T1 <0.90.

Risk factors for nausea and vomiting following vitrectomy in adults.

BACKGROUND AND OBJECTIVE: Postoperative nausea and vomiting (PONV) after ophthalmic surgery under general anaesthesia remains a complex and perturbing complication associated with several factors. Little information is available regarding the risk factors for nausea and vomiting after vitrectomy in adults. In this study, we evaluated the potential risk factors for PONV after vitrectomy in adult patients. METHODS: Univariate and multivariate analyses of clinical factors associated with PONV were undertaken in a retrospective case-control series of 247 adult patients undergoing vitrectomy under general anaesthesia. We examined PONV for the first 48 h. Factors examined were age, body mass index (BMI), smoking status, H2-blocker as premedication, type of general anaesthesia (sevoflurane and fentanyl or total intravenous (i.v.) anaesthesia with propofol and fentanyl), duration of surgery, and intraoperative fentanyl dose. RESULTS: Fifty-nine patients (24%) reported one or more episodes of PONV during the study period. Female gender (P < 0.01), lower BMI (P < 0.01) and general anaesthesia with inhalational anaesthetics (P < 0.01) were significantly related to nausea during the first 2 h postoperatively. Female gender (P < 0.01) was significantly related to nausea and vomiting throughout the study period. Other factors, including smoking status, did not alter the risk for nausea and/or vomiting. CONCLUSIONS: We conclude that female gender, lower BMI and inhalation anaesthesia are the main risk factors for PONV after vitrectomy in adults. Smoking status did not reduce the incidence of PONV in our patients.

Fibulin-1 suppression of fibronectin-regulated cell adhesion and motility.

Fibulin-1 is an extracellular matrix protein often associated with fibronectin (FN) in vivo. In this study, the ability of fibulin-1 to modulate adhesion, spreading and motility-promoting activities of FN was investigated. Fibulin-1 was found to have pronounced inhibitory effects on the cell attachment and spreading promoted by FN. Fibulin-1 was also found to inhibit the motility of a variety of cell types on FN substrata. For example, the FN-dependent haptotactic motility of breast carcinoma (MDA MB231) cells, epidermal carcinoma (A431), melanoma (A375 SM), rat pulmonary aortic smooth muscle cells (PAC1) and Chinese hamster ovary (CHO) cells was inhibited by the presence of fibulin-1 bound to FN-coated Boyden chamber membranes. Cells transfected to overproduce fibulin-1 displayed reduced velocity, distance of movement and persistence time on FN substrata. Similarly, the incorporation of fibulin-1 into FN-containing type I collagen gels inhibited the invasion of endocardial cushion mesenchymal cells migrating from cultured embryonic heart explants. By contrast, incorporation of fibulin-1 into collagen gels lacking FN had no effect on the migration of endocardial cushion cells. These results suggest that the motility-suppressive effects of fibulin-1 might be FN specific. Furthermore, such effects are cell-type specific, in that the migration of gingival fibroblasts and endothelial cells on FN substrata is not responsive to fibulin-1. Additional studies found that the mechanism for the motility-suppressive effects of fibulin-1 does not involve perturbations of interactions between alpha5beta1 or alpha4 integrins, or heparan sulfate proteoglycans with FN. However, fibulin-1 was found to inhibit extracellular signal regulated kinase (ERK) activation and to suppress phosphorylation of myosin heavy chain. This ability to influence signal transduction cascades that modulate the actin-myosin motor complex might be the basis for the effects of fibulin-1 on adhesion and motility.

[Anesthetic management of a patient with Brugada syndrome].

Brugada syndrome is characterized by right bundle-branch block, ST elevation in leads V 1 through V 3 and normal QT interval. Ventricular fibrillation frequently occurs in patients with Brugada syndrome. There have been few reports of anesthetic management of Brugada patients. We managed a 47-year-old man with Brugada syndrome, who underwent hemilaminectomy under general anesthesia, without untoward cardiovascular events. Potential problems in anesthetic management of patients with Brugada syndrome are also discussed.

Endoderm and heart development.

Since the first half of the 20th century, experimental embryologists have noted a relationship between endoderm cells and the development of cardiac tissue from mesoderm. During the past decade, the accumulation of evidence for an obligatory interaction between endoderm and mesoderm during the specification and terminal differentiation of myocardial, and more recently endocardial, cells has markedly accelerated. Moreover, the endoderm-derived molecules that may regulate these processes are being identified. It now appears that endoderm-derived growth factors regulate the formation of both myocardial and endocardial cells during specification, terminal differentiation, and perhaps morphogenesis of cells in the developing embryonic heart.

[Anesthetic management of tracheal stent insertion under total intravenous anesthesia--a report of two cases].

Tracheal stent insertion is a useful method for patients with malignant tracheal stenosis. Expandable metal stents were inserted in two patients with severe dyspnea due to tracheal stenosis caused by lung cancer and esophageal cancer. The tracheas were intubated after spraying the pharynx with 4% lidocaine solution. The respiration was assisted. Anesthesia was maintained by intravenous propofol. There were no episodes of coughing during and after the procedure.

[Anesthetic management of a patient with hypohidrotic ectodermal dysplasia].

Hypohidrotic ectodermal dysplasia (HED) is a rare congenital anomaly complex characterized by hypodontia, hypotricosis and hypohidrosis. There have been only a few reports of anesthetic management of patients with HED. We managed a 20-year-old man with HED, who underwent debridement and skin grafting under epidural anesthesia, without untoward events. Potential problems in anesthetic management of patients with HED are also discussed.

Identification of an autocrine signaling pathway that amplifies induction of endocardial cushion tissue in the avian heart.

Endocardial cushion tissue is formed by an epithelial-mesenchymal transformation of endocardial cells, a process which results from an inductive interaction between the myocardium and endocardium within the atrioventricular (AV) and outflow tract (OT) regions of the heart. We report here that a protein previously found to be required for myocardially induced transformation of endocardial cells in vitro, ES/130, is highly expressed within the AV and OT regions not only by myocardial cells, but also by the endocardium and its mesenchymal progeny. Given these findings and others, we have tested the hypothesis that endocardial cushion tissue secretes factors which autoregulate its transformation to mesenchyme. Endocardial cushion tissue was cultured and its conditioned growth medium was harvested and applied to nontransformed endocardial cells maintained in the absence of the inductive myocardium. This treatment resulted in endocardial cell invasion into three-dimensional collagen gels plus increased expression of proteins associated with endocardial cell transformation in vivo. Whereas endocardial cushion tissue was found to express ES/130 protein in vivo and in vitro, minimal detection of ES/130 in its conditioned growth medium was observed in immunoblots. Attempts to inhibit the mesenchyme-promoting activity of the conditioned medium with ES/130 antisense were unsuccessful. However, strong intracellular ES/130 expression was detected in endocardial cells, and this expression correlated with the ability of endocardial cells to transform. For example, the minority of endocardial cultures that failed to transform in response to conditioned medium treatment also failed to undergo increased expression of ES/130. These observations are interpreted to suggest that (i) endocardial cushion tissue secretes factors that promote its transformation to mesenchyme, and (ii) while endocardial cushion tissue appears to signal through secretion of factors other than or in addition to ES/130, intracellular ES/130 expression nevertheless may be a target endocardial cell response required for endocardial cell transformation.

Cord blood transplantation from sibling donors in Japan. Report of the national survey.

A joint national survey on cord blood transplantation (CBT) was conducted in Japan and 18 sibling CBTs were reported. Diseases of the patients were leukemia (ten), neuroblastoma (one), bone marrow failure (four) and inborn errors of metabolism (three). A volume of 50-141 ml of cord blood containing 27-197 x 10(7) nucleated cells was collected from sibling infants soon after delivery. HLA antigens were identical in 14 and one to three antigens mismatched in four. Engraftment of donor cord blood was achieved in 17 cases. Autologous hematopoiesis was recovered in one case. Days of engraftment were 13-29 days (median 19 days) for neutrophils (500/microliter), 18-67 days (median 30 days) for reticulocytes (2%) and 21-96 days (median 46 days) for platelets (50 x 10(3)/microliter). Acute GVHD was grade 0 in seven cases, grade I in five cases and grade II in one case in HLA-identical pairs, but became grade II in two cases and grade III in two cases in HLA-mismatched pairs. Chronic GVHD of limited type developed in two out of 17 evaluable cases, however both responded to immunosuppressive therapy. Altogether, 14 out of 18 patients are currently surviving 4-27 months following transplantation. Probabilities of overall survival and disease free survival were estimated to be 77.0 and 71.8% using Kaplan-Meier tests. These findings suggest the feasibility of cord blood transplantation from sibling donors and the possibility of unrelated cord blood transplantation. A cord blood banking system is necessary for the universal use of cord blood stem cells from unrelated donors.

Combined BMP-2 and FGF-4, but neither factor alone, induces cardiogenesis in non-precardiac embryonic mesoderm.

Previous work in this laboratory has shown that endoderm cells in the heart forming region (HFR endoderm) of the chicken embryo induce terminal cardiac differentiation in explanted precardiac mesoderm cells. Immunostaining patterns indicating that HFR endoderm cells express Drosophila decapentaplegic (dpp)-like antigens prompted a degenerate polymerase chain reaction (PCR) screen to identify cDNAs in the dpp subgroup of the transforming growth factor-beta (TGF-beta) family. Among 50 clones of PCR products that have been sequenced, over half have identity with bone morphogenetic protein-2 (BMP-2). No other TGF-beta cDNAs have been detected, suggesting that BMP-2 is the major dpp subgroup protein synthesized by HFR endoderm cells. However, BMP-2 protein did not promote survival of either precardiac or non-precardiac mesoderm cells in culture. Whereas FGF-4 supports cardiogenesis in precardiac mesoderm, it did not induce cardiogenesis in nonprecardiac mesoderm, although explant viability was maintained. In contrast to the isolated effects of these growth factors, treatment of non-precardiac mesoderm with combined BMP-2 and FGF-4 induced cardiogenesis in the majority of explants, as revealed by the formation of a rhythmically contractile multicellular vesicle that expresses sarcomeric alpha-actin. These findings suggest that BMP-2 and FGF-4 possess respective differentiative and proliferative activities, the combination of which specifies cells to the cardiac lineage.

Formation and early morphogenesis of endocardial endothelial precursor cells and the role of endoderm.

The formation of endocardial endothelium in quail embryos was investigated using in vivo and in vitro systems. Based on the expression of an quail endothelial marker, QH-1, the initial emergence of endothelial precursor cells in the embryo occurs at stage 7+ (two somites) in the posterior parts of the bilateral heart forming regions. Cells that expressed the QH-1 antigen were mesenchymal and positioned between the mesodermal epithelium of the heart region and the endoderm. By confocal microscopy, an asymmetrical distribution of QH-1 positive cells was observed between the two heart regions: specifically between 7+ and 8-, more precursor cells were seen in the right region than the left. Endothelial precursor cells did not appear outside of the heart forming regions until stage 8- (three somites). Free, mesenchymal-like endothelial precursor cells intrinsic to the heart regions also expressed two extracellular antigens, JB3, a fibrillin-like protein, and cytotactin, both associated with segments of the primary heart tube where endothelial cells "re-transform" back to a mesenchymal phenotype during cardiac cushion tissue formation. Between stages 8 and 9 (four to seven somites), (1) QH-1 positive cells within the heart forming region established vascular-like connections with QH-1 positive cells located outside of the heart region, as initially shown by Coffin and Poole (1988), (2) after fusion of the heart regions, a plexus of QH-1 positive cells was formed ventral to the foregut, and (3) the definitive endocardial lining of the primary heart tube formed directly from the ventral plexus of endothelial precursor cells. Because the QH-1 positive, endothelial precursor cells of each heart forming region were always in close association with anterior endoderm, we sought to determine if the endoderm mediated the formation of precursor cells committed to a cardiac endothelial lineage as reflected by their expression of QH-1, JB3 antigen, and cytotactin. To test this hypothesis, precardiac mesodermal explants were isolated from stage 5 heart forming regions prior to their expressing of either endocardial or myocardial markers and cultured on the surface of collagen gets in the presence or absence of endoderm. In the absence of endoderm, precardiac mesoderm of each stage 5 explant remained epithelial, formed contractile tissue, but did not exhibit any QH-1 positive cells or mesenchymal cells. Conversely, when cocultured with endoderm or endoderm conditioned medium, in addition to the formation of contractile tissue, the explant formed mesenchymal cells. The latter invaded the gel lattice and, as in vivo, expressed QH-1 antigen, JB3 antigen, and cytotactin. These findings suggest that endoderm induces mesoderm of the heart fields to undergo an epithelial to mesenchyme transformation that results in the segregation of myocardial and endocardial precursor cells.

[Anesthetic management of a patient with tuberous sclerosis].

Tuberous sclerosis is characterized by facial angiofibromatosis, epilepsy, and mental retardation. There have been only a few reports of anesthetic management of patients with tuberous sclerosis. We managed a 22-year-old patient with tuberous sclerosis using nitrous oxide (67%) in oxygen supplemented with fentanyl and midazolam. There were no untoward events related to anesthesia and surgery. Problems in managing patients with tuberous sclerosis are also discussed.

Epithelial-mesenchymal transformations in early avian heart development.

Cardiac morphogenesis proceeds from a sequential series of epithelial-mesenchymal transitions which begins by establishing bipotential heart-forming cells and later their segregation into endocardial and myocardial lineages. Cells within each lineage integrate to form two concentric epithelia which inductively interact to transform cells of the inner epithelium, the endocardium, into mesenchymal or 'cushion' cells. Noncardiogenic epithelia (dorsal mesocardium, epicardium, neural ectoderm and coelomic mesothelium) undergo transition into populations of extracardiac mesenchyme that combine over time with cushion tissue to remodel the simple tubular heart into a four-chambered organ. Model systems are described for studying the mechanisms of cardiac-related transformations including primary cultures of precardiac epithelia and a differentiation-inducible, avian stem cell line called QCE-6. Focus is centered on the molecular mechanism by which endocardial epithelium transforms into cushion mesenchyme. Experimental findings are reviewed and interpreted in the context of a hypothetical model that seeks to answer why only some cells within an epithelium transform and whether the transformation process is regulated by intrinsic or extrinsic mechanisms. The model proposes that epithelial cells competent to transform to mesenchyme express characteristic markers including receptors for extrinsic signals secreted by stimulator cells (e.g. myocardium). Candidate extrinsic signals include multicomponent complexes called adherons. If applied directly to cultured endocardium, myocardial adherons but not those secreted by L6 myoblasts, induce changes in gene expression within target endocardial cells for proteases and cell:cell and cell:matrix adhesion molecules that accompanied transformation to mesenchyme. A main component of myocardial adherons has been identified as ES antigens, one of which, ES/130, has been cloned, found to have a novel sequence and in culture assays shown to be required for endocardium to transform to mesenchyme. The spatiotemporal pattern of ES protein expression within the embryo suggests that common mechanisms may exist for embryonic epithelial-mesenchymal transformations.

Activin-A and FGF-2 mimic the inductive effects of anterior endoderm on terminal cardiac myogenesis in vitro.

We recently reported that the differentiation of cultured embryonic precardiac myocytes is specifically promoted by anterior lateral plate endoderm from Hamburger-Hamilton stage 6 chick embryos. Polypeptide growth factors are probable mediators of cardiogenesis during embryonic development. It was previously noted that activin-A is a major secretory product of endoderm cultured from chicken embryos. Also, fibroblast growth factor-like proteins are present in anterior endoderm of stage 6 chick embryos. Therefore, we have examined the cardiogenic effects of these growth factors on cultured precardiac mesoderm cells explanted from stage 6 embryos. Similar to the effects of anterior endoderm, low concentrations of activin-A, FGF-2 (bFGF), or insulin significantly increased the incidence of explants that exhibited synchronous contractions and expressed cardiac alpha-actin mRNA. By contrast, explants treated with transferrin, bovine serum albumin, or nerve growth factor never contracted and contained only cytoplasmic beta-actin transcripts. These results provide additional evidence that endoderm-secreted activin-A, FGF-2, and perhaps insulin participate in regulating terminal cardiac differentiation in the embryo.

Developmental expression of fibroblast growth factor receptor-1 (cek-1; flg) during heart development.

Previous work in this laboratory has indicated that fibroblast growth factor-2 (FGF-2; bFGF) regulates the initial stages of avian heart development in paracrine and autocrine fashion (Parlow et al. [1991] Dev. Biol. 146:139-147; Sugi et al. [1993] Dev. Biol. 157:28-37). Because these findings inferred the presence of a functional receptor for fibroblast growth factor (FGFR), we have immunochemically assessed the appearance of FGFR-1 (cek-1; flg) during development. Using a peptide-generated antibody, Western blots of total embryonic proteins revealed that FGFR-1 was barely detectable at pre-heart stages, followed by sequential increases in relative abundance that peaked at stage 24, followed by a decline at days 7-14. Western blots of proteins from isolated embryonic hearts demonstrated a similar developmental pattern, except that FGFR-1 expression was not decreased at later stages. The presence of FGFR-1 mRNA was verified by reverse transcription/polymerase chain reaction (RT/PCR) amplification. Immunohistochemical examination revealed punctate deposits of FGFR-1 in the precardiac endoderm at stage 6, followed by detection in the endoderm, foregut, and pre-cardiac splanchnic mesoderm at stage 8 and in the newly formed myocardium at the heart tube stage (9/10). By stage 13, FGFR-1 staining was observed only in the myocardium, a pattern which persisted at least until stage 30 (day 7), after which only isolated hearts were examined. After stage 30, staining was diminished in the ventricle, but not in the atrium. Staining of cardiac endothelial cells was not observed at any stage. A functional role for FGFR-1 was indicated by experiments in which anti-FGFR-1, but not pre-absorbed antiserum, retarded proliferation and multilayering of cardiogenic cells in an in vitro model of cardiac morphogenesis.

NCAM polypeptides in heart development: association with Z discs of forms that contain the muscle-specific domain.

Previous studies of neural cell adhesion molecule (NCAM) cDNAs have revealed an alternatively spliced set of small exons (12A, 12B, 12C, and 12D) that encode a region in the extracellular portion of the molecule known as the muscle-specific domain (MSD). The entire MSD region can be expressed in skeletal muscle, heart, and skin; only exons 12A and 12D have been found in brain. These studies did not reveal which NCAM polypeptides contain the MSD region or the immunohistochemical distribution of these NCAM molecules. To address these questions, we prepared antibodies against the oligopeptides encoded by exons 12A and 12B and by exons 12C and 12D, and we used these antibodies to study the forms of NCAM containing the MSD region expressed during embryonic chicken heart development. These antibodies recognize certain forms of NCAM found in the heart, but they do not recognize brain NCAM. In the heart, each of the splice variants of NCAM (large cytoplasmic domain, small cytoplasmic domain, and small surface domain) that differ in their mode of attachment to the plasma membrane or in the size of their cytoplasmic domain is expressed in a form that contains and in a form that lacks the MSD region. No microheterogeneity is observed in the size of NCAM molecules containing the MSD region, even at the level of cyanogen bromide fragments, suggesting that exons 12A-D are expressed as a single unit. Depending on the site and the stage of development, the percent of NCAM molecules containing the MSD region can vary from nearly 0 to 100%. In general, this percentage increases during development. In immunohistochemical studies of hearts from stage 18 embryos, forms of NCAM containing the MSD region colocalized with Z discs. No other adhesion molecules were found in this distribution at this early stage of development. Studies on isolated cells in vitro demonstrate that the colocalization with Z discs of NCAM molecules containing the MSD region does not depend on cell-cell contact, and they raise the possibility that this form of NCAM is involved in cell-extracellular matrix interactions. The association of NCAM molecules containing the MSD region with Z discs suggests that this form of NCAM is involved in early myofibrillogenesis.

Early endocardial formation originates from precardiac mesoderm as revealed by QH-1 antibody staining.

The formation of endocardial endothelium in quail embryos was investigated using in vivo and in vitro systems. At stage 7+ (2 somite), the initial emergence of endothelial cells within the bilateral heart forming region (HFR) was detected in quail embryos by immunohistochemistry with QH-1 (an anti-quail endothelial cell marker) and confocal microscopy. We consistently observed more QH-1 positive cells in the right HFR than the left. At stage 8 (4 somite), the HFR, including QH-1 positive cells, were located in the splanchnic mesoderm after formation of the coelom. During stage 8, the HFR migrated along the margin of anterior intestinal portal in association with the endoderm. By stage 8+ (5 somite), the two HFR had fused at the midline and formed a plexus of QH-1 positive endothelial precursor cells. The definitive endocardium developed as a single, hollow, tube within this plexus. Posteriorly, QH-1 positive cells of the HFR established vascular-like connections with QH-1 positive cells that had formed outside (peripheral to) the HFR. During migration and subsequent determination, the precardiac mesoderm is continuously associated with the basement membrane of the anterior endoderm. To determine the role of endoderm on endocardial endothelial cell formation and development, precardiac mesoderm from stage 5 embryos, which does not express QH-1 antigen, was explanted onto the surface of collagen gels. When co-cultured with endoderm, the outgrowth of free cells from the mesoderm was much more extensive, many of which invaded the gel and expressed the QH-1 antigen; mesoderm cultured without endoderm did not seed nor express QH-1 antigen. These findings suggest that the segregation of endothelial and myocardial lineages may occur by an endoderm-mediated, mesenchymal formation.