Search

Myocardial ischemia reperfusion injury (IRI) adversely affects cardiac performance and the prognosis of patients with acute myocardial infarction. Although myocardial signal transducer and activator of transcription (STAT) 3 is potently cardioprotective during IRI, the inhibitory mechanism responsible for its activation is largely unknown. The present study aimed to investigate the role of the myocardial suppressor of cytokine signaling (SOCS)-3, an intrinsic negative feedback regulator of the Janus kinase (JAK)-STAT signaling pathway, in the development of myocardial IRI. Myocardial IRI was induced in mice by ligating the left anterior descending coronary artery for 1 h, followed by different reperfusion times. One hour after reperfusion, the rapid expression of JAK-STAT-activating cytokines was observed. We precisely evaluated the phosphorylation of cardioprotective signaling molecules and the expression of SOCS3 during IRI and then induced myocardial IRI in wild-type and cardiac-specific SOCS3 knockout mice (SOCS3-CKO). The activation of STAT3, AKT, and ERK1/2 rapidly peaked and promptly decreased during IRI. This decrease correlated with the induction of SOCS3 expression up to 24 h after IRI in wild-type mice. The infarct size 24 h after reperfusion was significantly reduced in SOCS3-CKO compared with wild-type mice. In SOCS3-CKO mice, STAT3, AKT, and ERK1/2 phosphorylation was sustained, myocardial apoptosis was prevented, and the expression of anti-apoptotic Bcl-2 family member myeloid cell leukemia-1 (Mcl-1) was augmented. Cardiac-specific SOCS3 deletion led to the sustained activation of cardioprotective signaling molecules including and prevented myocardial apoptosis and injury during IRI. Our findings suggest that SOCS3 may represent a key factor that exacerbates the development of myocardial IRI.

Endothelial to mesenchymal transition (EMT) that occurs during cardiac outflow tract (OFT) development is critical for formation of the semilunar valves. Fibulin-1 (Fbln1) is an extracellular matrix protein that is present at several sites of EMT, including the OFT (i.e., E9.5-10.5). The aim of this study was to determine the role of Fbln1 in EMT during the earliest events of OFT development. Examination of proximal OFT cushions in Fbln1 null embryos detected hypercellularity at both E9.5 (93% increase; p = 0.002) and E10.5 (43% increase; p = 0.01) as compared to wild type, suggesting that Fbln1 normally suppresses OFT endocardial cushion EMT. This was supported by studies of proximal OFT cushion explants, which showed that explants from Fbln1 null embryos displayed a 58% increase in cells migrating from the explants as compared to wild type (p = 0.005). We next evaluated the effects of Fbln1 deficiency on the expression of factors that regulate proximal OFT EMT. At E9.5, Fbln1 null proximal OFT endocardium and EMT-derived mesenchyme showed increased TGFβ2 (58% increase; p = 0.01) and increased Snail1-positive nuclei (27% increase; p = 0.0003). Histological examination of OFT cushions in Fbln1 null embryos (E9.5) also detected cells present in the cushion that were determined to be erythrocytes based on round morphology, autofluorescence, and positive staining for hemoglobin. Erythrocytes were also detected in Fbln1 null OFT cushions at E10.5. Together, the findings indicate that Fbln1 normally suppresses proximal OFT EMT preventing proximal cushion hypercellularity and blood cell accumulation., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

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.

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.

The ability of anterior lateral plate mesoderm cells in the heart-forming region (HFR) of stage 6 chicken embryos to respond to cardiogenic stimuli from cells in adjacent germ layers has been investigated using explants cultured under defined conditions. Two types of explantation were evaluated: those in which two germ layers were explanted in contiguity, and those in which germ layers were isolated and co-cultured. Two parameters--contractility and expression of sarcomeric alpha-actin--were monitored to evaluate the terminal differentiation of cardiac myocytes. Contiguously explanted anterior endoderm/mesoderm became multilayered and underwent terminal differentiation within 2 days. By contrast, although contiguous anterior ectoderm/mesoderm or posterior endoderm/mesoderm co-explants also became multilayered, these explants did not differentiate, up to 5 days. To ascertain the cardiogenic potential of cells from different regions of the embryo, individual germ layers were isolated and co-cultured by placing the explants in separate areas of the culture chamber. These determinations demonstrated that anterior, but not posterior, endoderm effected differentiation of anterior mesoderm. As before, mesoderm in both types of co-culture survived and became multilayered; by contrast, mesoderm did not survive when cultured in isolation. These experiments provide evidence that anterior endoderm regulates the terminal differentiation, as opposed to growth, of presumptive cardiac myocytes in mesoderm cells from the anterior lateral plate. Finally, anterior endoderm was co-cultured with mesoderm from the posterior half of the embryo, which does not contain an HFR. The failure of these co-cultured explants to differentiate infers that pre-cardiac myoblasts in stage 6 anterior mesoderm are previously specified to respond to the terminal cardiogenic effects of endoderm.

The three-dimensional organization of cytoskeletal filaments associated with the myofibrils and sarcolemma of the myocardial cells of early chick embryos was studied by the rapid-freeze deep-etch method combined with immunocytochemistry. In the endoplasmic region of saponin-treated myocardial cells, 12-14 nm filaments formed a loose network surrounding nascent myofibrils. These 12-14 nm filaments attached to the myofibrils and some of them converged into Z disc regions. In the non-junctional cytocortical region thinner 8-11 nm filaments composed a dense network just beneath the sarcolemma. In myofibril terminating regions at the sarcolemma, i.e., the fascia adherens, 3-5 nm cross-bridges were observed among the thin filaments. In Triton-permeabilized and myosin subfragment 1 (S1)- treated samples, subsarcolemmal 8-11 nm filaments proved to be S1-decorated actin filaments under which there was a loose network of S1-undecorated filaments. Subsarcolemmal S1-decorated actin filaments had mixed polarity and attached to the sarcolemma at one end. A loose network of S1-undecorated filaments among myofibrils in the endoplasmic region was revealed to consist of desmin-containing intermediate filaments after immuno-gold staining for desmin. These networks connecting myofibrils with sarcolemma were assumed to play an important role in integrating and transmitting the contractile force of individual myofibrils within early embryonic myocardial cells.

Cytoskeletal filaments in myocardial cells of chick embryo (stage 18-20, day 3) were studied by immunocytochemical and rapid-freeze deep-etch methods. A three-dimensional network of cytoplasmic filaments surrounding nascent myofibrils was visualized in saponin-treated myocardial cells. The major part of the network was composed of 12 to 14 nm filaments in platinum replicas. To identify the filaments, the myocardial cells were permeabilized with Triton X-100 and treated with myosin subfragment-1 (S1) for actin or immunogold-labeled antibody for desmin. A large number of filaments in myofibrils and a few cytoplasmic filaments were decorated with S1. A loose network surrounding the myofibrils was not decorated with S1 but with gold particles. This finding means that the majority of filaments occupying the interfibrillar space were desmin-containing filaments.

Cellular growth and changes in subcellular components as well as in intercellular junction structure were investigated quantitatively in sinus and atrioventricular nodes in comparison with right atrial and left ventricular myocardial cells of the goat heart in fetuses aged 6, 8, 10, 12, 14, 16 and 18 gestational weeks, young animals aged 1 and 3 postnatal weeks, and adults. At the earliest stage examined (sixth gestational week) no clear difference was recognised, in the patterns of star diagrams representing cellular characteristics, between nodal cells and ordinary cardiac muscle cells, except for a larger number of myofibrils in the ordinary cells. Atrial specific granules also appeared in the atrial cells at this stage. In general, initial signs of differentiation in ordinary cardiac muscle cells were detected at the sixteenth gestational week when transverse tubule formation occurred in the ventricular muscle cells. Throughout the developmental period, no appreciable alterations in the ultrastructural features were found in the sinus nodal cells whereas the atrioventricular nodal cells revealed slight changes at the later fetal and adult stages.