Hiroko Matsui

Understanding heart development and congenital heart defects through developmental biology: A segmental approach

ABSTRACT  The heart is the first organ to form and function during development. In the pregastrula chick embryo, cells contributing to the heart are found in the postero‐lateral epiblast. During the pregastrula stages, interaction between the posterior epiblast and hypoblast is required for the anterior lateral plate mesoderm (ALM) to form, from which the heart will later develop. This tissue interaction is replaced by an Activin‐like signal in culture. During gastrulation, the ALM is committed to the heart lineage by endoderm‐secreted BMP and subsequently differentiates into cardiomyocyte. The right and left precardiac mesoderms migrate toward the ventral midline to form the beating primitive heart tube. Then, the heart tube generates a right‐side bend, and the d‐loop and presumptive heart segments begin to appear segmentally: outflow tract (OT), right ventricle, left ventricle, atrioventricular (AV) canal, atrium and sinus venosus. T‐box transcription factors are involved in the formation of the heart segments: Tbx5 identifies the left ventricle and Tbx20 the right ventricle. After the formation of the heart segments, endothelial cells in the OT and AV regions transform into mesenchyme and generate valvuloseptal endocardial cushion tissue. This phenomenon is called endocardial EMT (epithelial‐mesenchymal transformation) and is regulated mainly by BMP and TGFβ. Finally, heart septa that have developed in the OT, ventricle, AV canal and atrium come into alignment and fuse, resulting in the completion of the four‐chambered heart. Altered development seen in the cardiogenetic process is involved in the pathogenesis of congenital heart defects. Therefore, understanding the molecular nature regulating the ‘nodal point’ during heart development is important in order to understand the etiology of congenital heart defects, as well as normal heart development.

Heart development before beating

During heart development at the pregastrula stage, prospective heart cells reside in the posterior lateral region of the epiblast layer. Interaction of tissues between the posterior epiblast and hypoblast is necessary to generate the future heart mesoderm. Signaling regulating the interaction involves fibroblast growth factor (FGF)-8, Nodal, bone morphogenetic protein (BMP)-antagonist, and canonical Wnt and acts on the posterior epiblast to induce the expression of genes specific for the anterior lateral mesoderm. At the early gastrula stage, prospective heart cells accumulate at the posterior midline and migrate to the anterior region of the primitive streak. During gastrulation, future heart cells leave the primitive streak and migrate anterolaterally to form the left and right anterior lateral plate mesoderm including the precardiac mesoderm. At this stage, prospective heart cells receive endoderm-derived signals, including BMP, FGF, and Wnt-antagonist, and thereby become committed to the heart lineage. At the neurula stage, the left and right precardiac mesoderm move to the ventral midline and fuse, resulting in the formation of a single primitive heart tube. Therefore, a two-step signaling cascade, which includes tissue interaction between epiblast and hypoblast at the blastula stage and endoderm-derived signals during gastrulation, is required to generate a beating heart.

Induction of initial cardiomyocyte α-actin—smooth muscle α-actin—in cultured avian pregastrula epiblast: A role for nodal and BMP antagonist

During early cardiogenesis, endoderm‐derived bone morphogenetic protein (BMP) induces the expression of both heart‐specific transcription factors and sarcomeric proteins. However, BMP antagonists do not inhibit the expression of the “initial heart α‐actin”—smooth muscle α‐actin (SMA)—which is first expressed in the anterior lateral mesoderm and then recruited into the initial myofibrils (Nakajima et al. [2002] Dev. Biol. 245:291–303). Therefore, mechanisms that regulate the expression of SMA in the heart‐forming mesoderm are not well‐understood. Regional explantation experiments using chick blastoderm showed that the posterolateral region of the epiblast differentiated into cardiomyocytes. Posterior epiblast cultured with or without the associated hypoblast showed that interaction between the tissues of these two germ layers at the early pregastrula stage (stages X–XI) was a prerequisite for the expression of SMA. Posterior epiblast that is cultured without hypoblast could also be induced to express SMA if TGF‐β or activin was added to the culture medium. However, neither neutralizing antibodies against TGF‐βs nor follistatin perturbed the expression of SMA in cultured blastoderm. Adding BMP to the cultured blastoderm inhibited the expression of SMA, whereas BMP antagonists, such as chordin, were able to induce the expression of SMA in cultured posterior epiblast. Furthermore, adding lefty‐1, a nodal antagonist, to the blastoderm inhibited the expression of SMA, and nodal plus BMP antagonist up‐regulated the expression of SMA in cultured posterior epiblast. Results indicate that the interaction between the tissues of the posterior epiblast and hypoblast is necessary to initiate the expression of SMA during early cardiogenesis and that nodal and BMP antagonist may play an important role in the regulation of SMA expression. Developmental Dynamics 233:1419–1429, 2005.

Rho kinase inhibitor Y27632 affects initial heart myofibrillogenesis in cultured chick blastoderm

During early vertebrate development, Rho‐associated kinases (ROCKs) are involved in various developmental processes. Here, we investigated spatiotemporal expression patterns of ROCK1 protein and examined the role of ROCK during initial heart myofibrillogenesis in cultured chick blastoderm. Immunohistochemistry showed that ROCK1 protein was distributed in migrating mesendoderm cells, visceral mesoderm of the pericardial coelom (from which cardiomyocytes will later develop), and cardiomyocytes of the primitive heart tube. Pharmacological inhibition of ROCK by Y27632 did not alter the myocardial specification process in cultured posterior blastoderm. However, Y27632 disturbed the formation of striated heart myofibrils in cultured posterior blastoderm. Furthermore, Y27632 affected the formation of costamere, a vinculin/integrin‐based rib‐like cell adhesion site. In such cardiomyocytes, cell–cell adhesion was disrupted and N‐cadherin was distributed in the perinuclear region. Pharmacological inactivation of myosin light chain kinase, a downstream of ROCK, by ML‐9 perturbed the formation of striated myofibrils as well as costameres, but not cell–cell adhesion. These results suggest that ROCK plays a role in the formation of initial heart myofibrillogenesis by means of actin–myosin assembly, and focal adhesion/costamere and cell–cell adhesion. Developmental Dynamics 236:461–472, 2007.

ROCK1 Expression is Regulated by TGFβ3 and ALK2 During Valvuloseptal Endocardial Cushion Formation

During early heart development at the looped heart stage, endothelial cells in the outflow tract and atrioventricular (AV) regions transform into mesenchyme to generate endocardial cushion tissue. This endocardial epithelial–mesenchymal transition (EMT) is regulated by several regulatory pathways, including the transforming growth factor‐beta (TGFβ), bone morphogenetic protein (BMP), and Rho‐ROCK pathways. Here, we investigated the spatiotemporal expression pattern of ROCK1 mRNA during EMT in chick and examined whether TGFβ or BMP could induce the expression of ROCK1. At the onset of EMT, ROCK1 expression was up‐regulated in endothelial/mesenchymal cells. A three‐dimensional collagen gel assay was used to examine the mechanisms regulating the expression of ROCK1. In AV endocardium co‐cultured with associated myocardium, ROCK1 expression was inhibited by either anti‐TGFβ3 antibody, anti‐ALK2 antibody or noggin, but not SB431542 (ALK5 inhibitor). In cultured preactivated AV endocardium, TGFβ3 protein induced the expression of ROCK1, but BMP did not. AV endothelial cells that were cultured in medium supplemented with TGFβ3 plus anti‐ALK2 antibody failed to express ROCK1. These results suggest that the expression of ROCK1 is up‐regulated at the onset of EMT and that signaling mediated by TGFβ3/ALK2 together with BMP is involved in the expression of ROCK1. Anat Rec, 291:845‐857, 2008.

Induction of initial heart α-actin, smooth muscle α-actin, in chick pregastrula epiblast : The role of hypoblast and fibroblast growth factor-8

During heart development at the gastrula stage, inhibition of bone morphogenetic protein (BMP) activity affects the heart specification but does not impair the expression of smooth muscle α‐actin (SMA), which is first expressed in the heart mesoderm and recruited into initial heart myofibrils. Interaction of tissues between posterior epiblast and hypoblast at the early blastula stage is necessary to induce the expression of SMA, in which Nodal and Chordin are thought to be involved. Here we investigated the role of fibroblast growth factor‐8 (FGF8) in the expression of SMA. In situ hybridization and reverse transcription–polymerase chain reaction showed that Fgf8b is expressed predominantly in the nascent hypoblast. Anti‐FGF8b antibody inhibited the expression of SMA, cTNT, and Tbx5, which are BMP‐independent heart mesoderm/early cardiomyocyte genes, but not Brachyury in cultured posterior blastoderm, and combined FGF8b and Nodal, but neither factor alone induced the expression of SMA in association with heart specific markers in cultured epiblast. Although FGF8b did not induce the upregulation of phospho‐Smad2, anti‐FGF8b properties suppressed phospho‐Smad2 in cultured blastoderm. FGF8b was able to reverse the BMP‐induced inhibition of cardiomyogenesis. The results suggest that FGF8b acts on the epiblast synergistically with Nodal at the pregastrula stage and may play a role in the expression of SMA during early cardiogenesis.

Heart Myofibrillogenesis Occurs in Isolated Chick Posterior Blastoderm: A Culture Model

Early cardiogenesis including myofibrillogenesis is a critical event during development. Recently we showed that prospective cardiomyocytes reside in the posterior lateral blastoderm in the chick embryo. Here we cultured the posterior region of the chick blastoderm in serum-free medium and observed the process of myofibrillogenesis by immunohistochemistry. After 48 hours, explants expressed sarcomeric proteins (sarcomeric α-actinin, 61%; smooth muscle α-actin, 95%; Z-line titin, 56%; sarcomeric myosin, 48%); however, they did not yet show a mature striation. After 72 hours, more than 92% of explants expressed I-Z-I proteins, which were incorporated into the striation in 75% of explants or more (sarcomeric α-actinin, 75%; smooth muscle α-actin, 81%; Z-line titin, 83%). Sarcomeric myosin was expressed in 63% of explants and incorporated into A-bands in 37%. The percentage incidence of expression or striation of I-Z-I proteins was significantly higher than that of sarcomeric myosin. Results suggested that the nascent I-Z-I components appeared to be generated independently of A-bands in the cultured posterior blastoderm, and that the process of myofibrillogenesis observed in our culture model faithfully reflected that in vivo. Our blastoderm culture model appeared to be useful to investigate the mechanisms regulating the early cardiogenesis.

Suppression of Rat Stellate Cell Activation and Liver Fibrosis by a Japanese Herbal Medicine, Inchinko-to (TJ135)

Herbal medicine has been recognized as one of useful treatments for chronic liver diseases. Inchinko-to (TJ135) consists of Artemisia Capillaris Spike, Gardenia Fruit and Rhubarb Rhizome. Artemisia Capillaris Spike and Gardenia Fruit promote bile secretion [1,2]. Rhubarb Rhizome is used for constipation. Rhubarb Rhizome contains anthraquinone derivatives, such as aloe emodin and emodin. TJ135 is used for cholestasis, primary biliary cirrhosis [3] and hepatitis C [4] in Japan. Although therapeutic benefit of TJ135 was recently reported on acute liver injury induced by Fas-induced apoptosis of hepatocytes [5], its effect on liver fibrosis has never been studied so far. In the present study, we investigated the effect of TJ135 and its component on the activation of rat hepatic stellate cells in primary culture [6]. In vivo effect of TJ135 on an experimental liver fibrosis caused by thioacetamide (TAA) administration was also studied.

Amino acids, L-Cysteine and L-Methionine, Attenuate Activation of Rat Stellate Cells in Primary Culture.

Regulation of hepatic stellate cell activation is currently one of the focuses of clinical investigation in order to establish a useful therapeutic strategy for liver fibrosis. Because oxidative stress caused at the inflammatory site and reactive oxygen species derived from damaged hepatocytes has been thought to pull the trigger for the cell activation, it is reasonable to speculate that antioxidative substances are promising to attenuate the activation process. In fact, alpha-tocopherol and natural flavonoids have potential to inhibit collagen gene expression and DNA synthesis of stellate cells, respectively. Our laboratory demonstrated that N-acetyl-L-cysteine (NAC) inhibits DNA synthesis of rat stellate cells in response to serum and PDGF-BB [1]. NAC was found to downregulate the expression of PDGF receptor beta PDGFR beta), thereby hampering PDGF-BB-dependent phosphorylation of MAP kinase and Akt [2,3]. In addition, Kim et al. [4] reported that NAC induces cell cycle arrest at G1 phase through inducing p21. Suppression of dimethylnitrosamine-induced liver fibrosis by NAC was also reported. Taken together, these results suggested that reducing compounds with -SH suppliers would be promising candidates attenuating the activation of stellate cells in culture and also in vivo. NAC is an analogue of amino acid L-cysteine that has -SH base. L-Cysteine has been reported to suppress oxidative stress caused by smoking, alcohol intake and noxious metals. L-Methionine is a precursor of L-cysteine and plays important roles in the methylation of genes. Also, DL isoform of methionine has been used for the treatment of liver disease. However, pharmacological action of amino acids has been largely unknown. Thus, we tested in this study the effect of amino acids on rat hepatic stellate cells.