PubMed Health⌕ Search

Biomedical subjects

M J van den Hoff

Publications and source records attributed to M J van den Hoff.

At least 19 recordsLinked to original sources
PubMed2001

Formation of myocardium after the initial development of the linear heart tube.

Well after formation of the primary linear heart tube, the mesenchymal cardiac septa become largely myocardial, and myocardial sleeves are formed along the caval and pulmonary veins. This second wave of myocardium formation can be envisioned to be the result of recruitment of cardiomyocytes by differentiation from flanking mesenchyme and/or by migration from existing myocardium (myocardialization). As a first step to elucidate the underlying mechanism, we studied in chicken heart development the formation of myocardial cells within intra- and extracardiac mesenchymal structures. We show that the second wave of myocardium formation proceeds in a caudal-to-cranial gradient in vivo. At the venous pole, loosely arranged networks of cardiomyocytes are observed in the dorsal mesocardium from H/H19 onward, in the atrioventricular cushion region from H/H26 onward, and in the proximal outflow tract (conus) from H/H29 onward. The process is completed at H/H stage 43. Subsequently, we determined the potential of the different cardiac compartments to form myocardial networks in a 3D in vitro culture assay. This analysis showed that the competency to form myocardial networks in vitro is a characteristic of the myocardium that is flanked by intra- or extracardiac mesenchyme, i.e., the inflow tract, atrioventricular canal, and outflow tract. These cardiac compartments can be induced to form myocardial networks by a temporally released or secreted signal that is similar throughout the entire heart. Atrial and ventricular compartments are not competent and do not produce the inducer. Moreover, cardiac cushion mesenchyme was found to be able to (trans-)differentiate into cardiomyocytes in the in vitro culture assay. The combined observations suggest that a common mechanism and molecular regulatory pathway underlies the recruitment of mesodermal cells into the cardiogenic lineage during this second wave of myocardium formation through the entire heart.

Animals↗
PubMed2001

Transgenic mice overexpressing human KvLQT1 dominant-negative isoform. Part I: Phenotypic characterisation.

OBJECTIVES: The KCNQ1 gene encodes the KvLQT1 potassium channel, which generates in the human heart the slow component of the cardiac delayed rectifier current, I(Ks). Mutations in KCNQ1 are the most frequent cause of the congenital long QT syndrome. We have previously cloned a cardiac KCNQ1 human isoform, which exerts a strong dominant-negative effect on KvLQT1 channels. We took advantage of this dominant-negative isoform to engineer an in vivo model of KvLQT1 disruption, obtained by overexpressing the dominant-negative subunit under the control of the alpha-myosin heavy chain promoter. RESULTS: Three different transgenic lines demonstrated a phenotype with increasing severity. Functional suppression of KvLQT1 in transgenic mice led to a markedly prolonged QT interval associated with sinus node dysfunction. Transgenic mice also demonstrated atrio-ventricular block leading to occasional Wenckebach phenomenon. The atrio-ventricular block was associated with prolonged AH but normal HV interval in His recordings. Prolonged QT interval correlated with prolonged action potential duration and with reduced K(+) current density in patch-clamp experiments. RNase protection assay revealed remodeling of K(+) channel expression in transgenic mice. CONCLUSIONS: Our transgenic mouse model suggests a role for KvLQT1 channels not only in the mouse cardiac repolarisation but also in the sinus node automaticity and in the propagation of the impulse through the AV node.

Action Potentials↗
PubMed2001

Foetal rise in hepatic enzymes follows decline in c-met and hepatocyte growth factor expression.

BACKGROUND/AIMS: In the embryo, rapidly proliferating hepatocytes migrate from the liver primordium into the surrounding mesenchyme, whereas foetal hepatocytes are mitotically quiescent and accumulate hepatocyte-specific enzymes. We investigated the timing and topography of this behavioural switch. METHODS: The expression of the c-met receptor and its ligand, hepatocyte growth factor (HGF), was investigated in prenatal rat liver by in situ hybridization, immunohistochemistry and western-blot analysis. RESULTS: c-Met was expressed by hepatocytes and HGF by non-parenchymal liver cells. Their mRNA levels peaked during embryonic day (ED) 11-13. c-Met protein was weakly expressed in the entire liver during ED 11 and 12, but more abundantly at ED 13, when its expression withdrew to the hepatic periphery. Simultaneously, the periportal hepatocellular marker carbamoylphosphate synthetase began to accumulate in the centre of the liver. Although the definitive vascular architecture develops simultaneously, the downstream, pericentral hepatocytes began to express glutamine synthetase only 4 days later, suggesting a requirement for prior periportal hepatocyte maturation. Additionally, c-met protein appeared in the connective tissue surrounding the large veins. The c-met protein/mRNA ratio was substantially higher in non-epithelial cells (hepatic connective tissue, heart) than in endoderm-derived epithelia, including hepatocytes, indicating important post-transcriptional regulation. CONCLUSIONS: The decline in c-met expression reflects the end of the embryonic phase and heralds the onset of the fetal, maturational phase of liver development.

Animals↗
PubMed2001

Transient expression of phosphatidylserine at cell-cell contact areas is required for myotube formation.

Cell surface exposure of phosphatidylserine (PS) is shown to be part of normal physiology of skeletal muscle development and to mediate myotube formation. A transient exposure of PS was observed on mouse embryonic myotubes at E13, at a stage of development when primary myotubes are formed. The study of this process in cell cultures of differentiating C2C12 and H9C2 myoblasts also reveals a transient expression of PS at the cell surface. This exposure of PS locates mainly at cell-cell contact areas and takes place at a stage when the structural organization of the sarcomeric protein titin is initiated, prior to actual fusion of individual myoblast into multinucleated myotubes. Myotube formation in vitro can be inhibited by the PS binding protein annexin V, in contrast to its mutant M1234, which lacks the ability to bind to PS. Although apoptotic myoblasts also expose PS, differentiating muscle cells show neither loss of mitochondrial membrane potential nor detectable levels of active caspase-3 protein. Moreover, myotube formation and exposure of PS cannot be blocked by the caspase inhibitor zVAD(OMe)-fmk. Our findings indicate that different mechanisms regulate PS exposure during apoptosis and muscle cell differentiation, and that surface exposed PS plays a crucial role in the process of myotube formation.

Animals↗
PubMed2000

Presence of functional sarcoplasmic reticulum in the developing heart and its confinement to chamber myocardium.

During development fast-contracting atrial and ventricular chambers develop from a peristaltic-contracting heart tube. This study addresses the question of whether chamber formation is paralleled by a matching expression of the sarcoplasmic reticulum (SR) Ca(2+) pump. We studied indo-1 Ca(2+) transients elicited by field stimulation of linear heart tube stages and of explants from atria and outflow tracts of the prototypical preseptational E13 rat heart. Ca(2+) transients of H/H 11+ chicken hearts, which constitute the prototypic linear heart tube stage, were sensitive to verapamil only, indicating a minor contribution of Ca(2+)-triggered SR Ca(2+) release. Outflow tract transients displayed sensitivity to the inhibitors similar to that of the linear heart tube stages. Atrial Ca(2+) transients disappeared upon addition of ryanodine, tetracaine, or verapamil, indicating the presence of Ca(2+)-triggered SR Ca(2+) release. Quantitative radioactive in situ hybridization on sections of E13 rat hearts showed approximately 10-fold higher SERCA2a mRNA levels in the atria compared to nonmyocardial tissue and approximately 5-fold higher expression in compact ventricular myocardium. The myocardium of atrioventricular canal, outflow tract, inner curvature, and ventricular trabecules displayed weak expression. Immunohistochemistry on sections of rat and human embryos showed a similar pattern. The significance of these findings is threefold. (i) A functional SR is present long before birth. (ii) SR development is concomitant with cardiac chamber development, explaining regional differences in cardiac function. (iii) The pattern of SERCA2a expression underscores a manner of chamber development by differentiation at the outer curvature, rather than by segmentation of the linear heart tube.

Animals↗
PubMed1999

Myocardialization of the cardiac outflow tract.

During development, the single-circuited cardiac tube transforms into a double-circuited four-chambered heart by a complex process of remodeling, differential growth, and septation. In this process the endocardial cushion tissues of the atrioventricular junction and outflow tract (OFT) play a crucial role as they contribute to the mesenchymal components of the developing septa and valves in the developing heart. After fusion, the endocardial ridges in the proximal portion of the OFT initially form a mesenchymal outlet septum. In the adult heart, however, this outlet septum is basically a muscular structure. Hence, the mesenchyme of the proximal outlet septum has to be replaced by cardiomyocytes. We have dubbed this process "myocardialization." Our immunohistochemical analysis of staged chicken hearts demonstrates that myocardialization takes place by ingrowth of existing myocardium into the mesenchymal outlet septum. Compared to other events in cardiac septation, it is a relatively late process, being initialized around stage H/H28 and being basically completed around stage H/H38. To unravel the molecular mechanisms that are responsible for the induction and regulation of myocardialization, an in vitro culture system in which myocardialization could be mimicked and manipulated was developed. Using this in vitro myocardialization assay it was observed that under the standard culture conditions (i) whole OFT explants from stage H/H20 and younger did not spontaneously myocardialize the collagen matrix, (ii) explants from stage H/H21 and older spontaneously formed extensive myocardial networks, (iii) the myocardium of the OFT could be induced to myocardialize and was therefore "myocardialization-competent" at all stages tested (H/H16-30), (iv) myocardialization was induced by factors produced by, most likely, the nonmyocardial component of the outflow tract, (v) at none of the embryonic stages analyzed was ventricular myocardium myocardialization-competent, and finally, (vi) ventricular myocardium did not produce factors capable of supporting myocardialization.

Animals↗
PubMed1998

Uncoupling of S phase and mitosis in cardiomyocytes and hepatocytes lacking the winged-helix transcription factor Trident.

In order to maintain a stable karyotype, the eukaryotic cell cycle is coordinated such that only one round of S phase precedes each mitosis, and mitosis is not initiated until DNA replication is completed. Several checkpoints and regulatory proteins have been defined in lower eukaryotes that govern this coordination, but little is known about the proteins that are involved in mammalian cells. Previously, we have shown that the winged-helix transcription factor Trident - also known as HFH-11, FKL16 and WIN [1] [2] [3] - is exclusively expressed in cycling cells and is phosphorylated during mitosis [1] [4]. The cellular function of Trident has yet to be described, however. Here, we have shown that disruption of the Trident gene in mice resulted in postnatal death, most probably because of circulatory failure. Histological analysis of Trident -/- embryos from embryonic day 10 (E10) onwards revealed a specific, characteristic defect in the developing myocardium. The orientation of the myocytes was highly irregular and the nuclei of these disorganized cardiomyocytes were clearly polyploid with up to a 50-fold increase in DNA content. Polyploidy was also observed in embryonic hepatocytes. Our results indicate that expression of Trident is required to prevent multiple rounds of S phase in the heart and the liver. Trident therefore appears to have a role in preventing DNA re-replication during the G2 and M phases.

Animals↗
PubMed1998

Normal development of the outflow tract in the rat.

The outflow tract (OFT) provides the structural components forming the ventriculoarterial connection. The prevailing concept that this junction "rotates" to acquire its definitive topography also requires a concept of "counterrotation" and is difficult to reconcile with cell-marking studies. Rats between 10 embryonic days (EDs) and 2 postnatal days were stained immunohistochemically and by in situ hybridization. DNA replication was determined by incorporation of bromodeoxyuridine and apoptosis by the annexin V binding and terminal deoxynucleotidyl transferase-mediated dUTP-X nick end labeling (TUNEL) assays. Starting at ED12, cardiomyocytes in the distal (truncal) part of the OFT begin to shed their myocardial phenotype without proceeding into apoptosis, suggesting transdifferentiation. Myocardial regression is most pronounced on the dextroposterior side and continues until after birth, as revealed by the disappearance of the myocardial cuff surrounding the coronary roots and semilunar sinuses and by the establishment of fibrous continuity between mitral and aortic semilunar valves. Fusion of the endocardial ridges of the truncus on late ED13 is accompanied by the organization of alpha-smooth muscle actin-and nonmuscle myosin heavy chain-positive myofibroblasts into a central whorl and the appearance of the semilunar valve anlagen at their definitive topographical position within the proximal portion of the truncus. After fusion of the proximal (conal) portion of the endocardial ridges, many of the resident myofibroblasts undergo apoptosis and are replaced by cardiomyocytes. The distal myocardial boundary of the OFT is not a stable landmark but moves proximally over the spiraling course of the aortic and pulmonary routes, so that the semilunar valves develop at their definitive topographic position. After septation, the distal boundary of the OFT continues to regress, particularly in its subaortic portion. The myocardializing conus septum, on the other hand, becomes largely incorporated into the right ventricle. These opposite developments account for the pronounced asymmetry of the subaortic and subpulmonary outlets in the formed heart.

Actins↗
PubMed1998

Changing patterns of gene expression in the pulmonary trunk-banded rat heart.

A pressure-overload model in the rat by banding the pulmonary trunk (PT) was developed to investigate alterations in gene expression in left- and right-ventricular compartments during the transition from compensated right-ventricular (RV) hypertrophy to right heart failure. Right heart failure in rat is characterized by liver cirrhosis, hydrothorax and ascites. The diameter of constriction was found to determine the time course of heart failure development. Only the RV free wall and the right atrium increased in weight, without a difference between compensated and failing RV. An increase in circulating ANP revealed a hypertrophic response of the myocardium, while increased circulating ammonia levels discriminated between compensated hypertrophy and failure. As parameters for stress, fibrosis and Ca2+-handling, changes in the pattern and level of the mRNAs encoding atrial natriuretic peptide (ANP), collagenIIIalpha1, and sarcoplasmic endoplasmic reticular calcium ATPase 2 (SERCA2), phospholamban (PLB) and calsequestrin (CSQ) were studied by Northern blot and in situ hybridization analyses. Pulmonary trunk banding resulted in an induction of ANP mRNA, a moderate increase in collagenIII alpha1 mRNA and a decrease in SERCA2 and PLB mRNA levels in both the left and right ventricles, but changes were most pronounced in the myocardium surrounding the RV cavity. Increased ammonia blood levels are a promising prognostic marker to detect the development of right heart failure.

Ammonia↗
PubMed1997

Developmental changes in rat cardiac DNA, RNA and protein tissue base: implications for the interpretation of changes in gene expression.

During cardiac development the expression levels of many genes change as determined by Northern blot, dot blot, RNase protection, quantitative RT-PCR. Western blot or immunoprecipitation analyses. It is not always realized that the total amount of RNA or protein per gram of heart, dubbed tissue base, may change significantly during development as well. If this would be the case, this has to be taken into account. So far, the (changing) tissue base has not been established during cardiac development. To this end developmental profiles of cardiac DNA, RNA and protein concentration were determined in rats ranging in age from embryonic day 13 until neonatal day 121. The profiles show significant development changes in each parameter, that closely match the distinct growth phases of the developing heart and provide the parameters that are essential for an adequate interpretation of changes in the amount of a distinct mRNA and/or protein. In a comparison between in situ hybridization and Northern blot analysis it is demonstrated that the same developmental profile leads to an almost opposite conclusion depending on whether or not the changing tissue base is taken into account. These findings are of great interest for studies aimed at unravelling the molecular mechanisms underlying the regulation of gene expression during cardiac development.

Age Factors↗
PubMed1997

Towards quantitative in situ hybridization.

In situ hybridization analysis of tissue mRNA concentrations remains to be accepted as a quantitative technique, even though exposure of tissue sections to photographic emulsion is equivalent to Northern blot analysis. Because of the biological importance of in situ quantification of RNA sequences within a morphological context, we evaluated the quantitative aspects of this technique. In calibrated microscopic samples, autoradiographic signal (density of silver grains) was proportionate to the radioactivity present, to the exposure time, and to time of development of the photographic emulsion. Similar results were obtained with tissue sections, showing that all steps of the in situ hybridization protocol, before and including the detection of the signal, can be reproducibly performed. Furthermore, the integrated density of silver grains produced in liver and intestinal sections by the in situ hybridization procedure using 35S-labeled riboprobes is directly proportionate to the signal obtained by quantitative Northern blot analysis. The significance of this finding is that in situ quantification of RNA can be realized with high sensitivity and with the additional advantage of the possibility of localizing mRNA within the cells of interest. Application of this procedure on fetal and adult intestinal tissue showed that the carbamoylphosphate synthetase (CPS)-expressing epithelial cells of both tissues accumulated CPS mRNA to the same level but that whole-organ CPS mRNA levels decreased four-to fivefold in the same period, owing to a comparable decrease in the number of CPS-expressing cells in total intestinal tissue.

Animals↗
PubMed1996

The upstream regulatory region of the carbamoyl-phosphate synthetase I gene controls its tissue-specific, developmental, and hormonal regulation in vivo.

The carbamoyl-phosphate synthetase I gene is expressed in the periportal region of the liver, where it is activated by glucocorticosteroids and glucagon (via cyclic AMP), and in the crypts of the intestinal mucosa. The enhancer of the gene is located 6.3 kilobase pairs upstream of the transcription start site and has been shown to direct the hormone-dependent hepatocyte-specific expression in vitro. To analyze the function of the upstream region in vivo, three groups of transgenic mice were generated. In the first group the promoter drives expression of the reporter gene, whereas the promoter and upstream region including the far upstream enhancer drive expression of the reporter gene in the second group. In the third group the far upstream enhancer was directly coupled to a minimized promoter fragment. Reporter-gene expression was virtually undetectable in the first group. In the second group spatial, temporal, and hormonal regulation of expression of the reporter gene and the endogenous carbamoyl-phosphate synthetase gene were identical. The third group showed liver-specific periportal reporter gene expression, but failed to activate expression in the intestine. These results show that the upstream region of the carbamoyl-phosphate synthetase gene controls four characteristics of its expression: tissue specificity, spatial pattern of expression within the liver and intestine, hormone sensitivity, and developmental regulation. Within the upstream region, the far upstream enhancer at -6.3 kilobase pairs is the determinant of the characteristic hepatocyte-specific periportal expression pattern of carbamoyl-phosphate synthetase.

Animals↗
PubMed1995

The far-upstream enhancer of the carbamoyl-phosphate synthetase I gene is responsible for the tissue specificity and hormone inducibility of its expression.

The role of the proximal promoter and the far-upstream enhancer in the hepatocyte-specific and hormonal regulation of the carbamoyl-phosphate synthetase I (CPS) gene was investigated in transient transfection assays using primary rat hepatocytes, hepatoma cells, and fibroblasts. These experiments revealed that the activity of the promoter is comparable in all cells tested and is, therefore, not responsible for tissue-specific expression. The 5'-untranslated region of the mRNA is a major, non-tissue specific stimulator of expression in FTO-2B hepatoma cells, acting at the post-transcriptional level. A 469-base pair DNA fragment, 6 kilobase pairs upstream of the transcription start-site in the CPS gene, confers strong hormone-dependent tissue specific expression, both in combination with the CPS promoter and a minimized viral thymidine kinase promoter. Sequences similar to a cyclic AMP-responsive element and a glucocorticosteroid-responsive element were found in the isolated enhancer. Substitutional mutations in these sites strongly affected hormone-induced expression. Analysis of the interaction between the enhancer and parts of the CPS promoter revealed that, in addition to the TATA box, the GAG box, a motif similar to the GC box near the TATA motif, is instrumental in conferring the enhancer activity.

Animals↗