Regulation of intracellular cardiomyocyte calcium stores by peptides: a new approach to cardiac protection.

The control of cytosolic calcium is a major determinant not only of cardiac function, but also of the capability of myocardial tissue to survive damage. Increase of diastolic calcium leads rapidly to cell injury, and may be induced by a wide range of causes. In this review we describe the major points of calcium control in cardiac myocytes, mainly in mammalian ventricle, focusing on mechanisms of intracellular calcium influx during excitation, voltage gated channels of the sarcolemma and ryanodine receptors of the sarcoplasmic reticulum (SR), and efflux during relaxation, principally the sodium/calcium exchanger in membrane and the SR calcium complex. Mitochondria also depend on calcium concentration while also participating in its control. Moreover, we will outline receptor check points and their roles in physiology and pathology. We will focus on some new aspects of potential protective mechanisms that have been recently described and that involve peptide ligands and that in the case of the Neuregulin1beta/ErbB pathway are already reaching the clinical trial relevance.

The homologous rat chromogranin A1-64 (rCGA1-64) modulates myocardial and coronary function in rat heart to counteract adrenergic stimulation indirectly via endothelium-derived nitric oxide.

Chromogranin A (CGA), produced by human and rat myocardium, generates several biologically active peptides processed at specific proteolytic cleavage sites. A highly conserved cleavage N-terminal site is the bond 64-65 that reproduces the native rat CGA sequence (rCGA1-64), corresponding to human N-terminal CGA-derived vasostatin-1. rCGA1-64 cardiotropic activity has been explored in rat cardiac preparations. In Langendorff perfused rat heart, rCGA1-64 (from 33 nM) induced negative inotropism and lusitropism as well as coronary dilation, counteracting isoproterenol (Iso) - and endothelin-1 (ET-1) -induced positive inotropic effects and ET-1-dependent coronary constriction. rCGA1-64 also depressed basal and Iso-induced contractility on rat papillary muscles, without affecting calcium transients on isolated ventricular cells. Structure-function analysis using three modified peptides on both rat heart and papillary muscles revealed the disulfide bridge requirement for the cardiotropic action. A decline in Iso intrinsic activity in the presence of the peptides indicates a noncompetitive antagonistic action. Experiments on rat isolated cardiomyocytes and bovine aortic endothelial cells indicate that the negative inotropism observed in rat papillary muscle is probably due to an endothelial phosphatidylinositol 3-kinase-dependent nitric oxide release, rather than to a direct action on cardiomyocytes. Taken together, our data strongly suggest that in the rat heart the homologous rCGA1-64 fragment exerts an autocrine/paracrine modulation of myocardial and coronary performance acting as stabilizer against intense excitatory stimuli.

Replication and translation of epigenetic information.

Most cells in multicellular organisms contain identical genetic information but differ in their epigenetic information. The latter is encoded at the molecular level by post-replicative methylation of certain DNA bases (in mammals 5-methyl cytosine at CpG sites) and multiple histone modifications in chromatin. In addition, higher-order chromatin structures are generated during differentiation, which might impact on genome expression and stability. The epigenetic information needs to be "translated" in order to define specific cell types with specific sets of active and inactive genes, collectively called the epigenome. Once established, the epigenome needs to be "replicated" at each cell division cycle, i.e., both genetic and epigenetic information have to be faithfully duplicated, which implies a tight coordination between the DNA replication machinery and epigenetic regulators. In this review, we focus on the molecules and mechanisms responsible for the replication and translation of DNA methylation in mammals as one of the central epigenetic marks.

SALL1, the gene mutated in Townes-Brocks syndrome, encodes a transcriptional repressor which interacts with TRF1/PIN2 and localizes to pericentromeric heterochromatin.

The Townes-Brocks syndrome (TBS) is an autosomal dominantly inherited malformation syndrome presenting as an association of imperforate anus, triphalangeal and supernumerary thumbs, malformed ears and sensorineural hearing loss. Mutations in SALL1, a gene mapping to 16q12.1, were identified as a cause for TBS. To elucidate how SALL1 mutations lead to TBS, we have performed a series of functional studies with the SALL1 protein. Using epifluorescence and confocal microscopy it could be shown that a GFP-SALL1 fusion protein localizes to chromocenters and smaller heterochromatin foci in transiently transfected NIH-3T3 cells. Chromocenters consist of clustered pericentromeric heterochromatin and contain telomere sequences. Indirect immunofluorescence revealed a partial colocalization of GFP-SALL1 with M31, the mouse homolog of the Drosophila heterochromatic protein HP1. It was further demonstrated that SALL1 acts as a strong transcriptional repressor in mammalian cells. Transcriptional repression could not be relieved by the addition of the histone deacetylase inhibitor Trichostatin-A. In a yeast two-hybrid screen we identified PIN2, an isoform of telomere-repeat-binding factor 1 (TRF1), as an interaction partner of SALL1, and showed that the N-terminus of SALL1 is not necessary for the interaction with PIN2/TRF1. The interaction was confirmed in vitro in a GST-pulldown assay. The association of the developmental regulator SALL1 with heterochromatin is striking and unexpected. Our results propose an involvement of SALL1 in the regulation of higher order chromatin structures and indicate that the protein might be a component of a distinct heterochromatin-dependent silencing process. We have also provided new evidence that there is a close functional link between the centromeric and telomeric heterochromatin domains not only in Drosophila and yeast, but also in mammalian cells.

Non-random radial higher-order chromatin arrangements in nuclei of diploid human cells.

A quantitative comparison of higher-order chromatin arrangements was performed in human cell types with three-dimensionally (3D) preserved, differently shaped nuclei. These cell types included flat-ellipsoid nuclei of diploid amniotic fluid cells and fibroblasts and spherical nuclei of B and T lymphocytes from peripheral human blood. Fluorescence in-situ hybridization (FISH) was performed with chromosome paint probes for large (#1-5) and small (#17-20) autosomes, and for the two sex chromosomes. Other probes delineated heterochromatin blocks of numerous larger and smaller human chromosomes. Shape differences correlated with distinct differences in higher order chromatin arrangements: in the spherically shaped lymphocyte nuclei we noted the preferential positioning of the small, gene dense #17, 19 and 20 chromosome territories (CTs) in the 3D nuclear interior--typically without any apparent connection to the nuclear envelope. In contrast, CTs of the gene-poor small chromosomes #18 and Y were apparently attached at the nuclear envelope. CTs of large chromosomes were also preferentially located towards the nuclear periphery. In the ellipsoid nuclei of amniotic fluid cells and fibroblasts, all tested CTs showed attachments to the upper and/or lower part of the nuclear envelope: CTs of small chromosomes, including #18 and Y, were located towards the centre of the nuclear projection (CNP), while the large chromosomes were positioned towards the 2D nuclear rim. In contrast to these highly reproducible radial arrangements, 2D distances measured between heterochromatin blocks of homologous and heterologous CTs were strikingly variable. These results as well as CT painting let us conclude that nuclear functions in the studied cell types may not require reproducible side-by-side arrangements of specific homologous or non-homologous CTs. 3D-modelling of statistical arrangements of 46 human CTs in spherical nuclei was performed under the assumption of a linear correlation between DNA content of each chromosome and its CT volume. In a set of modelled nuclei, we noted the preferential localization of smaller CTs towards the 3D periphery and of larger CTs towards the 3D centre. This distribution is in clear contrast to the experimentally observed distribution in lymphocyte nuclei. We conclude that presently unknown factors (other than topological constraints) may play a decisive role to enforce the different radial arrangements of large and small CTs observed in ellipsoid and spherical human cell nuclei.

Remodeling of donor nuclei, DNA-synthesis, and ploidy of bovine cumulus cell nuclear transfer embryos: effect of activation protocol.

The purpose of this study was to investigate the effects of two activation protocols on nuclear remodeling, DNA synthesis during the first cell cycle, chromosome segregation after first mitosis and development to blastocyst of embryos produced by somatic nuclear transfer. Pronuclear formation was significantly higher when activation lasted 5 hr compared to 3 hr for both ethanol-cycloheximide and ionomycin-bohemine treatment. However, the presence of a single nucleus was significantly higher in embryos activated for 3 hr in bohemine. Initiation of DNA synthesis was delayed in ethanol-cycloheximide group, however, after 12 hr labeling 100% of embryos synthesized DNA in both groups. Embryos activated with ethanol-cycloheximide developed to blastocysts at a significantly higher rate than those activated with ionomycin-bohemine. Analysis of 2-cell embryos with DNA probes for chromosome 6, 7, and 15 by fluorescence in situ hybridization showed that at least 50% of NT embryos were of normal ploidy independent of the activation stimulus. The results presented in this study show differences between the protocols compared on the nuclear events during the first cell cycle and on the development to blastocyst. Mol. Reprod. Dev. 59: 371-379, 2001.