Paul J.R. Barton

Elevated expression of Nkx-2.5 in developing myocardial conduction cells.

A number of different phenotypes emerge from the mesoderm‐derived cardiomyogenic cells of the embryonic tubular heart, including those comprising the cardiac conduction system. The transcriptional regulation of this phenotypic divergence within the cardiomyogenic lineage remains poorly characterized. A relationship between expression of the transcription factor Nkx‐2.5 and patterning to form cardiogenic mesoderm subsequent to gastrulation is well established. Nkx‐2.5 mRNA continues to be expressed in myocardium beyond the looped, tubular heart stage. To investigate the role of Nkx‐2.5 in later development, we have determined the expression pattern of Nkx‐2.5 mRNA by in situ hybridization in embryonic chick, fetal mouse, and human hearts, and of Nkx‐2.5 protein by immunolocalization in the embryonic chick heart. As development progresses, significant nonuniformities emerge in Nkx‐2.5 expression levels. Relative to surrounding force‐generating (“working”) myocardium, elevated Nkx‐2.5 mRNA signal becomes apparent in the specialized cells of the conduction system. Similar differences are found in developing chick, human, and mouse fetal hearts, and nuclear‐localized Nkx‐2.5 protein is prominently expressed in differentiating chick conduction cells relative to adjacent working myocytes. This tissue‐restricted expression of Nkx‐2.5 is transient and correlates with the timing of spatio‐temporal recruitment of cells to the central and the peripheral conduction system. Our data represent the first report of a transcription factor showing a stage‐dependent restriction to different parts of the developing conduction system, and suggest some commonality in this development between birds and mammals. This dynamic pattern of expression is consistent with the hypothesis that Nkx‐2.5, and its level of expression, have a role in regulation and/or maintenance of specialized fate selection by embryonic myocardial cells. Anat Rec 263:307–313, 2001.

Up-regulation of c-jun mRNA in cardiac myocytes requires the extracellular signal-regulated kinase cascade, but c-Jun N-terminal kinases are required for efficient up-regulation of c-Jun protein.

Cardiac hypertrophy, an important adaptational response, is associated with up-regulation of the immediate early gene, c- jun, which encodes the c-Jun transcription factor. c-Jun may feed back to up-regulate its own transcription and, since the c-Jun N-terminal kinase (JNK) family of mitogen-activated protein kinases (MAPKs) phosphorylate c-Jun(Ser-63/73) to increase its transactivating activity, JNKs are thought to be the principal factors involved in c- jun up-regulation. Hypertrophy in primary cultures of cardiac myocytes is induced by endothelin-1, phenylephrine or PMA, probably through activation of one or more of the MAPK family. These three agonists increased c- jun mRNA with the rank order of potency of PMA approximately endothelin-1>phenylephrine. Up-regulation of c- jun mRNA by endothelin-1 was attenuated by inhibitors of protein kinase C (GF109203X) and the extracellular signal-regulated kinase (ERK) cascade (PD98059 or U0126), but not by inhibitors of the JNK (SP600125) or p38-MAPK (SB203580) cascades. Hyperosmotic shock (0.5 M sorbitol) powerfully activates JNKs, but did not increase c- jun mRNA. These data suggest that ERKs, rather than JNKs, are required for c- jun up-regulation. However, endothelin-1 and phenylephrine induced greater up-regulation of c-Jun protein than PMA and phosphorylation of c-Jun(Ser-63/73) correlated with the level of c-Jun protein. Up-regulation of c-Jun protein by endothelin-1 was attenuated by inhibitors of protein kinase C and the ERK cascade, probably correlating with a primary input of ERKs into transcription. In addition, SP600125 inhibited the phosphorylation of c-Jun(Ser-63/73), attenuated the increase in c-Jun protein induced by endothelin-1 and increased the rate of c-Jun degradation. Thus whereas ERKs are the principal MAPKs required for c- jun transcription, JNKs are necessary to stabilize c-Jun for efficient up-regulation of the protein.

Myocardial Dysfunction in Donor Hearts A Possible Etiology

BACKGROUND Potential cardiac donors show various degrees of myocardial dysfunction, and the most severely affected hearts are unsuitable for transplantation. The cause of this acute heart failure is poorly understood. We investigated whether alterations in calcium-handling proteins, beta-adrenoceptor density, or the inhibitory G protein Gialpha could account for this phenomenon in unused donor hearts (n=4 to 8). We compared these with end-stage failing hearts (n=14 to 16) and nonfailing hearts (n=3 to 12). METHODS AND RESULTS Myocardial samples were obtained from unused donor hearts displaying ejection fractions <30%. Both trabeculae and isolated myocytes responded as poorly as those from the group of failing hearts to increasing stimulation frequency with regard to inotropic function in vitro. Immunodetectable abundance of sarcoplasmic reticulum calcium-ATPase and sodium calcium exchanger were greater (177%; P<0.01) and smaller (29%; P<0.01), respectively, in the unused donor hearts relative to the failing group, which suggests that alterations of these proteins are not a common cause of contractile dysfunction in the 2 groups. Myocytes from the unused donor group were desensitized to isoprenaline to a similar degree as those from the failing heart group. However, beta-adrenoceptor density was reduced in the failing (P<0.001) but not in the unused donor heart group (P=0.37) relative to the nonfailing heart group (n=5). Gialpha activity was increased in samples from unused donor and failing hearts relative to nonfailing hearts (P<0.05). CONCLUSIONS Increased activity of the inhibitory G protein Gialpha is a significant contributory factor for impaired contractility in these acutely failing donor hearts.

Identification of novel, cardiac-restricted transcription factors binding to a CACC-box within the human cardiac troponin I promoter

OBJECTIVES The expression of the human cardiac troponin I (hTnIc) gene is developmentally regulated and tissue-specific. In analysing the putative binding elements within the proximal promoter, a CACC-box sequence overlapping a consensus Sp1 element has been identified. The aim of this study was to characterise the factors binding to this element and to determine their importance in the transcriptional activity of the promoter. METHODS A combination of supershift and competition electrophoretic mobility shift assays (EMSA) were used to identify the binding of factors to the overlapping CACC-box/Sp1 consensus element. The functional importance of this element was tested by transient transfection into primary neonatal rat cardiac myocytes in culture. RESULTS At least four factors were able to interact with this region including the zinc finger proteins Sp1, Sp3 and two potentially novel factors. Whereas both Sp1 and Sp3 bound to the consensus Sp1 element, and to a lesser extent the CACC-box, two of the complexes required the intact CACC-box for binding. Site-directed mutagenesis of this region showed that the CACC-box is essential for hTnIc promoter-reporter activity. Further characterisation using EMSA indicated that the factors binding the hTnIc CACC-box are unlikely to be zinc finger proteins as they are insensitive to the addition of divalent cation chelating agents. They were also unable to bind to other known CACC-box elements. These factors are present in both human and rat cardiac muscle but absent from a number of cell lines including several derived from skeletal muscle. CONCLUSION The human cardiac troponin I gene promoter requires an upstream CACC-box element for full activity. This element binds at least two complexes which represent novel, tissue-restricted DNA-binding activity present in the heart which we have named HCB1 and HCB2 for heart CACC-box binding factors.

Genes encoding troponin I and troponin T are organized as three paralogous pairs in the mouse genome

The troponin complex is located on the thin filament of striated muscle and is the calcium-sensitive molecular switch that regulates striated muscle contraction in response to alterations in intracellular calcium concentration. It is composed of three subunits: troponin C, which binds calcium; troponin T, which is involved in the attachment of the complex to tropomyosin; and troponin I, the inhibitory subunit. Multiple isoforms have been identified for each of the three subunits, and these are expressed with distinct patterns of tissue specificity and developmental regulation. In total, eight troponin genes have been identified in human and mouse: three for troponin I, three for troponin T, and two for troponin C (see Table 1). In human, both the cardiac troponin T (TNNT2) and the cardiac troponin I (TNNI3) genes have been directly implicated in familial hypertrophic cardiomyopathy (Thierfelder et al. 1994; Kimura et al. 1997), an autosomal dominant disease associated with myofibrillar disarray within the myocardium. We recently demonstrated that the six human troponin I and troponin T genes are organized as paralogous pairs at three chromosomal sites, each containing a troponin I and troponin T gene (Barton et al. 1997, 1999). While all sarcomeric proteins, including isoforms of actin, myosin, myosin light chain, and tropomyosin, are encoded by multigene families (Robert et al. 1985), only the myosin heavy chain genes have previously been identified as being linked (Weydert et al. 1985). These are grouped at two loci, one containing the aandb-cardiac genes, the other containing the skeletal muscle genes. Recent studies have shown that while myosin gene organization does not relate to their temporal or spatial pattern of expression, as is seen in the globin and homeobox gene clusters for example, it is highly conserved between mouse and human (Weiss et al. 1999). In order to further investigate the troponin I and T gene families, we have undertaken to examine their chromosomal organization in mouse. Initial localization of the slow skeletal troponin T gene ( Tnnt1) was achieved by using a representative set of DNA samples from the EUCIB resource. PCR primers were chosen from the 3 8 end of a cDNA (GenBank/EMBL accession number AJ131711), in a region predicted to lie within the 3 8 terminal exon (exon 14) based on the structure of the human gene (Barton et al. 1999). No recombinants between either Tnnt1andPkccor betweenTnnt1and the cardiac troponin I gene ( Tnni3) were observed on 33 common animals tested, thereby localizing the Tnnt1gene to the proximal region of chromosome (Chr) 7 close to Tnni3.Based on our studies in human (Barton et al. 1999), we predicted that the mouse Tnni3 gene would lie immediately upstream of, and head to tail with Tnnt1. This was confirmed to be the case in three independent PAC genomic clones identified by hybridization screening with the slow skeletal troponin T cDNA (see Fig. 1). Restriction digests showed that these clones are non-identical (data not shown), and Southern blot hybridization revealed that each contained both the 58 end of Tnni3 and the 38 end of Tnnt1 (see Fig. 1A). PCR reactions across the intergene region were carried out with a rightward primer derived from exon 8 of Tnni3 and a leftward primer corresponding to the 5 8 end of Tnnt1. This resulted in a 2.4-kb fragment with each of the three PAC recombinant clones (Fig. 1B). Taken together, these data show that the whole of both genes is contained within each of the three identified PAC recombinants, thatTnni3andTnnt1lie head to tail, and that they are separated by only 2.4 kb of intervening sequence. Initial localization of cardiac troponin T gene Tnnt2 was achieved by using a representative set of DNA samples from the EUCIB resource as described above. The results placed Tnnt2 in the distal region of Chr 1, in close proximity (three recombinants in 733 animals, 0.41 cM [0.08–1.19]) to the previously reported location of the slow skeletal troponin I ( Tnni1) gene (Gue ́net et al. 1996; data not shown). In order to estimate more precisely the distance betweenTnnt2 and Tnni1, we analyzed the segregation pattern of both genes in the recently described T31 mouse radiation hybrid panel (Van Etten et al. 1999). First, a mouse mRNA sequence for slow skeletal troponin I was deduced from EST alignments performed by using BLASTn and the mouse EST database screened initially with the human mRNA sequence (GenBank/ EMBL m19309). Detailed analysis of 50 of the identified sequences produced a consensus (GenBank/EMBL AJ242874) with a 564-bp open reading frame with >89% sequence identity to both the human and rat slow skeletal troponin I mRNAs (data not shown). The segregation pattern of the two genes in the T31 radiation hybrid panel was subsequently determined by PCR by using primer pairs for each gene, and the results were analyzed with the ‘rhmapper’ software (http:www.genome.wi.mit.edu/cgibin/mouse_rh/rhmap-auto/rhmapper.cgi) placing both genes between the markersD1Mit538 and D1Mit348 (Fig. 2). The data confirm the location ofTnnt2 in the distal region of Chr 1 and placeTnni1proximal to it. The distance between the two genes is 2.2 cR, which corresponds to an approximate physical distance of 185 kb. On the consensus MGD map, Tnni1 is located 60 cM from the centromere in a region homologous to human 18q21-22. With the EUCIB resource, the position of Tnni1 is 64.7 cM from the centromere. By using the T31 radiation panel, we found that Tnni1 and Tnnt2 map betweenD1Mit538 and D1Mit348 in a region homologous to human Chr 1q32, consistent with the known locaCorrespondence to: PJR Barton; E-mail: p.barton@ic.ac.uk

Efficiency of a high-titer retroviral vector for gene transfer into skeletal myoblasts

BACKGROUND Genetic transformation of skeletal myoblasts for myocardial repair is dependent on an efficient gene transfer system that integrates the genes of interest into the genome of the target cell and its progeny. The aim of this investigation was to evaluate the use of a new retrovirally based gene transfer system for this purpose. METHODS MFGnlslacZ retroviral vector, packaged in high-titer, split-genome packaging cell line (FLYA4) was used to transduce the skeletal myoblast cell line L6. L6 cells, cultured in 10% fetal calf serum, were transduced with the MFGnlslacZ vector by means of filtered supernatant from FLYA4 cells. Transduced L6 cells were divided into four groups. Group I cells were fixed as myoblasts 3 days after transduction. Group II cells were allowed to differentiate into myotubes. Group III cells were split every 3 days for 4 months. Group IV cells were split as in group III but then allowed to differentiate into myotubes. All samples were fixed and stained for beta-galactosidase activity. The effects on gene transfer of transforming growth factor-beta, insulin-like growth factor-I, and platelet-derived growth factor were determined by spectrophotometric assay of beta-galactosidase activity in cells transduced in the presence or absence of serum with 0 to 200 ng/ml of each growth factor. RESULTS Morphometric analysis showed that 66.3% +/- 3% to 69.6% +/- 6% of cells in group I to IV expressed the lacZ reporter gene. In the presence of serum, transforming growth factor-beta significantly inhibited gene transfer, whereas insulin-like growth factor-I and platelet-derived growth factor significantly enhanced gene transfer. In absence of serum, however, only platelet-derived growth factor enhanced retrovirally mediated gene transfer into skeletal myoblasts. CONCLUSION MFG retroviral vectors packaged in FLYA4 cells are efficient in gene transfer into skeletal myoblasts and result in transgenic expression that is maintained after repeated cell division, differentiation, or both. Platelet-derived growth factor enhances retrovirally mediated gene transfer into skeletal myoblasts.

Rapamycin (Sirolimus) Inhibits Heart Cell Growth In Vitro

Abstract. The success of infant and neonatal heart transplantation has increased dramatically since the mid-1980s. This success is due both to improved medical and surgical techniques and better pharmacological management of rejection episodes. We report here the results of in vitro studies designed to investigate the effect of rapamycin (a macrolide antibiotic with potent antirejection activity) on the proliferation of fetal cardiac myocytes. Our data suggest that rapamycin inhibits the proliferation of these cells, which is an effect that appears to be irreversible. This inhibition is relevant to the use of rapamycin in the treatment of rejection episodes in the infant and neonate, in which cardiac myocyte development is incomplete.

Myocardial molecular biology: an introduction

The recent publication of draft copies of the human genome sequence from both public and private sector consortia has fuelled anticipation that eventually, once all genes have been identified, we will be able to ascertain which of them are involved in human diseases, including those affecting the cardiovascular system. Understanding the molecular biology behind both inherited and acquired disorders is now viewed as essential to provide a full picture of the aetiology and progression of disease. Within the past decade considerable advances have been made in identifying the genetic basis of myocardial disorders such as familial hypertrophic cardiomyopathy and dilated cardiomyopathy, as well as the molecular signalling pathways and gene regulatory events that characterise acquired disease such as pressure overload induced cardiac hypertrophy. Furthermore, by defining the molecular processes underlying normal development we may be able to manipulate immature cell phenotypes such as those of embryonic stem cells or skeletal myoblasts to replace damaged, terminally differentiated cells such as cardiac myocytes. In this review we outline the basic principals of gene expression, the different mechanisms by which expression is regulated and how these can be examined experimentally. The blueprint for any organism is contained within its genome in the form of chromosomes and is written in the universal four “base” language of adenine (A), guanine (G), cytosine (C), and thymine (T). Chromosomes are built of chromatin , double stranded DNA wrapped around a multi-protein complex core comprised of histone proteins. This DNA contains the language (DNA or nucleotide sequence) that can be read and translated into proteins, and these areas of DNA are called genes .1, 2 In higher organisms, ranging from yeast to plants and man, practically all genes are interrupted, with sequences coding for protein (coding exons ) separated by regions of non-coding DNA called introns . The …

Regulation and Organization of Human Troponin Genes

In spite of considerable advances, understanding the mechanisms that regulate correct temporal and spatial gene expression during development remains one of the major challenges of molecular biology. During cardiac development, intricate patterns of gene expression underlie the processes of precursor cell specification and differentiation. Subsequent alterations in expression can be identified that correlate both with the formation of distinct cell types, including ventricular, atrial, and conduction system myocytes, and with the general process of myocyte maturation. One approach to identifying mechanisms that regulate these processes is through the dissection of specific regulatory pathways required for expression of particular genes, and this has led to the identification of a number of important transcriptional pathways. We have chosen to investigate the regulation of expression of troponin genes in the human heart as a route to identifying such pathways.