Raffaella Di Lisi

An atrioventricular canal domain defined by cardiac troponin I transgene expression in the embryonic myocardium.

During early cardiac development the atrial myocardium is continuous with the ventricular myocardium throughout the atrioventricular canal. The atrioventricular canal undergoes complex remodelling involving septation, formation of atrioventricular valves and insulation between atria and ventricles except at the level of the atrioventricular node. Understanding of these processes has been hampered by the lack of markers specific for this heart region. We have generated transgenic mice expressing β-galactosidase under the control of the cardiac troponin I gene that show transgene expression mainly confined to the atrioventricular canal myocardium during early embryonic development. With further development β-galactosidase positive cells are observed in the atrioventricular node and in the lower rim of both right and left atria, supporting the view that atrioventricular canal myocardium contributes to the atrioventricular node and is in part incorporated into the lower rim of the atria. These results identify the atrioventricular canal myocardium as a distinct transcriptional domain.

Developmental expression of the SH3BGR gene, mapping to the Down syndrome heart critical region.

The SH3BGR gene has been recently isolated and mapped to chromosome 21 within the Down syndrome (DS) congenital heart disease (CHD) minimal region. As a first step to evaluate the possible involvement of SH3BGR in CHD that affect 40% of DS patients, we have analyzed by in situ hybridization the expression pattern of the mouse homolog gene (Sh3bgr), during development. Our results show that Sh3bgr is already expressed at embryonic day 7.75 (E7.75) in the precardiogenic mesoderm and that from E8.5 to E10.5 its expression is restricted to the heart. In subsequent developmental stages, Sh3bgr transcripts are also detected in skeletal muscle and in some visceral smooth muscles including urinary bladder and gut wall, but not in vascular smooth muscle. Our results, demonstrating that Sh3bgr is expressed in earliest stages of mouse heart development, support a possible role of this gene in heart morphogenesis and, consequently, in the pathogenesis of CHD in DS.

GATA elements control repression of cardiac troponin I promoter activity in skeletal muscle cells

BackgroundWe reported previously that the cardiac troponin I (cTnI) promoter drives cardiac-specific expression of reporter genes in cardiac muscle cells and in transgenic mice, and that disruption of GATA elements inactivates the cTnI promoter in cultured cardiomyocytes. We have now examined the role of cTnI promoter GATA elements in skeletal muscle cells.ResultsMutation or deletion of GATA elements induces a strong transcriptional activation of the cTnI promoter in regenerating skeletal muscle and in cultured skeletal muscle cells. Electrophoretic mobility shift assays show that proteins present in nuclear extracts of C2C12 muscle cells bind the GATA motifs present in the cTnI promoter. However, GATA protein complex formation is neither reduced nor supershifted by antibodies specific for GATA-2, -3 and -4, the only GATA transcripts present in muscle cells.ConclusionThese findings indicate that the cTnI gene promoter is repressed in skeletal muscle cells by GATA-like factors and open the way to further studies aimed at identifying these factors.

Molecular Genetics of Congenital Heart Disease: A Problem of Faulty Septation

The transition from the single circulation of the embryo to the double circulation of the neonatal and adult heart involves the transformation of the primitive heart tube through a complex morphogenetic process, resulting in completely separated right and left heart chambers and distinct pulmonary and systemic circulations. Septation of heart chambers starts at early stages in embryogenesis and is completed only at birth with the closure of the foramen ovale. Defects in heart septation, including atrial septal defects, atrioventricular canal septal defects, ventricular septal defects, and conotruncal septal defects, represent a major cause of congenital heart disease. The molecular basis of faulty heart septation is now the object of intensive investigation. Mutant genes coding for 2 transcription factors, TBX5 and NKX2.5, have been recently identified by positional cloning in families with high incidence of atrial or ventricular septal defects. Mutations of the TBX5 gene cause the Holt-Oram syndrome, a developmental disorder affecting the heart and the upper limb, the most frequent cardiac abnormalities being atrial and/or ventricular septal defects and conduction defects.1 2 Mutations of the homeobox transcription factor NKX2.5 cause nonsyndromic congenital heart disease, in particular, atrial septal defects and atrioventricular node dysfunction.3 Most mutations so far identified in the TBX5 and NKX2.5 genes induce the formation of truncated proteins resulting in haploinsufficiency. On the other hand, the atrioventricular canal septal defects frequently associated with Down syndrome are probably due to gene overexpression rather than deficiency, namely the presence of 3 copies of chromosome 21 genes. The study of rare patients with partial chromosome 21 trisomy and atrioventricular canal septal defects has allowed the definition of a 2.5-Mb critical region at 21q22.2-q22.3, which is responsible for cardiac malformations: this region should therefore contain one or more genes whose overexpression interferes with correct atrioventricular canal septation.4 Progress …

Heart morphogenesis is not affected by overexpression of the Sh3bgr gene mapping to the Down syndrome heart critical region

Congenital heart disease (CHD) is the most common birth defect in humans and is present in 40% of newborns affected by Down syndrome (DS). The SH3BGR gene maps to the DS-CHD region and is a potential candidate for the pathogenesis of CHD, since it is selectively expressed in cardiac and skeletal muscle. To determine whether overexpression of Sh3bgr in the murine heart may cause abnormal cardiac development, we have generated transgenic mice using a cardiac- and skeletal-muscle-specific promoter to drive the expression of a Sh3bgr transgene. We report here that heart morphogenesis is not affected by overexpression of Sh3bgr.

A Cardiac-Specific Troponin I Promoter. Distinctive Patterns of Regulation in Cultured Fetal Cardiomyocytes, Adult Heart and Transgenic Mice

Different types of regulatory genes are involved in cardiac muscle development and cardiac gene regulation, including ubiquitous factors, such as SRF, SP1 and TEF-1, and genes coding for specific transcription factors: MADS-box transcription factor genes, such as the MEF2 genes which are also involved in the specification of skeletal and smooth muscle, homeobox genes, such as Nkx2.5, zinc-finger genes of the GATA family, such as GATA 4–6, and bHLH genes, such as dHAND and eHAND [1,2]. Gene regulation seems to require combinatorial interactions between cardiac-specific and ubiquitous factors: for example a physical interaction between Nkx2.5 and SRF is involved in the activation of the cardiac a-actin gene [3]. The study of cardiac gene regulation is complicated by the specific pattern of transcription of each gene, both with respect to temporal specificity during development and spatial specificity in the various heart chambers, presumably reflecting a modular regulation via multiple cis-acting elements [4]. Multiple approaches, including promoter analyses in cultured cells, in adult heart and in transgenic mice, are required to dissect the activity in time and space of cardiac regulatory genes and their combinatorial interactions.