Petra E.M.H. Habets

Distinct Regulation of Developmental and Heart Disease–Induced Atrial Natriuretic Factor Expression by Two Separate Distal Sequences

Nppa, encoding atrial natriuretic factor, is expressed in fetal atrial and ventricular myocardium and is downregulated in the ventricles after birth. During hypertrophy and heart failure, Nppa expression is reactivated in the ventricles and serves as a highly conserved marker of heart disease. The Nppa promoter has become a frequently used model to study mechanisms of cardiac gene regulation. Nevertheless, the regulatory sequences that provide the correct developmental pattern and ventricular reactivation during cardiac disease remain to be defined. We found that proximal Nppa fragments ranging from 250 bp to 16 kbp provide robust reporter gene activity in the atria and correct repression in the atrioventricular canal and the nodes of the conduction system in vivo. However, depending on fragment size and site of integration into the genome of mice, the fetal ventricular activity was either absent or present in an incorrect pattern. Furthermore, these fragments did not provide ventricular reactivation in heart disease models. These results indicate that the proximal promoter does not provide a physiologically relevant model for ventricular gene activity. In contrast, 2 modified bacterial artificial chromosome clones with partially overlapping genomic Nppa sequences provided appropriate reactivation of the green fluorescent protein reporter during pressure overload–induced hypertrophy and heart failure in vivo. However, only 1 of these bacterial artificial chromosomes provided correct fetal ventricular green fluorescent protein activity. These results show that distinct distal regulatory sequences and divergent regulatory pathways control fetal ventricular activity and reactivation of Nppa during cardiac disease, respectively.

Regulatory modules in the developing heart

Fragments of regulatory DNA of cardiac genes drive reporter gene expression in sometimes unexpected subdomains of the heart. These patterns have revealed that the regulatory DNA of genes consists of distinct subfragments (regulatory modules) that are active in different regions of the developing heart. In this review we give an overview of the activity of regulatory modules in vivo. Furthermore, we investigated the relationship between the activity domains of the regulatory modules, the building blocks of the heart and the developmental patterning of the myocardium. Most of the regulatory modules show a domain of activity broader than the morphological boundary of a cardiac compartment and seem to respond to a patterning program along the antero-posterior axis.

Species-specific differences of myosin content in the developing cardiac chambers of fish, birds, and mammals

Key morphogenetic events during heart ontogenesis are similar in different vertebrate species. We report that in primitive vertebrates, i.e., cartilaginous fishes, both the embryonic and the adult heart show a segmental subdivision similar to that of the embryonic mammalian heart. Early morphogenetic events during cardiac development in the dogfish are long‐lasting, providing a suitable model to study changes in pattern of gene expression during these stages. We performed a comparative study among dogfish, chicken, rat, and mouse to assess whether species‐specific qualitative and/or quantitative differences in myosin heavy chain (MyHC) distribution arise during development, indicative of functional differences between species. MyHC RNA content was investigated by means of in situ hybridisation using an MyHC probe specific for a highly conserved domain, and MyHC protein content was assessed by immunohistochemistry. MyHC transcripts were found to be homogeneously distributed in the myocardium of the tubular and embryonic heart of dogfish and rodents. A difference between atrial and ventricular MyHC content (mRNA and protein) was observed in the adult stage. Interestingly, differences in the MyHC content were observed at the tubular heart stage in chicken. These differences in MyHC content illustrate the distinct developmental profiles of avian and mammalian species, which might be ascribed to distinct functional requirements of the myocardial segments during ontogenesis. The atrial myocardium showed the highest MyHC content in the adult heart of all species analysed (dogfish (S. canicula), mouse (M. musculus), rat (R. norvegicus), and chicken (G. gallus)). These observations indicate that in the adult heart of vertebrates the atrial myocardium contains more myosin than the ventricular myocardium. Anat Rec 268:27–37, 2002.

Expression Systems to Analyze Transgenes in the Heart

Recent insights from molecular and cellular biology have contributed to a better understanding of potential causes of cardiac hypertrophy and heart failure. Especially, the use of transgenic mice has become increasingly useful in determining the molecular mechanisms of cardiac hypertrophy and heart failure. As shown in Table 15-1, there is a diverse and rapidly growing list of genes that can trigger features of hypertrophy and associated cardiomyopathy after their cardiac-specific (over)expression. The transgenic mouse models listed in this table 15-1 have been produced by pronuclear injection into fertilised oocytes. This technique results in a variable number of transgene copies that integrate into random genomic loci. The variable copy number and the random integration may independently affect the level of expression of the transgene, the pattern and the tissue specificity1-3. For example, dense chromatin at the site of integration may result in (partial) silencing of the DNA construct, the presence of regulatory sequences (e.g., enhancers) at the site of integration can result in ectopic expression patterns and the variable number of integrated copies can result in variation in expression level. The severity of these problems increases strongly when decreasing the size of the regulatory DNA fragment that is used to drive the transgene.