TISSUE-SPECIFIC EXPRESSION OF THE HUMAN BRAIN NATRIURETIC PEPTIDE GENE IN CARDIAC MYOCYTES

Brain natriuretic peptide (BNP) is a cardiac hormone constitutively ex pressed in the adult heart. To identify the cis-acting elements involv ed in regulation of the human BNP gene, we subcloned the full-length p romoter (-1818 to +100) and deletions thereof upstream from a lucifera se reporter gene and transiently transfected them into primary culture s of neonatal rat atrial and ventricular myocytes and myocardial fibro blasts. Luciferase activity of the full-length construct was higher in ventricular (39 064+/-8488 relative light units, n=11) and atrial (11 225+/-1907, n=17) myocytes than myocardial fibroblasts (329+/-113, n= 5). Maximal promoter activity in ventricular and atrial myocytes was m aintained by sequences positioned between -1818 and -1283 relative to the transcription start site. Deletion to -1175 resulted in a decrease , whereas further deletion to -500 effected an increase in reporter ac tivity in both cell types. In ventricular and atrial myocytes, deletio n from -500 to -40 reduced luciferase activity 20-fold and 2-fold, res pectively, whereas in myocardial fibroblasts, deletion to -40 upregula ted the BNP promoter 2-fold. Of note, deleting 16 bp between -127 and -111 reduced luciferase activity 7-fold and 4-fold in ventricular and atrial myocytes, respectively, but had essentially no effect on lucife rase activity in fibroblasts. Placement of sequences lying between -12 7 and -40 upstream from a heterologous thymidine kinase promoter resul ted in reporter expression that was 7.4-fold greater than the vector a lone in ventricular myocytes, approximately 2-fold greater in atrial m yocytes, and equivalent to the vector alone in fibroblasts. For study of activity of the human BNP promoter in adult myocytes, either 408 or 97 bp of 5' flanking sequence coupled to the luciferase reporter gene was injected into the apex of adult male Sprague-Dawley rat hearts. A fter 7 days, luciferase activity in the injected myocardium was 9.8-fo ld higher for the longer construct (70 683+/-14 744 versus 7223+/-3920 , n=4, P<.01), consistent with our in vitro data. These data indicate that (1) the full-length human BNP promoter is more active in ventricu lar versus atrial myocytes and essentially inactive in fibroblasts, (2 ) the distal BNP promoter contains both positive and negative regulato ry elements, (3) a region of the proximal BNP promoter located between -127 and -40 confers tissue specificity, and (4) the BNP promoter is active after injection into the adult rat heart.