Detection of C-type natriuretic peptide compared with brain and atrial natriuretic peptide transcripts in human heart by the polymerase chain reaction

Abstract

Nemer 1 1 Laboratory of Molecular Genetics, Clinical Research Institute, Montreal 2 Medizinische Klinik II, Klinikum Groghadern, Universit/it Mtinchen C-type natriuretic peptide (CNP), initially isolated from porcine brain [3], is the most recently characterized member of the natriuretic peptide family. The human CNP gene consists of at least two exons and an intron [4]. Whereas synthesis of atrial and brain natriuretic peptides (ANP and BNP) has been demonstrated mainly in the heart, but also in various other organs [2, 5], CNP mRNA in man has so far been found only in the central nervous system. We have investigated human cardiac tissues for CNP expression and compared the abun- dance of CNP transcripts with that of ANP and BNP. Atrial and ventricular tissues were obtained from a 76-year-old female patient with congestive heart failure at autopsy. Extrac- tion of total RNA, assessment of RNA integrity, RNA quantifi- cation and cDNA synthesis by reverse transcriptase were per- formed as described in detail elsewhere [1]. The sense and anti- sense primers used for PCR amplification corresponded to se- quences in the first and second exon of the ANP, BNP and CNP genes, respectively. Thus, amplification of the cDNA and genomic DNA templates yielded fi'agments of 445 bp and 576 bp for ANP, 291 bp and 522 bp for BNP, and 358 bp and 802 bp for CNP. Various amounts (60-180 ng) of cDNA and 85 ng of human genomic DNA were coamplified, allowing for quantitation of the cDNA relative to total cDNA [1]. Amplifi- cation products were separated by agarose gel electrophoresis, transferred to nylon membranes (Hybond, Amersham) and hy- bridized to end-labelled oligonucleotides, corresponding to se- quences located between the two sequences used as PCR primers. Quantification was performed by image analysis densitometry of the hybridization bands. CNP transcripts of similar size were detected in brain, atrial and ventricular cDNA. Omission of cDNA served as negative control (Fig. 1). As summarized in Table 1, CNP transcripts were about three orders of magnitude less abundant than ANP and BNP transcripts in both atrial and ventricular tissue. We here present evidence for CNP expression in the human heart. Genomic