A novel alternative spliced variant of the brain natriuretic peptide gene is superior to BNP as a molecular marker of ventricular dysfunction

Abstract

Background and Aim: Two mRNA isoforms of BNP (i.e., immature and mature transcripts) were found to be expressed in both normal and diseased porcine heart. Given that there were indications suggesting that heart failure (HF) may be associated with production of various isoforms of BNP, we searched for novel variants of alternative splicing of BNP in the porcine model of HF. Methods: HF was induced in 6-day-old neonatal piglets by a single i.v. injection of 2 mg/kg of Doxorubicin (Dox). To profile the cardiac gene expression changes associated with Dox-induced HF, a non-radioactive mRNA differential display (DDRT-PCR) was used. A novel alternative spliced variant of the BNP gene (designated E2-BNP) was identified, characterized, cloned and expressed. E2-BNP mRNA levels were determined by semi-quantitative and qRT-PCR. Results: On day 24 after Dox-administration, experimented piglets developed the features of diastolic HF. Using DDRT-PCR, the band corresponding to the E2-BNP was identified as being overexpressed in failing versus normal piglet myocardium. The sequence of this band displayed a 100% homology with exon 1 and exon 3 sequences of BNP. Further RT-PCR amplification of BNP (through exons 1-3) from the failing piglet myocardium cDNA produced three products, two of which were identical to the reported BNP immature and mature transcripts, whereas the third one was characterized by the precise deletion of the exon 2. Skipping of exon 2 ( E2) causes a frame-shift from the beginning of exon 3, generating a coding sequence for a new protein with no C-terminal homology to known natriuretic peptides. In newborn piglets (n=5), the E2-BNP mRNA was 15 fold more abundant in the left ventricle (LV) than in the right ventricle (RV). During development, the E2-BNP gene expression is rapidly downregulated in both ventricles being, at postnatal day 30, 10-20-fold lower as compared to newborn piglets. By contrast, in 30-day-old piglets with Dox-induced HF (n=12) the E2-BNP mRNA levels were found to be consistently and significantly augmented in both ventricles being much more up-regulated in the RV (40±2.6 fold) than in the LV (10±1.6 fold) as compared with age-matched controls. The average fold-increase of the BNP mRNA content in the failing porcine RV and LV was only 6.3±0.5 and 5.1±0.3, respectively. Conclusion: As such, variation in E2-BNP mRNA transcript abundance within the ventricular wall can be used to diagnose heart conditions in large-animal models of HF. In addition, the E2-BNP seems to be superior to BNP as a molecular marker for HF.