Exon-skipping brain natriuretic peptide variant is overexpressed in failing myocardium and attenuates brain natriuretic peptide production in vitro

Abstract

[Abstract] Brain natriuretic peptide/natriuretic peptide precursor B (NPPB) is one of the most studied genes in relation to heart failure (HF) conditions. However, it is still unclear as to whether alternative splicing could create NPPB mRNA variants, which may be expressed in normal and diseased myocardium. We aimed to identify and characterize a novel alternatively spliced variant of porcine and human NPPB resulting from exon 2 skipping (designated as ΔE2-NPPB). A variety of conventional molecular, biochemical and immunochemical methods were used to examine the expression and functional consequences of ΔE2-NPPB in vitro and in vivo. The pig ΔE2-NPPB mRNA is effectively translated into stable protein in cell-based assays but, in contrast to normally spliced NPPB, the ΔE2-NPPB protein is not secreted into the media. Co-transfection assays demonstrate that ΔE2-NPPB attenuates production and secretion of normally spliced NPPB, suggesting a negative feedback loop of NPPB signaling through generation of ΔE2-NPPB. The inhibitory effects of ΔE2-NPPB on the expression of NPPB are associated with sequence elements residing in exon 3 of ΔE2-NPPB. In piglets, ΔE2-NPPB gene expression is downregulated in both ventricles after birth, but it is markedly re-activated in the postnatal myocardium in experimental diastolic heart failure. In addition, we demonstrate that the exon-skipped NPPB variants are expressed in the postnatal and adult human myocardium and upregulated at end-stage HF due to dilated cardiomyopathy. Our work uncovers an important role of alternative exon skipping in the regulation of NPPB gene expression, thereby pinpointing a putative new mechanism for post-transcriptional regulation of NPPB production and secretion.