Abstract 3152: Soluble Natriuretic Peptide Receptor-related Fragment (sNRF), An Alternative Product of the Natriuretic Peptide Receptor Gene, Regulates Receptor Function
In heart failure, the beneficial effects of natriuretic peptides (NPs) are blunted, in part, due to NP receptor (NPRA) unresponsiveness caused by an unknown mechanism. We have recently identified a novel human cDNA transcript that encodes a cytosolic protein comprising the C terminus of NPRA (NPRA820–1061) that we have named sNRF. sNRF expression blocks NP-induced PKG membrane translocation. In primary cultures of neonatal rat cardiac fibroblasts (CF), sNRF expression inhibits ligand-induced NPRA phosphorylation and reverses NP’s inhibitory effects on alpha-smooth muscle actin expression, a key marker of CF differentiation. Thus, sNRF appears to be an endogenous protein that regulates NPRA and downstream NP action. The 5’ end of the sNRF cDNA sequence extends 215 nt into intron 15 of the NPRA gene, followed by exons 16–22 of the fully spliced NPRA mRNA sequence. To verify the presence of sNRF mRNA in human tissue, we analyzed mRNA extracted from human heart on a Northern blot with a probe comprised of the 215-nt intron 15 sequence. A doublet was identified at approximately 1.5 kb, consistent with the size predicted for an alternative NPRA gene-derived transcript initiating in intron 15. To determine if the sNRF mRNA was differentially expressed in human heart disease, total mRNA was extracted from seventeen hearts explanted from patients undergoing cardiac transplantation because of a variety of cardiac diseases. Quantitative real-time RT-PCR using primers corresponding to the intron 15 sequence and the 3’ end of NPRA exon 17 revealed significant and highly variable amounts of endogenous sNRF mRNA. The size of the PCR product was that predicted from the presence of intron 15 and the splicing out of intron 16, thus excluding the possibility that the PCR product was derived from a partially processed NPRA pre-mRNA. A separate analysis of poly(A)+ RNA from a normal heart and from an explanted heart from a patient with dilated cardiomyopathy showed a 5.7-fold increase in sNRF mRNA in the diseased heart. We conclude that sNRF expression inhibits critical NPRA functions. sNRF mRNA is expressed in the human heart and appears to be increased in the clinical setting of heart failure. Whether sNRF inhibition reverses NP resistance in heart failure remains to be determined.