Pro-atrial natriuretic peptide1-126 is processed in the human circulation to a mature GC-A activating peptide with therapeutic potential in heart failure

Abstract

Purpose: Atrial natriuretic peptide (ANP), a stimulator of cGMP through its receptor, GC-A, circulates in various molecular forms in humans. Importantly, mature ANP1-28 and Urodilatin, which is ANP1-28 plus 4 additional N-terminus added amino acids, are used as drugs for acute decompensated heart failure (HF). GC-A activation in the heart is characterized by anti-hypertrophic and anti-fibrotic responses in vivo and in vitro. To date it remains unclear if the precursor; proANP1-126 is processed by convertase corin into active forms in the circulation of normal and HF with the possibility that if converted could serve as a long-acting ANP like peptide for HF. Studies report that corin is reduced in the circulation in HF and thus proANP processing might be impaired. Methods: Fresh serum was obtained from normals (n=13) and patients with NYHA III to IV HF (n=14). Samples were incubated with or without Histidine-tagged exogenous proANP1-126 in the C-terminus to facilitate isolation of processed forms which were isolated by immunoprecipitation and detected by Western Blot (WB), and then sequenced. GC-A or GC-B expressing HEK293 cells were treated with/without mature ANP1-28, proANP1-126, the isolated peptides from proANP1-126 in serum, and the cGMP activity determined by specific assay was measured so as to test biological activity of processed ANP. In addition, cGMP activity and TGF beta-1 stimulated Collagen type I (Col I) mRNA expression measured by qPCR in human cardiac fibroblasts (CFs) in which corin protein exist were examined with or without treatment with ANP1-28 or proANP1-126. Results: ProANP1-126 was processed into ANP1-28 in normal serum; processed ANP1-28 appeared at 5 min and then decreased but remained present at 180 min. In HF serum proANP1-126 was processed at the same rate as in normals The processed peptide from proANP1-126 as well as unprocessed proANP1-126 stimulated cGMP production in GC-A cells, but processed peptide had significantly higher cGMP stimulation than unprocessed proANP1-126. In addition, proANP1-126 had equivalent cGMP activating and Col I mRNA inhibiting actions compared to ANP in CFs. Conclusions: Exogenous proANP1-126 is processed into biologically active GC-A peptides in both normal and HF human serum. ProANP1-126 and its processed form possess significant cGMP activating and anti-fibrotic actions in vitro in human CFs. Importantly, proANP1-126 appears to have a longer half-life than mature ANP1-28 ex vivo, suggesting the emerging concept that proANP1-126 may have a therapeutic opportunity as a long acting GC-A agonist for HF.