Pro-B-type natriuretic peptide-1-108 processing and degradation in human heart failure

Abstract

Background: We have reported that pro-B-type natriuretic peptide (BNP)-1-108 circulates and is processed to mature BNP1-32 in human blood. Building on these findings, we sought to determine whether proBNP1-108 processed forms in normal circulation are biologically active and stimulate cGMP, and whether proBNP1-108 processing and activity are altered in human heart failure (HF) compared with normal. Because BNP1-32 is deficient whereas proBNP1-108 is abundant in HF, we hypothesize that proBNP1-108 processing and degradation are impaired in HF patients ex vivo.

Methods and results: We measured circulating molecular forms, including BNP1-32, proBNP1-108, and N-terminal-proBNP, and all were significantly higher in patients with HF when compared with that in normals. Fresh serum samples from normals or patients with HF were incubated with or without exogenous nonglycosylated proBNP1-108 tagged with 6 C-terminal Histidines to facilitate peptide isolation. His-tag proBNP1-108 was efficiently processed into BNP1-32/3-32 at 5 minutes in normal serum, persisted for 15 minutes, then disappeared. Delayed processing of proBNP1-108 was observed in HF samples, and the degradation pattern differed depending on left ventricular function. The 5-minute processed forms from both normal and HF serums were active and generated cGMP via guanylyl cyclase-A receptors; however, the 180-minute samples were not active. The proBNP1-108 processing enzyme corin and BNP-degrading enzyme dipeptidyl peptidase-4 were reduced in HF versus normal, perhaps contributing to differential BNP metabolism in HF.

Conclusions: Exogenous proBNP1-108 is processed into active BNP1-32 and ultimately degraded in normal circulation. The processing and degradation of BNP molecular forms were altered but complete in HF, which may contribute to the pathophysiology of HF.

Keywords: B-type natriuretic peptide; enzymes; heart failure.

Figure 1. ProBNP1-108 processing and degradation in normal human serum ex vivo

A: A schema of ex vivo study method. Exogenous proBNP1-108 was incubated in fresh human serum for indicated times at 37°C. Unprocessed or processed proBNP1-108 were isolated by IMPT using His-tag beads, and detected by WB with 6xHis antibody. B: Representative WB for His-tag proBNP1-108 incubated in serum samples from normal subjects for indicated times. C and D: Densitometric analysis of unprocessed proBNP1-108 (C) and processed form (BNP1-32/3-32) (D) at indicated times. Values are mean ± SEM. *p<0.05 vs 0 min, 1-way ANOVA with Bonferroni multiple comparison test.

Figure 2. Ex vivo ProBNP1-108 processing and degradation in HF and circulating corin and DPPIV levels

A: Representative WB of proBNP1-108 processing in HF serum. B and C: Densitometric analysis of unprocessed proBNP1-108 (B) and processed form (BNP1-32/3-32) (C) at indicated times from normal vs HF patients. Values are mean ± SEM. *p<0.05 vs 0 min, 1-way ANOVA with Bonferroni multiple comparison test. †p<0.05 vs normals in indicated times, 2-way ANOVA with Bonferroni multiple comparison test. D and E: Circulating corin (D) and DPPIV (E) levels in normal vs HF serum. Values are mean ± SEM.

Figure 3. ProBNP1-108 processing and degradation based on %EF and high proBNP1-108 concentration

A and B: Sub-analysis of ex vivo ProBNP1-108 processing and degradation in HF by %EF. Densitometric analysis of unprocessed proBNP1-108 (A) and processed form (BNP1-32/3-32) (B) at indicated times; EF<50% (closed square), EF≥50% (grayed circle), or normals (opened circle with breaking line). Values are mean ± SEM. p values were shown in graphs analyzed by 2-way ANOVA with Bonferroni multiple comparison test. No significant difference between groups at indicated times, 2-way ANOVA with Bonferroni multiple comparison test. C and D: Ex vivo ProBNP1-108 processing and degradation in normals with or without pretreatment with proBNP1-108. Representative WB for His-tag proBNP1-108 incubated in serum samples from normal subjects (n=4) for indicated times. Samples were pretreated with or without 500 pg/ml non-glycosylated proBNP1-108 (C) or glycosylated proBNP1-108 (D) before treatment with His-tag proBNP1-108.

Figure 4. cGMP response in GC-A or GC-B expressing HEK293 cells

A: GC-A or GC-B transfected HEK cells were treated with synthetic BNP1-32 and proBNP1-108 at 10⁻⁸M concentration for 10 min. B: GC-A or GC-B transfected HEK cells were treated with isolated peptides from 10⁻⁸M proBNP1-108 incubated serums as indicated for 10 min. Values are mean ± SEM from 3 samples from normals or HF patients. *p<0.05 vs no treatment. †p<0.05 vs ProBNP1-108 in PBS.