Abstract 12744: O-glycosylated Pro-B-type Natriuretic Peptide is Cleaved Intracellularly by Furin in Ventricular and Atrial Myocytes: Importance of the Distance between the O-glycosylation and Cleavage Sites
Object: We investigated the molecular mechanism underlying the processing of proBNP, levels of which are increased in heart failure.
Methods: Rat neonatal atrial and ventricular myocytes were cultured separately. We examined: (1) molecular forms of secreted and intracellular BNP in atrial and ventricular myocytes; (2) levels of corin and furin mRNA in atrial and ventricular myocytes and the effect their knockdown on proBNP processing; (3) molecular forms of BNP in plasma from rats and humans; (4) the impact of the distance between the glycosylation and cleavage sites in wild-type and mutant human proBNP expressed in rat myocytes transfected with lentiviral vectors. We measured rat and human BNP and proBNP in the medium and plasma by specific immunoassay.
Results: BNP was the major molecular form secreted by atrial (75±9%) and ventricular (85±9%) myocytes, and was the major intracellular form in atrial myocytes (60±5%), but not in ventricular myocytes (42±4%). Transfection of furin siRNA reduced proBNP processing in both atrial and ventricular myocytes and the relative levels of furin mRNA correlated with the relative fraction of BNP in the medium of atrial and ventricular myocytes (r=0.93, r=0.95, p≤ 0.001). BNP was the major molecular form in rat plasma (90±10%), whereas proBNP was the major form in human plasma (72±8%). The relative fraction of human BNP in rat myocytes expressing human proBNP was 60±5%, but increasing the distance between the glycosylation and cleavage sites through mutation, increased the processed fraction correspondingly (Figure). There are nine amino acids between the glycosylation and cleavage sites in rat proBNP, whereas only five amino acids in human proBNP.
Conclusion: These results suggest that proBNP is processed into BNP and N-terminal proBNP intracellularly, most likely by furin. The level of proBNP processing is lower in humans than rats, most likely due to the smaller distance between the O-glycosylation and cleavage sites in humans.
- © 2013 by American Heart Association, Inc.