Regulation of atrial natriuretic peptide secretion in cardiomyocytes by Rab3a and zDHHC9-mediated palmitoylation of Rab3gap1.

Abstract

Natriuretic peptides are secreted by the heart in response to cardiovascular stress to reduce blood pressure and volume and thus there is interest in bolstering natriuretic peptide levels to improve outcomes in heart failure. We uncovered novel functions for the Golgi S-acyltransferase, zDHHC9, in regulating atrial natriuretic peptide (ANP) secretion in cardiomyocytes. zDHHC9 palmitoylates Rab3 GTPase activating protein 1 (Rab3gap1) in cardiomyocytes both in vitro and in vivo, resulting in enhanced Rab3gap1 retention at the Golgi, elevation of Rab3a-GTP levels, and a profound expansion of Rab3a-positive vesicles at the cardiomyocyte periphery in contrast to predominant Golgi localization in control cardiomyocytes. However, these zDHHC9-induced effects were associated with impairment of ANP release, suggesting zDHHC9-mediated disruption of Rab3a nucleotide cycling interferes with exocytosis of ANP. Consistent with this, overexpression of wildtype Rab3gap1 that promotes Rab3a GTP:GDP turnover greatly accelerated ANP secretion whereas overexpression of a Rab3gap1 GAP-defective mutant lacked this effect. Phenylephrine (PE) treatment induced Rab3gap1 palmitoylation, Rab3a-GTP levels, and Rab3a-containing post-Golgi vesicles, but this was associated with a substantial and well-established increase in ANP biosynthesis and secretion. Knockdown of zDHHC9 blunted PE-induced Rab3a GTP-loading and expansion of Rab3a-associated vesicles but evoked even greater PE-stimulated ANP release, suggesting Rab3gap1 palmitoylation provides an intrinsic regulatory mechanism to modulate cardiomyocyte exocytosis and ANP secretion. Importantly, Rab3a localized to atrial granules in vivo and transgenic mice with cardiomyocyte-specific overexpression of zDHHC9 exhibited dysregulated atrial granule morphology, accumulation of ANP in atrial cardiomyocytes, and reduced serum ANP levels, suggesting a defect in ANP release in zDHHC9-overexpressing hearts. Collectively, these data implicate zDHHC9-mediated palmitoylation of Rab3gap1 in disruption of Rab3a GTP:GDP cycling in cardiomyocytes required for proper ANP release, thereby highlighting novel roles for zDHHC9, Rab3gap1, and Rab3a in modulating ANP secretion.