A Single Membrane Protein Required for Atrial Secretory Granule Formation

Abstract

Conflicts:The authors declare no conflict of interest.Classification: Biological Sciences (major); Cell Biology (minor) Abstract:The discovery of atrial secretory granules and the natriuretic peptides stored in them identified the atrium as an endocrine organ. Although neither atrial nor brain natriuretic peptide (ANP, BNP) is amidated, the major membrane protein in atrial granules is Peptidylglycine -Amidating Monooxygenase (PAM), an enzyme essential for amidated peptide biosynthesis. Mice lacking cardiomyocyte PAM (Pam Myh6-cKO/cKO ) are viable, but a gene dosage-dependent drop in atrial ANP and BNP content occurred. Ultrastructural analysis of adult Pam Myh6-cKO/cKO atria revealed a 20-fold drop in the volume fraction of secretory granules and a decrease in peripherally localized Golgi complexes. When primary cultures of Pam 0-Cre-cKO/cKO atrial myocytes (PAM floxed, no Cre recombinase) were transduced with Cre-GFP lentivirus, PAM protein levels dropped, followed by a decline in proANP levels. Expression of exogenous PAM in Pam Myh6-cKO/cKO atrial myocytes produced a dose-dependent increase in proANP content. Strikingly, rescue of proANP content did not require the monooxygenase activity of PAM. Unlike many prohormones, atrial proANP is stored intact and its basal secretion is stimulated by drugs that inhibit Golgi-localized Arf activators. Increased basal secretion of proANP was a major contributor to its reduced levels in Pam Myh6-cKO/cKO myocytes; the inability of these drugs to inhibit basal proANP secretion by Pam Myh6-cKO/cKO myocytes revealed a role for COPI-mediated recycling of PAM to the endoplasmic reticulum. Analysis of atrial coated vesicles and the ability PAM to make fluorescently-tagged proANP accumulate in the cis-Golgi region of cells lacking secretory granules revealed a non-catalytic role for PAM in soluble cargo trafficking early in the secretory pathway. Significance:Transmission electron microscopy of atrial cardiomyocytes revealed dense granules resembling those in endocrine cells and neurons, leading to the discovery of the natriuretic peptides stored in these granules. Subsequent studies revealed features unique to atrial granules, including high level expression of Peptidylglycine -Amidating Monooxygenase (PAM), an enzyme required for the synthesis of many neuropeptides, but not for the synthesis of natriuretic peptides. The discovery that atrial myocytes lacking PAM are unable to produce granules and that PAM lacking its monooxygenase activity can rescue granule formation provides new information about the proANP secretory pathway. A better understanding of the unique features of atrial cell biology should provide insight into atrial fibrillation, the most common cardiac arrhythmia, atrial amyloidosis and heart failure. RESULTS Secretory granules are rarely seen in the atria of Pam Myh6-cKO/cKO miceWe turned to transmission electron microscopy to determine whether the drop in proANP levels observed in the atria of Pam Myh6-cKO/cKO mice (3) were accompanied by a decrease in the num...

Commentary on: Peptidylglycine ?-amidating Monooxygenase is Required for Atrial Secretory Granule Formation

“…Atrial levels of proANP and proBNP protein declined in the absence of PAM (Figure 2D). Using primary cultures of atrial myocytes from Pam cKO/ cKO mice and lentiviral delivery of Cre-recombinase, we established that loss of PAM caused a reduction in proANP content (Figure 3A) [13]. Using primary cultures of atrial myocytes from Pam Myh6-cKO/cKO mice and lentiviral delivery of PAM, we demonstrated that expression of PAM in cells which had not previously expressed PAM was sufficient to rescue proANP content to near normal (Figure 3B).…”

“…Using primary cultures of atrial myocytes from Pam Myh6-cKO/cKO mice and lentiviral delivery of PAM, we demonstrated that expression of PAM in cells which had not previously expressed PAM was sufficient to rescue proANP content to near normal (Figure 3B). PAM lacking one of its essential copper binding sites is enzymatically inactive; strikingly, expression of inactive PAM also restored the proANP content of Pam Myh6-cKO/cKO atrial myocytes to near normal (Figure 3B) [13].…”

“…Mice unable to express PAM in their cardiomyocytes (Pam Myh6-cKO/cKO ) are viable [12]. Electron microscopy revealed the almost total absence of secretory granules in the atria of Pam Myh6-cKO/cKO mice (Figure 2C), with few other changes in their ultrastructure [13]. Atrial levels of proANP and proBNP protein declined in the absence of PAM (Figure 2D).…”

“…The apparent lack of a similar cardiovascular phenotype in the Pam Myh6-cKO/cKO mice [12] suggests that the increased basal secretion of proANP, which is thought to occur via a Golgi bypass pathway in isolated Pam Myh6-cKO/cKO atrial cardiac myocytes, is sufficient to counteract these changes. Neither plasma levels of BNP nor secretion of BNP by Pam Myh6-cKO/cKO atrial myocytes in culture has been assessed, but atrial proBNP was even more depleted than proANP [12,13]. BNP is expressed in both atrial and ventricular myocytes in mice (in a ratio of approx.…”