S-Palmitoylation Mediates Caveolae Localization and Limits Cysteine Oxidation of GC-1 in Cardiomyocytes

Objectives: The nitric oxide (NO) receptor soluble guanylyl cyclase (GC-1) can localize to cell membrane microdomains, including caveolae. In failing hearts, GC-1 relocalizes away from caveolae and becomes oxidized. How this occurs is unknown. We hypothesized that S-palmitoylation, a reversible, enzyme-catalyzed, post-translational modification that enhances membrane affinity of protein substrates, mediates caveolae-localization and modulates redox regulation of GC-1. Methods: Adult male C57BL/6 mice were subjected to transverse aortic constriction (TAC) versus thoracotomy (Sham). Modified acyl-biotin exchange (ABE) assays were performed to assess S-palmitoylation of GC-1 in failing TAC vs non-failing Sham hearts. Using bioinformatics, we identified 5 conserved cysteines in the GC-1α subunit as potential S-palmitoylation sites. We created cysteine-serine mutants of GC-1α at each and all 5 sites (C3S, C15S, C176S, C497S, C595S, CS*). Mutants and WT GC-1α were expressed in neonatal rat cardiomyocytes (NRCM) and their palmitoylation status compared by ABE. We assessed localization and oxidation of GC-1α (CS* vs WT) by confocal microscopy and redox Western, respectively. Results: In TAC hearts, S-palmitoylation of GC-1α was half of that in Sham ( p =0.02). Likewise, palmitoyl-transferase ZDHHC16 was downregulated in TAC vs Sham by 31% ( p =0.01) at the transcript level and by 56% ( p =0.03) at the protein level. In NRCMs, S-palmitoylation of CS* was markedly diminished (6.9%, p <0.001), while that of C15S, C176S, and C497S were modestly diminished (23.8%, p=0.03; 23.2%, p=0.01; 22.8%, p=0.01), compared to WT (54.7%). Moreover, S-palmitoylation of WT but not CS* was decreased by palmitoylation inhibitor 2-BP (17.8%, p<0.0001). Confocal microscopy showed more diffuse cytosolic distribution of CS* compared to WT. Redox Westerns revealed increased oxidation of CS* vs WT upon H 2 O 2 treatment (1.7-fold increase, p=0.008). Conclusions: Our data support that GC-1α is S-palmitoylated at C15, C176, and C497. S-palmitoylation of GC-1α at these 3 cysteines appears to protect GC-1α against oxidation and to mediate its membrane localization.