title: Ischaemic preconditioning preferentially increases protein S-nitrosylation in subsarcolemmal mitochondria.
abstract: Nitric oxide (NO) and protein S-nitrosylation (SNO) have been shown to play important roles in ischaemic preconditioning (IPC)-induced acute cardioprotection. The majority of proteins that show increased SNO following IPC are localized to the mitochondria, and our recent studies suggest that caveolae transduce acute NO/SNO cardioprotective signalling in IPC hearts. Due to the close association between subsarcolemmal mitochondria (SSM) and the sarcolemma/caveolae, we tested the hypothesis that SSM, rather than the interfibrillar mitochondria (IFM), are major targets for NO/SNO signalling derived from caveolae-associated eNOS. Following either control perfusion or IPC, SSM and IFM were isolated from Langendorff perfused mouse hearts, and SNO was analysed using a modified biotin switch method with fluorescent maleimide fluors. In perfusion control hearts, the SNO content was higher in SSM compared with IFM (1.33 ± 0.19, ratio of SNO content Perf-SSM vs. Perf-IFM), and following IPC SNO content significantly increased preferentially in SSM, but not in IFM (1.72 ± 0.17 and 1.07 ± 0.04, ratio of SNO content IPC-SSM vs. Perf-IFM, and IPC-IFM vs. Perf-IFM, respectively). Consistent with these findings, eNOS, caveolin-3, and connexin-43 were detected in SSM, but not in IFM, and IPC resulted in a further significant increase in eNOS/caveolin-3 levels in SSM. Interestingly, we did not observe an IPC-induced increase in SNO or eNOS/caveolin-3 in SSM isolated from caveolin-3(-/-) mouse hearts, which could not be protected with IPC. In conclusion, these results suggest that SSM may be the preferential target of sarcolemmal signalling-derived post-translational protein modification (caveolae-derived eNOS/NO/SNO), thus providing an important role in IPC-induced cardioprotection.
authors.affiliation: Systems Biology Center, National Heart Lung and Blood Institute, National Institutes of Health, 10 Center Drive, Bldg10/Rm8N206, Bethesda, MD 20892, USA sun1@mail.nih.gov.
authors.forename: Junhui
authors.initials: J
authors.lastname: Sun
authors.affiliation: Systems Biology Center, National Heart Lung and Blood Institute, National Institutes of Health, 10 Center Drive, Bldg10/Rm8N206, Bethesda, MD 20892, USA.
authors.forename: Tiffany
authors.initials: T
authors.lastname: Nguyen
authors.affiliation: Systems Biology Center, National Heart Lung and Blood Institute, National Institutes of Health, 10 Center Drive, Bldg10/Rm8N206, Bethesda, MD 20892, USA Proteomics Core Facility, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Angel M
authors.initials: AM
authors.lastname: Aponte
authors.affiliation: Systems Biology Center, National Heart Lung and Blood Institute, National Institutes of Health, 10 Center Drive, Bldg10/Rm8N206, Bethesda, MD 20892, USA.
authors.forename: Sara
authors.initials: S
authors.lastname: Menazza
authors.affiliation: Systems Biology Center, National Heart Lung and Blood Institute, National Institutes of Health, 10 Center Drive, Bldg10/Rm8N206, Bethesda, MD 20892, USA Department of Pathology, Johns Hopkins Medical Institutions, Baltimore, MD 21205, USA.
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Kohr
authors.affiliation: Department of Anesthesiology, VA San Diego Healthcare System and University of California at San Diego, La Jolla, CA 92093, USA.
authors.forename: David M
authors.initials: DM
authors.lastname: Roth
authors.affiliation: Department of Anesthesiology, VA San Diego Healthcare System and University of California at San Diego, La Jolla, CA 92093, USA.
authors.forename: Hemal H
authors.initials: HH
authors.lastname: Patel
authors.affiliation: Systems Biology Center, National Heart Lung and Blood Institute, National Institutes of Health, 10 Center Drive, Bldg10/Rm8N206, Bethesda, MD 20892, USA.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Murphy
authors.affiliation: Department of Pathology, Johns Hopkins Medical Institutions, Baltimore, MD 21205, USA.
authors.forename: Charles
authors.initials: C
authors.lastname: Steenbergen
mesh_terms: D000818:Animals; D051244:Caveolin 3 / Q000235:genetics / Q000378:metabolism*; D019157:Ischemic Preconditioning, Myocardial / Q000379:methods; D008297:Male; D008810:Mice, Inbred C57BL; D008929:Mitochondria, Heart / Q000378:metabolism*; D009206:Myocardium / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000235:genetics; D012508:Sarcolemma / Q000378:metabolism*
chemical_list: D051244:Caveolin 3; D052250:Nitric Oxide Synthase Type III; C496315:Nos3 protein, mouse
keywords: Caveolae; Ischaemic preconditioning; Protein S-nitrosylation; Subsarcolemmal and interfibrillar mitochondria