Abstract 20279: Signalosomes Orchestrate Ouabain-Induced Intracellular Inotropic Signaling

• Published in: Circulation (New York, N.Y.) Vol. 134; no. Suppl_1 Suppl 1; p. A20279
• Main Authors: Garlid, Anders O, Wang, Ding, Ping, Peipei, Garlid, Keith D
• Format: Journal Article
• Language: English
• Published: by the American College of Cardiology Foundation and the American Heart Association, Inc 11.11.2016
• ISSN: 0009-7322; 1524-4539

IntroductionReceptor-mediated, intracellular signaling leading to cardioprotection and inotropy is orchestrated by signalosome. The caveolar vesicles bud off the plasma membrane, move to mitochondria, and use a terminal kinase to phosphorylate an outer membrane protein. This activates an inner membrane PKCε that opens the mitochondrial ATP-sensitive K channel (mitoKATP), which is required for positive inotropy induced by ouabain, Ca, and dobutamine.HypothesisWe propose that positive inotropic signaling is mediated by a signalosome mechanism, whereby activation of its receptor, Na,K-ATPase, instigates the formation of a caveolar signaling complex that opens mitoKATP.MethodsWe isolate signalosomes from Langendorff perfused rat hearts treated with ouabain, use light scattering to assay their ability to open mitoKATP in untreated isolated mitochondria, and investigate their molecular composition, assembly, and regulation using immunocytochemistry, quantitative proteomics, and bioinformatic approaches.ResultsWe characterized the signalosome fraction from ouabain-perfused rat hearts as caveolar in nature and enriched with caveolins 1 and 3, Src, PKCε and both α1 and α2 subunits of Na,K-ATPase. Protection and signalosome formation in the ex vivo perfused heart occurred at sub-inotropic concentrations (10 uM) of ouabain, indicating that the α2 subunit is the signaling component of the pump (p < 0.05, n = 7). Electron microscopy and immunogold labeling confirmed vesicles that were highly decorated with caveolin-3 and ~140 nM in diameter (137 ± 25 nm; n = 48). Ouabain signalosomes from heart opened mitoKATP in mitochondria isolated from untreated hearts and liver through the terminal kinases Src and PKCε, which together phosphorylate an endogenous outer membrane p38 MAPK.ConclusionWe conclude that (1) ouabain binding to the α2-subunit of Na,K-ATPase causes formation of signalosomes that move to mitochondria and activate mitoKATP; (2) terminal kinases Src and PKCε act on mitochondria; (3) they phosphorylate an outer membrane p38 MAPK; and (4) in addition to cardioprotection and inotropy, this is a general mechanism of cell signaling, given that signalosomes from rat heart open mitoKATP in rat liver mitochondria.