title: Diazoxide postconditioning induces mitochondrial protein S-nitrosylation and a redox-sensitive mitochondrial phosphorylation/translocation of RISK elements: no role for SAFE.
abstract: Postconditioning (PostC) can be obtained either with brief cycles of ischemia/reperfusion (I-PostC) or with a direct targeting of mitochondria with Diazoxide (pharmacological PostC, P-PostC). I-PostC may induce the activation of RISK and SAFE pathways and may favor nitric oxide production with S-Nitrosylation of proteins and redox signaling. It is not clear whether Diazoxide can lead to similar effects. We compared the effects of I-PostC and P-PostC on (a) kinases of RISK- and SAFE pathway, (b) S-Nitrosylation of mitochondrial proteins and (c) reduction of death signals (PKCδ, cleaved caspase-3 and Beclin-1) in cytosolic and mitochondrial fractions. Isolated rat hearts underwent (1) perfusion without ischemia (Sham), (2) ischemia/reperfusion (30-min ischemia plus 2-h reperfusion), (3) I-PostC (5 intermittent cycles of 10-s reperfusion and 10-s ischemia immediately after the 30-min ischemia), (4) P-PostC (Diazoxide 30 μM in the first of 3-min of reperfusion) or (5) I-PostC + MPG or P-PostC + MPG (MPG, 2-mercaptopropionylglycine 300 μM). Using Western blot and biotin switch assay, we found that P-PostC induced a redox sensible phosphorylation/translocation of Akt, ERK1/2 and GSK3β into the mitochondria, but not of phospho-STAT3, which was translocated into the mitochondria by I-PostC only. Either I-PostC or P-PostC increased mitochondrial S-Nitrosylated proteins (e.g., VDAC) and reduced the levels of phospho-PKCδ, cleaved caspase-3 and Beclin-1. Therefore, direct targeting of mitochondria with Diazoxide (a) activates the RISK pathway via a redox signaling, (b) favors discrete mitochondrial protein S-Nitrosylation, including VDAC and (c) decreases signals of death. Intriguingly, phospho-STAT3 translocation is induced by I-PostC, but not by P-PostC, thus suggesting a redox-independent mechanism in the SAFE pathway.
authors.affiliation: Dipartimento di Scienze Cliniche e Biologiche, Facoltà di Medicina e Chirurgia S. Luigi Gonzaga, Università di Torino, Regione Gonzole 10, 10043, Orbassano, TO, Italy.
authors.forename: C
authors.initials: C
authors.lastname: Penna
authors.forename: M-G
authors.initials: MG
authors.lastname: Perrelli
authors.forename: F
authors.initials: F
authors.lastname: Tullio
authors.forename: C
authors.initials: C
authors.lastname: Angotti
authors.forename: A
authors.initials: A
authors.lastname: Camporeale
authors.forename: V
authors.initials: V
authors.lastname: Poli
authors.forename: P
authors.initials: P
authors.lastname: Pagliaro
mesh_terms: D000818:Animals; D015153:Blotting, Western; D003981:Diazoxide / Q000494:pharmacology*; D006321:Heart / Q000187:drug effects*; D057775:Ischemic Postconditioning / Q000379:methods*; D008297:Male; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D024101:Mitochondrial Proteins / Q000378:metabolism; D015428:Myocardial Reperfusion Injury / Q000378:metabolism; D009924:Organ Culture Techniques; D010084:Oxidation-Reduction / Q000187:drug effects; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction / Q000187:drug effects* / Q000502:physiology; D014665:Vasodilator Agents / Q000494:pharmacology*
chemical_list: D024101:Mitochondrial Proteins; D014665:Vasodilator Agents; D011494:Protein Kinases; D003981:Diazoxide