Abstract 3032: Cardiac-Specific Over-Expression of H11 Kinase/Hsp22 Regulates Mitochondrial Oxidative Phosphorylation by a Nitric Oxide-Dependent Mechanism Reproducing Ischemic Preconditioning
Ischemic preconditioning (IPC) represents the “gold standard” method of cardioprotection, but it is triggered by repetitive episodes of ischemia/reperfusion (IR), which limits its clinical application, reflecting the need of alternative approaches to activate the mechanisms of IPC. H11 Kinase/Hsp 22 (H11K) is a small heat shock protein up-regulated by IR, which provides cardioprotection equal to IPC through a nitric oxide (NO)-dependent mechanism. A main target of NO-mediated IPC is the mitochondria (M), where NO reduces oxygen consumption (VO2) and radical oxygen species (ROS) production during ischemia. We tested the hypothesis that H11K over-expression modulates M function through NO similarly to IPC. Fresh M were isolated from hearts of transgenic mice (TG) with cardiac-specific over-expression (5-fold) of H11K and compared to wild type mice (WT). Upon equal loading (normalized by PGC1 α abundance and citrate synthase activity) and in presence of either glucose or lipids, M from TG compared to WT showed a significant (P<0.05) increase in both VO2 (substrate-dependent state 2: 139±11% of WT) and ATP synthesis (ADP-dependent state 3: 119±8% of WT). When oxygen demand was increased by the uncoupling agent carbonyl cyanide 4-TMP, mitochondrial respiratory chain (MRC) activity in TG was 150±11% of WT (P<0.05), while releasing less ROS (68±6% of WT; P<0.05), demonstrating improved MRC efficiency. Pretreatment of TG mice with the NO synthase inhibitor L-NG-nitroarginine methyl ester (L-NAME, 20 mg/kg) abolished the increase in MRC states 2 and 3 compared to WT. In hypoxia, a decrease in MRC activity was observed in both WT and TG but it was more important in TG than WT (state 3: 85±3 vs 57±6% of normoxic values in WT and TG, respectively; P<0.05), and such difference was abolished by L-NAME. Two-dimensional gel electrophoresis/mass spectrometry of purified M showed an enrichment in pyruvate dehydrogenase, NADH dehydrogenase and ATP synthase in TG versus WT (P<0.05). Therefore, H11K over-expression stimulates MRC activity and efficiency in normoxia, and decreases MRC in hypoxia through NO-dependent mechanisms. These characteristics replicate those of IPC, thereby placing H11K as a potential tool for prophylactic treatment of IR.