Abstract
ObjectivesStress-inducible heat shock protein 22 (Hsp22) confers protection against ischemia through induction of the inducible isoform of nitric oxide synthase (iNOS). Hsp22 overexpression in vivo stimulates cardiac mitochondrial respiration, whereas Hsp22 deletion in vivo significantly reduces respiration. We hypothesized that Hsp22-mediated regulation of mitochondrial function is dependent upon its mitochondrial translocation together with iNOS.Methods and ResultsAdenoviruses harboring either the full coding sequence of Hsp22 (Ad-WT-Hsp22) or a mutant lacking a N-terminal 20 amino acid putative mitochondrial localization sequence (Ad-N20-Hsp22) were generated, and infected in rat neonatal cardiomyocytes. Compared to β-Gal control, WT-Hsp22 accumulated in mitochondria by 2.5 fold (P<0.05) and increased oxygen consumption rates by 2-fold (P<0.01). This latter effect was abolished upon addition of the selective iNOS inhibitor, 1400W. Ad-WT-Hsp22 significantly increased global iNOS expression by about 2.5-fold (P<0.01), and also increased iNOS mitochondrial localization by 4.5 fold vs β-gal (P<0.05). Upon comparable overexpression, the N20-Hsp22 mutant did not show significant mitochondrial translocation or stimulation of mitochondrial respiration. Moreover, although N20-Hsp22 did increase global iNOS expression by 4.6-fold, it did not promote iNOS mitochondrial translocation.ConclusionTranslocation of both Hsp22 and iNOS to the mitochondria is necessary for Hsp22-mediated stimulation of oxidative phosphorylation.
Highlights
The heart depends on oxidative phosphorylation to supply the large amount of ATP required for its continuous contractile activity[1]
Translocation of both heat shock protein 22/H11 Kinase (Hsp22) and isoform of nitric oxide synthase (iNOS) to the mitochondria is necessary for Hsp22-mediated stimulation of oxidative phosphorylation
The NP-40 digestion showed that Hsp22 was located primarily within the membrane fraction, whereas only a residual amount was found in the matrix and inter-membrane space (IMS)
Summary
The heart depends on oxidative phosphorylation to supply the large amount of ATP required for its continuous contractile activity[1]. Cardiac-specific overexpression of Hsp in a transgenic (TG) mouse provides protection against myocardial ischemia that is powerful to ischemic preconditioning [5] through the induction of the inducible isoform of nitric oxide synthase (iNOS) [5], the effector of the second window of ischemic preconditioning [6]. It has been shown in Drosophila that Hsp localization in mitochondria is due to a translocation mechanism that depends on its N-terminal domain [9]. The TG mouse is characterized by an inhibition of the mitochondrial pathway of apoptosis [5]. These observations support the hypothesis that mitochondrial localization of Hsp might promote both cardiac cell survival and oxidative metabolism. The main purpose of our study was to interrogate the physiological consequence of this mechanism on mitochondrial respiration
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