Sevoflurane-induced cardioprotection depends on PKC-α activation via production of reactive oxygen species R.A. Bouwman, R.J.P. Musters, B.J. van Beek-Harmsen,

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Sevoflurane-induced cardioprotection depends on PKC-α activation via production of reactive oxygen species R.A. Bouwman, R.J.P. Musters, B.J. van Beek-Harmsen, J.J. de Lange, R.R. Lamberts, S.A. Loer, C. Boer British Journal of Anaesthesia Volume 99, Issue 5, Pages 639-645 (November 2007) DOI: 10.1093/bja/aem202 Copyright © 2007 British Journal of Anaesthesia Terms and Conditions

Fig 1 Overview of the randomized experimental design for trabeculae subjected to an I/R-protocol and contractile function measurements (A–E) or immunofluorescent analysis of PKC-α translocation (F–I). SI/R, simulated ischaemia and reperfusion; SEVO, sevoflurane; MPG, n-(2-mercaptopropionyl)-glycine (300 μM); 5-HD, 5-hydroxydecanoic acid sodium (100 μM); ROT, rottlerin (1 μM). British Journal of Anaesthesia 2007 99, 639-645DOI: (10.1093/bja/aem202) Copyright © 2007 British Journal of Anaesthesia Terms and Conditions

Fig 2 The role of PKC-α in sevoflurane-induced cardioprotection. Recovery of the developed force (Fdev,rec) is expressed as a fraction of the initial developed force (Fdev,start). Pretreatment with sevoflurane increased the post-ischaemic contractile force recovery and this protective effect was attenuated in trabeculae treated with Go6976 (50 nM). SI/R, simulated ischaemia and reperfusion; SEVO, sevoflurane; GO, Go6976. Data are mean (sem); time control, n = 6; SI/R, n = 9; SI/R + SEVO, n = 8; SI/R + SEVO + GO, n = 10; SI/R + GO, n = 5. British Journal of Anaesthesia 2007 99, 639-645DOI: (10.1093/bja/aem202) Copyright © 2007 British Journal of Anaesthesia Terms and Conditions

Fig 3 PKC-α translocation in response to sevoflurane in cross-sections of isolated rat trabeculae. (a) In all panels, green represents specific PKC-α staining, blue represents DAPI staining of the nuclei and red represents WGA staining of the sarcolemma. In control trabeculae, PKC-α is predominantly located in the cytosol. After sevoflurane exposure, PKC-α is detected in circumscript dots located within the cytosol (SEVO, white arrows). This sevoflurane-induced PKC-α translocation is effectively attenuated by Go6976 (50 nM) (SEVO + GO). Go6976 itself had no intrinsic effect on PKC-α translocation. (b) PKC-α colocalizes with the mitochondria after sevoflurane-induced preconditioning. In these panels, green represents specific PKC-α staining, blue the DAPI staining of the nuclei, and red the specific antibody against mitochondrial heat-shock protein GRP75. After sevoflurane preconditioning, PKC-α (PKC-α panel, white arrows) translocates towards mitochondria (GRP75 panel, white arrows) indicated by yellow in co-localization analyses (PKC-α + GRP75 panel, white arrows). SEVO, sevoflurane; GO, Go6976. British Journal of Anaesthesia 2007 99, 639-645DOI: (10.1093/bja/aem202) Copyright © 2007 British Journal of Anaesthesia Terms and Conditions

Fig 4 Sevoflurane-induced PKC-α tranlocation in relation to common mediators of cardioprotective signal transduction pathways. In all panels, green represents specific PKC-α staining, blue the DAPI staining of the nuclei, and red the WGA staining of the sarcolemma. Sevoflurane-induced a translocation of PKC-α from the cytosol towards the mitochondria (Fig. 3). This translocation was reduced by scavenging ROS during preconditioning with MPG (300 μM) (SEVO + MPG panel). Interestingly, sevoflurane-induced PKC-α translocation was not attenuated by 5HD (100 μM) (SEVO + 5HD panel) or by ROT (1 μM) (SEVO + ROT panel). British Journal of Anaesthesia 2007 99, 639-645DOI: (10.1093/bja/aem202) Copyright © 2007 British Journal of Anaesthesia Terms and Conditions