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. 2017 Oct 6;7:12749. doi: 10.1038/s41598-017-13096-7

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Schematic overview of the different protocols. All hearts were exposed to 20 min perfusion with either glucose only, or multi-substrate perfusate to stabilize the heart. Hearts of group 1 were then homogenized and mitochondria were isolated for determination of mitochondrial hexokinase amount and activity. Hearts of group 2 were exposed to 30 min baseline perfusion after which they were frozen and used for cardiac energetics and glycogen determination. Hearts of group 3 were exposed to 35 min baseline perfusion with or without IPC, followed by 25 min no-flow ischemia. Then hearts were homogenized and mitochondria isolated for determination of HKII amount and activity and Akt phosphorylation. Hearts of group 4 were exposed to 35 min baseline perfusion with or without IPC, 25 min ischemia and 45 min reperfusion. LDH was sampled at 5, 10, 15, 30 and 45 min reperfusion to determine cardiac damage. Hearts of group 5 were exposed to 20 min baseline perfusion with multi-substrate buffer, followed by 25 min ischemia and 10 min reperfusion. White: baseline perfusion with multi-substrate perfusate, white dotted: baseline perfusion with both glucose-only and multi-substrate perfusate, grey: no-flow ischemia, black: reperfusion.