Cyclophilin D ablation is associated with increased end-ischemic mitochondrial hexokinase activity

Abstract

Both the absence of cyclophilin D (CypD) and the presence of mitochondrial bound hexokinase II (mtHKII) protect the heart against ischemia/reperfusion (I/R) injury. It is unknown whether CypD determines the amount of mtHKII in the heart. We examined whether CypD affects mtHK in normoxic, ischemic and preconditioned isolated mouse hearts. Wild type (WT) and CypD^-/- mouse hearts were perfused with glucose only and subjected to 25 min ischemia and reperfusion. At baseline, cytosolic and mtHK was similar between hearts. CypD ablation protected against I/R injury and increased ischemic preconditioning (IPC) effects, without affecting end-ischemic mtHK. When hearts were perfused with glucose, glutamine, pyruvate and lactate, the preparation was more stable and CypD ablation^-resulted in more protection that was associated with increased mtHK activity, leaving little room for additional protection by IPC. In conclusion, in glucose only-perfused hearts, deletion of CypD is not associated with end-ischemic mitochondrial-HK binding. In contrast, in the physiologically more relevant multiple-substrate perfusion model, deletion of CypD is associated with an increased mtHK activity, possibly explaining the increased protection against I/R injury.

Figure 1

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.

Figure 2

Baseline characteristics of WT and CypD^−/− hearts. (a) Total lactate dehydrogenase (LDH) activity of CypD^−/− hearts normalized to protein content. (b) Hexokinase activity in whole heart normalized to protein content. (c) Mitochondrial citrate synthase (CS) activity normalized to protein content as parameter of mitochondrial capacity. (d) Mitochondrial HK activity as ratio to CS activity. (e) Cytosolic HK activity normalized to protein content. (f) Representative Western blot images of mitochondrial HKII/VDAC and (g) cytosolic HKII/tubulin. (h) The amount of mitochondrial HKII as ratio to VDAC normalized to WT. (i) The amount of cytosolic HKII as ratio to tubulin normalized to wt. n = 6 per group. Data are shown as mean + SEM. Differences between groups were analysed using Mann-Whiney U test *p < 0.05.

Figure 3

CypD ablation decreases I/R injury and CypD^−/− hearts are protected by IPC in hearts perfused with glucose only. (a) Lactate dehydrogenase (LDH) release during reperfusion as a percentage of total LDH in the heart. (b) Rate pressure product (RPP) at the end of reperfusion as percentage of baseline values. (c) End-diastolic pressure (EDP) at the end of reperfusion. (d) Developed left ventricular pressure (DLVP) at the end of reperfusion. N = 7–13 per group. Data are shown as mean + SEM. Mann-Whitney U tests were performed to test for significance between 1) control treated WT and CypD^−/− animals 2) control and IPC treated animals in WT and 3) CypD^−/− animals. *p < 0.05.

Figure 4

CypD and IPC have no effect on end-ischemic mtHKII in hearts perfused with glucose only. (a) End-ischemic mitochondrial HK activity as ratio to CS activity. (b) Cytosolic HK activity normalized to protein content. (c) Representative Western blot images of mitochondrial HKII/VDAC and (d) cytosolic HKII/tubulin. The amount of end-ischemic mitochondrial (e) and cytosolic (f) HKII as ratio of VDAC and tubulin respectively, normalized to WT control. n = 6 per group. Data are shown as mean + SEM. Mann-Whitney U tests were performed to test for significance between 1) control treated WT and CypD^−/− animals 2) control and IPC treated animals in WT and 3) CypD^−/− animals. p < 0.05.

Figure 5

Metabolic substrates influence time of contracture during ischemia. Diastolic pressure (a + c) and time of contracture (b + d) during ischemia and perfusion pressure (Pperf) at the end of baseline (e + f) in the presence of glucose only (a + b + e) or glucose, pyruvate, lactate and L-glutamine (c + d + f). (g) Glycogen content in the heart after 30 min basal perfusion with the different perfusates. n = 12–19 (a–f) and n = 6–7 (g) per group. Data are shown as mean + SEM. For TOC Mann-Whitney U tests were performed to test for significance between 1) control treated WT and CypD^−/− animals 2) control and IPC treated animals in WT and 3) CypD^−/− animals. Glycogen data weas analysed using an Independent T-test. *p < 0.05.

Figure 6

CypD ablation reduces I/R injury in hearts perfused with glucose, pyruvate, lactate and glutamine. (a) Lactate dehydrogenase (LDH) release during reperfusion as a percentage of total LDH in the heart. (b) Rate pressure product (RPP) at the end of reperfusion as percentage of baseline values. (c) End-diastolic pressure (EDP) at the end of reperfusion. (d) Developed left ventricular pressure (DLVP) at the end of reperfusion. n = 6–7 per group. Data are shown as mean + SEM. Mann-Whitney U tests were performed to test for significance between 1) control treated WT and CypD^−/− animals 2) control and IPC treated animals in WT and 3) CypD^−/− animals. *p < 0.05.

Figure 7

CypD and IPC have no effect on end-ischemic mtHKII in hearts perfused with glucose, pyruvate, lactate and glutamine. (a) End-ischemic mitochondrial HK activity as ratio to CS activity. (b) Cytosolic HK activity normalized to protein content. (c) Representative Western blot images of mitochondrial HKII/VDAC and (d) cytosolic HKII/tubulin. The amount of end-ischemic mitochondrial (e) and cytosolic (f) HKII as ratio of VDAC and tubulin respectively, normalized to WT control. (g) Representative Western blot image and quantification (h) of whole homogenate pAkt/Akt, normalized to WT control. n = 6 per group. Data are shown as mean + SEM. Mann-Whitney U tests were performed to test for significance between 1) control treated WT and CypD^−/− animals 2) control and IPC treated animals in WT and 3) CypD^−/− animals. *p < 0.05.