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.
Highlights
Ischemia and reperfusion cause oxidative stress, elevated phosphate concentrations, adenine nucleotide depletion and calcium overload
In the present study we examine in the intact mouse heart whether 1) mitochondrial HK association depends on the presence of cyclophilin D (CypD), 2) CypD effects on I/R injury are mirrored by alterations in end-ischemia mitochondrial hexokinase activity (mtHK), and 3) the suggested loss of ischemic preconditioning (IPC) cardioprotection with CypD ablation prevents end-ischemia mtHK increases
In this study we observed that 1) CypD is not necessary for hexokinase II (HKII) to bind to mitochondria in the heart, 2) deletion of the mitochondrial CypD enzyme augments hexokinase activity at the mitochondria during cardiac ischemia in the all substrates series, 3) CypD is not always mandatory for IPC protective effects, 4) glucose as sole metabolic substrate is insufficient for the Langendorff perfused mouse
Summary
Oregon Health and Science University, Oregon, USA This mouse was first described by Basso et al.[4] and has a deletion in the Ppif gene, coding for CypD. A heterozygous CypD+/− mouse was created by crossing CypD−/− males with C57BL/6 J females (Harlan). A WT and CypD−/− line were created from these CypD+/− animals. Cardiac energetics experiment were performed in 20–25 g male C57BL/6 J mice (Harlan). Isolated heart perfusion was performed as described previously with slight modifications[23,24]. A water-filled polyethylene balloon was inserted in the left ventricular cavity and end diastolic pressure (EDP) was set at ~4–8 mmHg. Hearts were continuously submerged in 37 °C KHB. Isolated hearts were Langendorff perfused for ~20 min to reach stable conditions after which the hearts were exposed to different protocols
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