Abstract 15296: Acute Dissociation of Hexokinase from Mitochondria abrogates Normal Functioning, Ischemic Tolerance and Ischemic Preconditioning of the Intact Beating Heart
Cellular studies have demonstrated a prosurvival role for association of hexokinase (HK) with mitochondria. Intact heart studies suggest a role for mitochondrial-HK binding (mitoHK) in ischemic preconditioning (IPC). However, it is unknown whether ischemic tolerance, IPC and normal functioning of the intact heart are affected by chronic and/or acute disruption of mitoHK association. We hypothesize that mitoHK is an important determinant of the (patho)physiology of the intact, beating heart. We performed 30 min ischemia (I) and 45 min reperfusion (R) in isolated hearts (n=7 each group) of wild-type (WT) and HKII+/− mice, with IPC consisting of 3 cycles of 5 min ischemia preceding IR. Cell death was determined by LDH release. HKII+/− hearts demonstrated increased cell death as compared to WT hearts (25.0 ± 1.8 vs 18.0 ± 2.6 μmol/g/reperfusion, respectively; P<0.05), without affecting IPC, indicating that a chronic decrease in HKII increases IR injury without affecting the magnitude of IPC. Subsequently, we treated WT hearts for 15 min with a TAT-HK or TAT-scrambled peptide. The TAT-HK peptide contains the binding motif of HK to mitochondria, thereby acutely disrupting mitoHK association. Cryomicrotome fluorescence 15 μm image analysis of hearts treated with a TAT-HK-FITC peptide demonstrated equal cellular loading throughout the heart. A low concentration of this TAT-HK peptide (200 nM), decreased mitoHK by 20% and did not affect baseline cardiac physiology, but dramatically increased IR injury (7.2 ± 2.2 (TAT-scrambled) vs. 45.2 ± 2.8 μmol/g/reperfusion (TAT-HK); P<0.05), and abrogated IPC protective effects on LDH release. In addition, just 15 min aerobic perfusion with 10 μM TAT-HK peptide completely halted cardiac pressure development concomitant with large LDH release. Electron microscopic analysis, together with HKI and II immunogold labelling of 15 min 10 μM TAT-HK treated hearts, demonstrated a decrease of total HK bound to mitochondria from 37.8 ± 1.8% to 25.8 ± 0.4%, and massive cellular desintegration with 78 ± 21% of all mitochondria being damaged.
Conclusion: The data suggest mitochondrial-hexokinase association being a major determinant of normal functioning, ischemic tolerance and ischemic preconditioning of the intact, beating heart.
- © 2010 by American Heart Association, Inc.