Disruption of hexokinase II-mitochondrial binding blocks ischemic preconditioning and causes rapid cardiac necrosis.

RATIONALE: Isoforms I and II of the glycolytic enzyme hexokinase (HKI and HKII) are known to associate with mitochondria. It is unknown whether mitochondria-bound hexokinase is mandatory for ischemic preconditioning and normal functioning of the intact, beating heart. OBJECTIVE: We hypothesized that reducing mitochondrial hexokinase would abrogate ischemic preconditioning and disrupt myocardial function. METHODS AND RESULTS: Ex vivo perfused HKII(+/-) hearts exhibited increased cell death after ischemia and reperfusion injury compared with wild-type hearts; however, ischemic preconditioning was unaffected. To investigate acute reductions in mitochondrial HKII levels, wild-type hearts were treated with a TAT control peptide or a TAT-HK peptide that contained the binding motif of HKII to mitochondria, thereby disrupting the mitochondrial HKII association. Mitochondrial hexokinase was determined by HKI and HKII immunogold labeling and electron microscopy analysis. Low-dose (200 nmol/L) TAT-HK treatment significantly decreased mitochondrial HKII levels without affecting baseline cardiac function but dramatically increased ischemia-reperfusion injury and prevented the protective effects of ischemic preconditioning. Treatment for 15 minutes with high-dose (10 µmol/L) TAT-HK resulted in acute mitochondrial depolarization, mitochondrial swelling, profound contractile impairment, and severe cardiac disintegration. The detrimental effects of TAT-HK treatment were mimicked by mitochondrial membrane depolarization after mild mitochondrial uncoupling that did not cause direct mitochondrial permeability transition opening. CONCLUSIONS: Acute low-dose dissociation of HKII from mitochondria in heart prevented ischemic preconditioning, whereas high-dose HKII dissociation caused cessation of cardiac contraction and tissue disruption, likely through an acute mitochondrial membrane depolarization mechanism. The results suggest that the association of HKII with mitochondria is essential for the protective effects of ischemic preconditioning and normal cardiac function through maintenance of mitochondrial potential.

Helium-induced preconditioning in young and old rat heart: impact of mitochondrial Ca(2+) -sensitive potassium channel activation.

BACKGROUND: The noble gas helium induces cardiac preconditioning. Whether activation of mitochondrial K(+) channels is involved in helium preconditioning (He-PC) is unknown. The authors investigated whether He-PC (1) is mediated by activation of Ca(2+) -sensitive potassium channels, (2) results in mitochondrial uncoupling, and (3) is age dependent. METHODS: Anesthetized Wistar rats were randomly assigned to six groups (n = 10 each). Young (2-3 months) control (Con) and aged (22-24 months) control animals (Age Con) were not treated further. Preconditioning groups (He-PC and Age He-PC) inhaled 70% helium for 3 x 5 min. The Ca(2+) -sensitive potassium channel blocker iberiotoxin was administered in young animals, with and without helium (He-PC+Ibtx and Ibtx). Animals underwent 25 min of regional myocardial ischemia and 120 min of reperfusion. In additional experiments, cardiac mitochondria were isolated, and the respiratory control index was calculated (state 3/state 4). RESULTS: Helium reduced infarct size in young rats from 61 +/- 7% to 36 +/- 14% (P < 0.05 vs. Con). Infarct size reduction was abolished by iberiotoxin (60 +/- 11%; P < 0.05 vs. He-PC), whereas iberiotoxin alone had no effect (59 +/- 8%; not significant vs. Con). In aged animals, helium had no effect on infarct size (Age Con: 59 +/- 7% vs. Age He-PC: 58 +/- 8%; not significant). Helium reduced respiratory control index in young animals (2.76 +/- 0.05 to 2.43 +/- 0.15; P < 0.05) but not in aged animals (Age Con: 2.87 +/- 0.17 vs. Age He-PC: 2.87 +/- 0.07; not significant). Iberiotoxin abrogated the helium effect on respiratory control index (2.73 +/- 0.15; P < 0.05 vs. He-PC) but had no effect itself on mitochondrial respiration (2.75 +/- 0.05; not significant vs. Con). CONCLUSION: Helium causes mitochondrial uncoupling and induces preconditioning in young rats via Ca(2+) -sensitive potassium channel activation. However, these effects are lost in aged rats.