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 ischemiareperfusion injury and prevented the protective effects of ischemic preconditioning. Treatment for 15 minutes with high-dose (10 mol/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. (Circ Res. 2011;108:1165-1169.)

Rationale: Cardiomyocytes switch substrate utilization from fatty acid to glucose under ischemic conditions; however, it is unknown how perturbations in glycolytic enzymes affect cardiac response to ischemia/reperfusion (I/R). Hexokinase (HK)II is a HK isoform that is expressed in the heart and can bind to the mitochondrial outer membrane.Objective: We sought to define how HKII and its binding to mitochondria play a role in cardiac response and remodeling after I/R. Methods and Results:We first showed that HKII levels and its binding to mitochondria are reduced 2 days after I/R. We then subjected the hearts of wild-type and heterozygote HKII knockout (HKII ؉/؊ ) mice to I/R by coronary ligation. At baseline, HKII ؉/؊ mice have normal cardiac function; however, they display lower systolic function after I/R compared to wild-type animals. The mechanism appears to be through an increase in cardiomyocyte death and fibrosis and a reduction in angiogenesis; the latter is through a decrease in hypoxia-inducible factor-dependent pathway signaling in cardiomyocytes. HKII mitochondrial binding is also critical for cardiomyocyte survival, because its displacement in tissue culture with a synthetic peptide increases cell death. Our results also suggest that HKII may be important for the remodeling of the viable cardiac tissue because its modulation in vitro alters cellular energy levels, O 2 consumption, and contractility. Conclusions:These results suggest that reduction in HKII levels causes altered remodeling of the heart in I/R by increasing cell death and fibrosis and reducing angiogenesis and that mitochondrial binding is needed for protection of cardiomyocytes. (Circ Res. 2011;108:60-69.)

The glycolytic enzyme hexokinase (HK) is suggested to play a role in ischemic preconditioning (IPC). In the present study we determined how ischemic preconditioning affects HK activity and HKI and HKII protein content at five different time points and three different subcellular fractions throughout cardiac ischemia-reperfusion. Isolated Langendorff-perfused rat hearts (10 groups of 7 hearts each) were subjected to 35 min ischemia and 30 min reperfusion (control groups); the IPC groups were pretreated with 3 times 5-min ischemia. IPC was without effect on microsomal HK activity, and only decreased cytosolic HK activity at 35 min ischemia, which was mimicked by decreased cytosolic HKII, but not HKI, protein content. In contrast, mitochondrial HK activity at baseline and during reperfusion was elevated by IPC, without changes during ischemia. No effect of IPC on mitochondrial HK I protein content was observed. However, mitochondrial HK II protein content during reperfusion was augmented by IPC, albeit not following the IPC stimulus. It is concluded that IPC results in decreased cytosolic HK activity during ischemia that could be explained by decreased HKII protein content. IPC increased mitochondrial HK activity before ischemia and during reperfusion that was only mimicked by increased HK II protein content during reperfusion. IPC was without effect on the phosphorylation status of HK before ischemia. We conclude that IPC is associated with 1) a biphasic response of increased mitochondrial HK activity before and after ischemia, 2) decreased cytosolic HK activity during ischemia, and 3) cellular redistribution of HKII but not HKI.

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.

The interaction of hexokinase with mitochondria has emerged as a powerful mechanism in protecting many cell types against cell death. However, the role of mitochondrial hexokinase (mitoHK) in cardiac ischemia-reperfusion injury has as of yet received little attention. In this review we examine whether increased binding of hexokinase to the mitochondrion is also an integral component of cardioprotective signalling. We discuss observations in cardiac mitochondrial activation that directed us to the hypothesis of hexokinase cellular redistribution with reversible, cardioprotective ischemia, summarize the data showing that many cardioprotective interventions, such as ischemic preconditioning, insulin, morphine and volatile anesthetics, increase mitochondrial hexokinase binding within the intact heart, and discuss similarities between mitochondrial hexokinase association and ischemic preconditioning. Although most data indicate that mitochondrial hexokinase may indeed be an integral part of cardioprotection, a definitive proof for a causal relation between the amount of mitoHK and cardiac ischemia-reperfusion injury in the intact heart is eagerly awaited. When such relationship is indeed observed, the association of hexokinase with mitochondria will offer an opportunity to develop new therapies to combat ischemic cardiac diseases.

In the present study, we examined whether standard chow (SDS versus Purina 5001; both low fat, high carbohydrate) and reductions in hexokinase (HK) II (wild-type versus HKII þ/2 mice) affect (1) growth parameters, (2) HK levels in cardiac and skeletal muscle and (3) low-flow cardiac ischaemia -reperfusion (IR) injury. Total HK activity and HKI and HKII expressions were determined, and low-flow IR injury was examined in isolated hearts subjected to 40 min 5% low-flow ischaemia and 120 min reperfusion. Standard chow, but not HKII reductions, significantly affected body weight, heart weight and cardiac hypertrophy. Both standard chow and reduced HKII diminished total cardiac and skeletal muscle HK activity. For the heart, the Purina chow-induced decrease in total HK activity was through decreases in HKI expression, whereas for skeletal muscle post-translational mechanisms are suggested. Both standard chow and reduced HKII demonstrated a non-significant trend for affecting cardiac IR damage. However, the low-flow ischaemia model was associated with mild sublethal injury only ( 1% cell death). In conclusion, standard chow affects body weight, heart weight and HK activity and HKI expression in the heart, without altering HKII expression. This implicates standard chow as an important factor in genomic, physiological research models and demonstrates that large differences in fat or carbohydrates in the diet are not necessary to affect growth. In a cardiac low-flow IR model, resulting in only mild injury, standard chow or reduced HKII does not significantly affect IR damage.Keywords: Nutrition, body weight, cardiology, pathology, perfused organ Laboratory Animals 2011; 45: 160-166. DOI: 10.1258/la.2011.010096To our knowledge, standard chow has received little attention in the field of animal experimentation. In contrast, much attention has been given to diets that differ largely in fat or carbohydrates, comparing e.g. low-fat with high-fat diets or low-carbohydrate with high-carbohydrate diets. However, often these diets not only differ in their fat or carbohydrate composition but also in many other components because diets are obtained from different suppliers. 1 -4 Uncertainty then exists as to whether observed effects can be ascribed to alterations in the main constituent (fat or carbohydrate) or to differences in the background standard chow used. Obviously, information is needed concerning the effects that standard chow per se may have on growth and other physiological parameters. We examined this question using wild-type (WT) and HK þ/2 mice. The HK þ/2 mice were shown to have a 50% reduction in hexokinase (HK) II activity in adipose, skeletal and heart tissues, without an effect on HKI activity.5 However, our own initial results with the HK þ/2 mice on HK activity did not show a significant difference between WT and HK þ/2 hearts. Interestingly, a recent study 1 demonstrated that difference in cardiac HKII content between WT and HK þ/2 hearts disappeared when animal nutrition was changed from low-fat to h...

Cellular studies have demonstrated a protective role of mitochondrial hexokinase against oxidative insults. It is unknown whether HK protective effects translate to the in vivo condition. In the present study, we hypothesize that HK affects acute ischemia–reperfusion injury in skeletal muscle of the intact animal. Male and female heterozygote knockout HKII (HK+/-), heterozygote overexpressed HKII (HKtg), and their wild-type (WT) C57Bl/6 littermates mice were examined. In anesthetized animals, the left gastrocnemius medialis (GM) muscle was connected to a force transducer and continuously stimulated (1-Hz twitches) during 60 min ischemia and 90 min reperfusion. Cell survival (%LDH) was defined by the amount of cytosolic lactate dehydrogenase (LDH) activity still present in the reperfused GM relative to the contralateral (non-ischemic) GM. Mitochondrial HK activity was 72.6 ± 7.5, 15.7 ± 1.7, and 8.8 ± 0.9 mU/mg protein in male mice, and 72.7 ± 3.7, 11.2 ± 1.4, and 5.9 ± 1.1 mU/mg in female mice for HKtg, WT, and HK+/-, respectively. Tetanic force recovery amounted to 33 ± 7% for male and 17 ± 4% for female mice and was similar for HKtg, WT, and HK+/-. However, cell survival was decreased (p = 0.014) in male HK+/- (82 ± 4%LDH) as compared with WT (98 ± 5%LDH) and HKtg (97 ± 4%LDH). No effects of HKII on cell survival was observed in female mice (92 ± 2% LDH). In conclusion, in this mild model of acute in vivo ischemia–reperfusion injury, a partial knockout of HKII was associated with increased cell death in male mice. The data suggest for the first time that HKII mediates skeletal muscle ischemia–reperfusion injury in the intact male animal.

We previously demonstrated that hexokinase (HK) II plays a key role in the pathophysiology of ischemia-reperfusion (I/R) injury of the heart (Smeele et al. Circ Res 108: 1165-1169, 2011; Wu et al. Circ Res 108: 60-69, 2011). However, it is unknown whether HKII also plays a key role in I/R injury and healing thereafter in skeletal muscle, and if so, through which mechanisms. We used male wild-type (WT) and heterozygous HKII knockout mice (HKII(+/-)) and performed in vivo unilateral skeletal muscle I/R, executed by 90 min hindlimb occlusion using orthodontic rubber bands followed by 1 h, 1 day, or 14 days reperfusion. The contralateral (CON) limb was used as internal control. No difference was observed in muscle glycogen turnover between genotypes at 1 h reperfusion. At 1 day reperfusion, the model resulted in 36% initial cell necrosis in WT gastrocnemius medialis (GM) muscle that was doubled (76% cell necrosis) in the HKII(+/-) mice. I/R-induced apoptosis (29%) was similar between genotypes. HKII reduction eliminated I/R-induced mitochondrial Bax translocation and oxidative stress at 1 day reperfusion. At 14 days recovery, the tetanic force deficit of the reperfused GM (relative to control GM) was 35% for WT, which was doubled (70%) in HKII(+/-) mice, mirroring the initial damage observed for these muscles. I/R increased muscle fatigue resistance equally in GM of both genotypes. The number of regenerating fibers in WT muscle (17%) was also approximately doubled in HKII(+/-) I/R muscle (44%), thus again mirroring the increased cell death in HKII(+/-) mice at day 1 and suggesting that HKII does not significantly affect muscle regeneration capacity. Reduced HKII was also associated with doubling of I/R-induced fibrosis. In conclusion, reduced muscle HKII protein content results in impaired muscle functionality during recovery from I/R. The impaired recovery seems to be mainly a result of a greater susceptibility of HKII(+/-) mice to the initial I/R-induced necrosis (not apoptosis), and not a HKII-related deficiency in muscle regeneration.