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Rationale: We have shown that partial dissociation of hexokinase II (HKII) from mitochondria in the intact heart using low-dose transactivating transcriptional factor (TAT)-HKII (200 nmol/L) prevents the cardioprotective effects of ischemic preconditioning, whereas high-dose TAT-HKII (10 μmol/L) administration results in rapid myocardial dysfunction, mitochondrial depolarization, and disintegration. In this issue of Circulation Research, Pasdois et al argue that the deleterious effects of TAT-HKII administration on cardiac function are likely because of vasoconstriction and ensuing ischemia., Objective: To investigate whether altered vascular function and ensuing ischemia recapitulate the deleterious effects of TAT-HKII in intact myocardium., Methods and Results: Using a variety of complementary techniques, including mitochondrial membrane potential (ΔΨm) imaging, high-resolution optical action potential mapping, analysis of lactate production, nicotinamide adenine dinucleotide epifluorescence, lactate dehydrogenase release, and electron microscopy, we provide direct evidence that refutes the notion that acute myocardial dysfunction by high-dose TAT-HKII peptide administration is a consequence of impaired vascular function. Moreover, we demonstrate that low-dose TAT-HKII treatment, which abrogates the protective effects of ischemic preconditioning, is not associated with ischemia or ischemic injury., Conclusions: Our findings challenge the notion that the effects of TAT-HKII are attributable to impaired vascular function and ensuing ischemia, thereby lending further credence to the role of mitochondria-bound HKII as a critical regulator of cardiac function, ischemia-reperfusion injury, and cardioprotection by ischemic preconditioning.

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.

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, O₂ 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.

The oxidative capacity of mitochondria isolated from myocardium is undiminished after myocardial stunning, which is remarkable because stunning affects many other cellular functions. The aim of the present study was to assess the mitochondrial oxidative response in intact rather than isolated myocardium. The mean response time of mitochondrial O2 consumption to heart rate steps (tmito) was measured before and after 15-minute ischemia or high-flow hypoxia in isolated rabbit hearts. The tmito was calculated from the time course of venous O2 tension to steps in heart rate, with corrections made for diffusion and vascular transport delay. Isovolumic hearts were perfused with Tyrode's solution at 37 degrees C. Developed left ventricular pressure at 35 minutes of reperfusion was decreased significantly to 67 +/- 3% after ischemia (mean +/- SEM, n = 8) and to 79 +/- 6% after hypoxia (n = 8) relative to the control condition (n = 8), without increased cellular creatine kinase release. Before ischemia or hypoxia, tmito was 4.3 +/- 0.3 seconds. During reperfusion after ischemia or hypoxia, the increase in tmito (by 62 +/- 10% and 64 +/- 18%, respectively) was significantly larger than that in time controls (24 +/- 12% increase). The major determinant of decreased contractility and slower mitochondrial response appeared to be O2 deprivation and/or reintroduction rather than other consequences of stopped flow. O2 consumption at a given rate-pressure product was not increased after ischemia or hypoxia, indicating undiminished cardiac contractile economy. Brief ischemia or hypoxia, resulting in stunning, was associated with a slowing of the in vivo mitochondrial oxidative response, indicating that energy transfer and/or signaling between energy-consuming sites and mitochondria is affected in stunned myocardium.