Disruption of Hexokinase II–Mitochondrial Binding Blocks Ischemic Preconditioning and Causes Rapid Cardiac NecrosisNovelty and Significance
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.
Hexokinase (HK), a glycolytic enzyme, is overexpressed in cancer cells. The enzyme binds to mitochondria and appears to regulate mitochondria-associated cell death induced by oxidative stress such as ischemia-reperfusion (I/R) injury.1,–,5 Mitochondrial HK (mitoHK) probably confers on cancer cells their resilience against cell death.2 Ischemic preconditioning (IPC), which is one of the most powerful interventions known to protect the heart against I/R injury, is believed to operate through complex mitochondrial signaling cascades.6,7 Indeed, translocation and activation of mitochondrial kinases have been suggested to be involved in preconditioning.8 Although various cardioprotective interventions, including IPC, cause the redistribution of hexokinase to mitochondria,9,–,11 it remains unclear whether hexokinase translocation and binding to mitochondria constitutes a requirement for or is simply an epiphenomenon of IPC. Furthermore, although hexokinase is known to associate with mitochondria in many tissues and organs within the body, the functional significance of this association in relation to normal cardiac physiology remains unclear.
In the present work, we used a combination of genetic and pharmacological tools to address the role of mitoHK in hearts under normal and stress conditions. We show for the first time that the physical binding of hexokinase II (HKII) to the mitochondria not only plays an essential role in IPC, but the attachment is also crucial for normal cardiac function, through maintenance of mitochondrial polarization.
An expanded Methods section is available in the Online Data Supplement at http://circres.ahajournals.org.
The soluble peptides TAT-CON (TAT control peptide), TAT-HK, TAT-HK-FITC, and TAT were produced by Pepscan Presto (Lelystad, Netherlands). Peptides were administered during the last 15 minutes before the 30-minute ischemic period and during the first 5 minutes of reperfusion.
Data are presented as mean±SEM. Data were analyzed by ANOVA followed by Fisher post hoc test.
Low-Dose TAT-HK Prevents the Cardioprotective Effects of IPC by Decreasing MitoHKII Levels
We first examined hearts from HKII+/− mice. Although genetic reduction of HKII decreased ischemic tolerance, no effects on IPC were observed (Online Figure I). To determine whether an acute pharmacological reduction in mitoHKII levels can alter IPC, isolated hearts were perfused with a cell-permeable peptide that contained the HKII mitochondrial binding motif. This peptide has been shown in cellular studies to decrease HKII association with mitochondria.12 Cellular uptake of this peptide was confirmed in the intact heart with FITC labeling. Fluorescence imaging of isolated hearts perfused with various concentrations of the peptide showed a progressive increase in fluorescence intensity with increasing peptide doses. As shown, homogeneous fluorescence could be readily observed even after prolonged periods of peptide washout (Figure 1A).
Baseline perfusion of hearts with a low concentration (200 nmol/L) of TAT-HK for 15 minutes did not cause major changes in cardiac contractility (rate-pressure product decreased slightly from 101±3% in TAT-CON–treated hearts to 92±3% in TAT-HK–treated hearts) or perfusion pressure. Using electron microscopy analysis and immunogold labeling for HKI and HKII, we observed a significant decrease (by 40%) in mitoHKII (Figure 1B), with no alterations in mitoHKI levels (Figure 1C). Representative electron microscopy images indicated an intact, well-preserved cardiac ultrastructure with clear localization of HKI and HKII at the outer mitochondrial membrane or in the cytosol in hearts treated with TAT-CON (Figures 2A–C) and TAT-HK (Figures 2D–F). Similar effects of the 200-nmol/L TAT-HK peptide compared with the TAT-CON peptide were observed at 5 minutes of reperfusion (Online Figure II).
Next, hearts were subjected to I/R with and without preceding IPC. The acute decrease in mitoHKII levels in TAT-HK–treated hearts was associated with more injury, as reflected by a more pronounced rise in release of lactate dehydrogenase (Figure 1D) and cardiac contracture at the end of reperfusion (Figure 1E). Remarkably, IPC in hearts with acutely decreased mitoHKII levels was completely ineffective in mediating protection against I/R injury, except for a nominal improvement in rate-pressure product recovery (Figure 1F).
TAT-HK–treated hearts also displayed increased cytosolic cytochrome C, which indicates damage and mitochondrial leakage (Figure 1G). Analysis of mitochondrial monomeric Bax (Figure 1H) suggested that the increased I/R damage in TAT-HK–treated hearts could not be explained by increased Bax translocation to mitochondria. Similar results were obtained for mitochondrial Bax oligomers (Online Figure III).
High-Dose TAT-HK Decreases MitoHKI and MitoHKII, Damages Mitochondria, and Results in Acute Mitochondrial Depolarization
To determine the pathophysiological consequences of severe disruption of mitochondrial HK binding to the intact myocardium, hearts were treated with a high dose (10 μmol/L) of TAT-HK for 15 minutes. The TAT-CON peptide was without effect on cardiac structure (Figure 2G through 2I); however, TAT-HK treatment caused a significant reduction in mitoHKII (Figure 3A) but not mitoHKI (Figure 3B) levels. Electron microscopy analysis demonstrated extensive structural disruption of cardiac tissue, with 78% of mitochondria exhibiting swelling or gross damage (Figures 2J through 2L and 3C). Profound cardiac dysfunction was observed, with rate-pressure product reduced to zero within 15 minutes of peptide perfusion (rate-pressure product 1±1% versus 93±2% for TAT-HK and TAT-CON, respectively), and perfusion pressure was doubled (81±7 versus 161±13 mm Hg, for TAT-CON and TAT-HK, respectively).
Mechanisms underlying the detrimental effects of acute dissociation of HK from mitochondria on cardiac structure and function of the intact heart are not clear. We hypothesized that infusion of the TAT-HK peptide altered the mitochondrial membrane potential (ΔΨm). ΔΨm was unaffected by treatment with the TAT-CON peptide; however, administration of the TAT-HK peptide resulted in a rapid and sustained decrease in ΔΨm (Figure 3D). Regions of mitochondrial depolarization developed heterogeneously across the epicardial surface of the heart, which indicates global mitochondrial de-energization at the intact heart level (Figure 3D; Online Figure IV).
Finally, we examined in isolated cardiomyocytes whether mitochondrial depolarization in and of itself may indeed cause cell death and whether TAT-HK is still detrimental when mitochondria are already depolarized. Similar to our findings in the intact heart, treatment of myocytes with 10 μmol/L TAT-HK resulted in cell death (Figure 3E). Remarkably, mitochondrial depolarization caused by incubation of myocytes with a submaximal dose (1 μmol/L) of the uncoupling agent carbonyl cyanide 3-chlorophenylhydrazone (CCCP) resulted in a degree of cell death comparable to that elicited by treatment with the TAT-HK peptide (Figure 3F). Interestingly, cotreatment of CCCP incubated with 10 μmol/L TAT-HK did not cause additional cell death. Similar data were obtained for tetramethylrhodamine ethyl ester fluorescence analysis in cardiomyocytes (Online Figure V). These data suggest that the development of cell death in the heart during detachment of HK from mitochondria is most likely caused by a mechanism that involves mitochondrial depolarization.
MitoHKII and Ischemic Tolerance
TAT-HK peptide–mediated mitochondrial HKII dissociation is associated with an extensive decrease in the tolerance of the heart to an I/R insult. Because it is unlikely that total cellular HK activity is altered with this peptide, the increased cell death can only be ascribed to decreases in mitoHKII and not to decreases in cardiac HK activity. One mechanism through which detachment of HK from mitochondria could cause cell death is through the translocation of proapoptotic Bax to mitochondria3,4; however, this was not observed in the present experiments. Although the large increase in release of lactate dehydrogenase suggests that the detrimental effects of HKII detachment from mitochondria may be mediated through necrosis, further experiments are needed to establish this more firmly.
MitoHKII and IPC
The present study indicates that a basal level of HKII binding to mitochondria is required for IPC-mediated cardioprotection against I/R injury; however, the observation that IPC was still effective in the HKII+/− hearts but not in TAT-HK–treated hearts, with both models having similar reductions in mitoHKII (30% to 40%), suggests that it is not just a reduction of overall HKII binding to mitochondria that prevents IPC. Although not investigated, it is possible that the TAT-HK peptide used in the present study occupied mitochondrial binding sites that were specifically targeted for IPC-induced translocation of HKII to mitochondria. This is commensurate with the observation that cardioprotective signaling was associated with a protein kinase C-ε mitochondrial protein complex that contained HK in the amount of only 1% of total HK present.13 Further experiments are required to clarify this issue.
MitoHKII and Normal Cardiac Function
The present data demonstrate that HKII detachment from mitochondria results in acute mitochondrial depolarization, which is the likely mechanism for cardiac dysfunction. Together with our observation that CCCP mediated cell death through mitochondrial depolarization, without directly affecting mitochondrial permeability transition pore opening, suggests that mitochondrial membrane depolarization per se may be the execution trigger for HKII-related cardiac dysfunction. Previous literature has indicated that mitochondrial membrane depolarization may induce opening of the mitochondrial permeability transition pore, especially when pH is above 7.0.14 Overall, the present data suggest that mitochondria-bound HKII is required for the polarization and stability of mitochondrial energetics within the intact heart.
Sources of Funding
This work was supported by an Academic Medical Center institutional research grant (CJZ), a Society of Cardiovascular Anesthesiologists midcareer research grant (CJZ), and Dutch Heart Foundation grant No. NHS 2010B011 (CJZ).
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Brief UltraRapid Communications are designed to be a format for manuscripts that are of outstanding interest to the readership, report definitive observations, but have a relatively narrow scope. Less comprehensive than Regular Articles but still scientifically rigorous, BURCs present seminal findings that have the potential to open up new avenues of research. A decision on BURCs is rendered within 7 days of submission.
Non-standard Abbreviations and Acronyms
- carbonyl cyanide 3-chlorophenylhydrazone
- heterozygote hexokinase II–deficient mice
- ischemic preconditioning
- mitochondria-bound hexokinase
- transactivating transcriptional factor from human immunodeficiency virus 1
- Received March 16, 2011.
- Revision received April 11, 2011.
- Accepted April 15, 2011.
- © 2011 American Heart Association, Inc.
- Pastorino JG,
- Shulga N,
- Hoek JB
- Gottlob K,
- Majewski N,
- Kennedy S,
- Kandel E,
- Robey RB,
- Hay N
- Wu R,
- Smeele KM,
- Wyatt E,
- Ichikawa Y,
- Eerbeek O,
- Sun L,
- Potini V,
- Hollmann MW,
- Nagpal V,
- Heikkinen S,
- Laakso M,
- Jujo K,
- Wasserstrom JA,
- Zuurbier CJ,
- Ardehali H
- Murphy E,
- Steenbergen C
- Halestrap A
- Miura T,
- Tanno M,
- Sato T
- Zuurbier CJ,
- Eerbeek O,
- Meijer AJ
- Gürel E,
- Smeele KM,
- Eerbeek O,
- Koeman A,
- Demirci C,
- Hollmann MW,
- Zuurbier CJ
- Chiara F,
- Castellaro D,
- Marin O,
- Petronilli V,
- Brusilow WS,
- Juhaszova J,
- Sollott SJ,
- Forte M,
- Bernardi P,
- Rasola A
- Baines CP,
- Song CX,
- Zheng YT,
- Wang GW,
- Zhang J,
- Wang OL,
- Guo Y,
- Bolli R,
- Cardwell EM,
- Ping P
- Bernardi P
Novelty and Significance
What Is Known?
The glycolytic enzyme hexokinase (HK) translocates to the mitochondria during insulin stimulation, ischemia, and ischemic preconditioning.
The association of hexokinase with the mitochondria is thought to both promote glycolysis and prevent mitochondrial permeability transition pore opening and may be an important end effector for cardioprotection by ischemic preconditioning.
What New Information Does This Article Contribute?
Acute targeted disruption of HK-mitochondria binding with low doses of a cell-permeable peptide homologous to the binding region of HK had no effect on basal cardiac function but compromised ischemic tolerance and blocked ischemic preconditioning.
Genetic reduction of HKII (by 30% at the mitochondria) also compromised ischemic tolerance but had no effect on ischemic preconditioning.
Administration of the same peptide at a higher dose caused rapid and dramatic mitochondrial damage, necrosis, and cardiac dysfunction.
Detachment of HK from mitochondria appears to cause acute necrosis through mitochondrial depolarization, swelling, and rupture rather than Bax-mediated apoptosis.
The data suggest that the glycolytic enzyme HK is an important guardian of the mitochondrion in the beating, intact heart. It could be argued that its key integrative role between glycolysis and mitochondria makes it an ideal candidate as a metabolic sensor or cell death switch; its upregulation in cancer cells as part of the Warburg effect provides some evidence for this. Our findings establish the essential importance of HK in maintaining mitochondrial (and hence myocyte) viability and provide further evidence that upregulation of HK-mitochondria binding may underlie the protective effect of ischemic preconditioning.