Hexokinase II and Reperfusion Injury
TAT-HK2 Peptide Impairs Vascular Function in Langendorff-perfused Rat Hearts
Rationale: Mitochondrial-bound hexokinase II (HK2) was recently proposed to play a crucial role in the normal functioning of the beating heart and to be necessary to maintain mitochondrial membrane potential. However, our own studies confirmed that mitochondria from ischemic rat hearts were HK2-depleted, yet showed no indication of depolarization and responded normally to ADP.
Objective: To establish whether the human TAT-HK2 peptide used to dissociate mitochondrial-bound HKII in the Langendorff-perfused heart may exert its effects indirectly by impairing coronary function.
Methods and Results: Ischemic preconditioning was blocked in rat hearts perfused with 2.5 µmol/L TAT-HK2 before ischemia or at the onset of reperfusion. However, TAT-HK2 also decreased the phosphocreatine:ATP ratio that correlated with reduced rate pressure product and increased diastolic pressure. These effects were preceded by increased aortic pressure (Langendorff constant flow) or decreased coronary flow (Langendorff constant pressure), which was also observed, albeit less pronounced, at 200 nmol/L TAT-HK2 and was prevented by coperfusion with the NO-donor diethylamine NONOate. Mitochondria from TAT-HK2–perfused hearts showed no loss of bound HK2, unlike mitochondria from ischemic hearts where the expected loss was prevented by ischemic preconditioning.
Conclusions: In the perfused rat heart, TAT-HK2 should be used with caution and careful attention to dosage because some of its effects may be mediated by vasoconstriction of the coronary vasculature rather than dissociation of HK2 from myocyte mitochondria.
The predominant hexokinase isoform expressed in the myocardium is hexokinase (HK2), some of which is bound to mitochondria (mitoHK2)1 where it acts as an important regulator of mitochondria-induced cell death.2–5 It dissociates during prolonged ischemia.6,7 This is associated with greater reactive oxygen species production,7 which, together with calcium overload, induces mitochondrial permeability transition pore opening, a critical event in reperfusion injury.8 Ischemic preconditioning (IP) prevents mitoHK2 loss during ischemia,6 and recently Smeele et al9 showed that cardioprotection by IP in the perfused heart could be blocked by a peptide (TAT-HK2) containing the human HK2 mitochondrial binding motif, which others have reported to dissociate HK2 from mitochondria.10,11 They concluded that mitoHK2 is important for normal heart function by keeping mitochondria energetically charged. However, our own studies showed that HK2-deficient mitochondria from ischemic hearts responded normally to ADP while exhibiting greater reactive oxygen species emission.7 Thus, we decided to reevaluate the effect of TAT-HK2 perfusion of normoxic hearts on mitochondrial function. We found that human TAT-HK2 has profound effects on the coronary vasculature while not actually inducing dissociation of HK2 from myocyte mitochondria for which it was used.
Sources of reagents used are given in Online Data Supplement, which also contains details of all methods used.
Langendorff perfusions of rat hearts (male Wistar; 225–300 g) were performed with constant flow (12 mL/min) or constant pressure (80 mm Hg) using the protocols summarized in Figure 1A and 1B, respectively. After the required perfusion protocol, hearts were either freeze-clamped to prepare heart powder (stored at −80°C for later analysis), used for the preparation of density-gradient purified mitochondria, or stained to determine infarct size.
MitoHK2 was determined by Western blotting and enzymatic assay of its specific activity. Phosphocreatine (PCr) and ATP were determined enzymatically after extraction from frozen heart powder.
Data are presented as means±SE. Statistical significance was evaluated using 1-way ANOVA (Kaleidagraph, 4.03), and differences were considered significant at P<0.05.
TAT-HK2 Perfusion Impaired Hemodynamic Function and Increased Infarct Size
Hearts were perfused with 2.5 μmol/L TAT-HK2 or control peptide (TAT-CON) before ischemia. TAT-HK2 perfusion of the control group (CP) decreased rate pressure product (RPP) from 26600±970 to 13900±2600 (n=6; P<0.01) after 8 minutes, whereas TAT-CON had no effect (Figure 2A). In the IP group, TAT-HK2 also decreased RPP (Figure 2C). In both groups, RPP impairment during TAT-HK2 perfusion was accompanied by increased aortic pressure (Figure 2B and 2D) caused by elevated diastolic pressure (Online Table I). TAT-HK2 perfusion inhibited RPP recovery in the IP group while having little effect in the CP group (Figure 2C and 2A, respectively). At the onset of reperfusion, aortic pressures in the CP and IP groups perfused with TAT-HK2 were higher than the corresponding TAT-CON–perfused controls (Figure 2B and 2D). TAT-HK2 perfusion also increased infarct size (% of the whole heart) from 12±4 to 30±4 in the IP group but had no significant effect in the CP group (Figure 2E). TAT-HK2 was equally effective at inhibiting RPP recovery when present only at the onset of reperfusion (Figure 2C) and strongly increased aortic pressure from 67±14 to 120±3 mmHg throughout its perfusion (Figure 2D; n=5). This was accompanied by a greater infarct size (Figure 3E), whereas TAT-CON had no effect (data not shown).
TAT-HK2 Perfusion Decreased PCr Content in Normoxic Hearts
The effects of TAT-HK2 perfusion on the bioenergetic status of hearts were determined by measuring their PCr and ATP content. After stabilization, hearts were perfused with the TAT peptides before freeze-clamping and metabolite assay. RPP impairment accompanying TAT-HK2 perfusion correlated with a decrease in the PCr:ATP ratio (Figure 2F), reflecting a decreased PCr without significant change in ATP (Online Figure IA). We also determined the l-lactate content in these hearts and observed that its accumulation increased in the TAT-HK2–treated hearts compared with the controls (Online Figure IIA), and this increase correlated with a decrease in the PCr:ATP ratio (Online Figure IIB). TAT-CON perfusion was without effect on these parameters. These data suggest that the myocardium was becoming hypoxic during TAT-HK2 perfusion.
TAT-HK2 Perfusion Induces Hypoxia in the Perfused Heart
We hypothesized that TAT-HK2 might cause vasoconstriction of the coronary vessels, resulting in reduced coronary flow in hearts perfused at constant pressure. In the presence of TAT-HK2, RPP depression was accompanied by decreased coronary flow from 9.2±0.2 to 3.8±0.9 mL/min per g; both parameters returned to baseline after washout of TAT-HK2 (Figure 3A and 3B). It should be noted that as the coronary flow decreased during perfusion with TAT-HK2, the peptide concentration increased by ≈80% from the initial concentration of 2.5 µmol/L (Online Table II). This was not the case in the constant flow mode. When TAT-HK2 was coperfused with the NO-donor diethylamine NONOate (0.5 µmol/L), neither RPP nor coronary flow was decreased (Figure 3C and 3D). Thus, we concluded that TAT-HK2 caused vasoconstriction of the coronary vessels, decreasing perfusion quality and causing hypoxia.
TAT-HK2 Peptide Perfusion Failed to Dissociate HK2 Bound to Mitochondria
Mitochondria were isolated from 6 different groups of hearts, and the specific activity of bound HK isoforms (Figure 4A) and HK2 protein content (Western blotting) were determined (Figure 4B and 4C). TAT-HK2 perfusion had no effect on either parameter, whereas ischemia significantly reduced HK2 binding measured by both techniques, and this decrease was largely prevented by IP as reported previously.6,7
There is evidence that mitoHK2 stabilizes mitochondria against mitochondrial permeability transition pore opening12 and that ischemia dissociates mitoHK2.6,7 Thus, it would seem likely that mitoHK2 dissociation plays a role in mitochondrial permeability transition pore opening during reperfusion, and we have suggested that this may be mediated by greater oxidative stress.7 Indeed, IP prevents mitoHK2 dissociation during ischemia,6 and this is accompanied by less oxidative stress and less mitochondrial permeability transition pore opening.13 Dissociation of mitoHK2 should prevent IP, which is what Smeele et al9 observed using immunogold electron microscopy. However, when we performed such studies using Western blotting of isolated mitochondria (as used by the authors in previous studies),6 we observed the expected decrease in bound HK2 after ischemia but not after treatment with TAT-HK2 at either 2.5 μmol/L (Figure 4) or 200 nmol/L (Online Figure IV).
TAT-HK2 Exerts Effects on Heart Function Independently of mitoHK2 Dissociation
Our data imply that TAT-HK2 may have effects on heart function that are independent of HK2 binding to myocyte mitochondria. We performed the majority of our studies with 2.5 μmol/L TAT-HK2, a concentration intermediate between 200 nmol/L and 10 μmol/L used by Smeele et al.9 In HeLa cells, dissociation of mitoHK2 required incubation with 20 µmol/L TAT-HK2 for 1 hour.10 For Rat1a cells 30 minutes was used to induce dissociation, but several hours were required to induce mitochondrial depolarization and apoptosis.14 This is much longer than the 2 to 4 minutes of exposure to 2.5 µmol/L TAT-HK2 that induced the changes in hemodynamic function we observed and which were not accompanied by mitoHK2 dissociation. We have repeated our experiments using the peptide at 200 nmol/L which Smeele et al9 reported inhibited IP, and these data are reported in Online Figures II–IV. Interestingly, Smeele et al9 themselves noted that RPP decreased to 92±3% in hearts treated with 200 nmol/L TAT-HK2, whereas at 10 µmol/L the peptide abolished heart function after 15 minutes of perfusion, which was associated with mitochondrial depolarization and ultrastructural damage. Here, we demonstrate that 2.5 μmol/L TAT-HK2 impaired vascular function as revealed by an increased aortic pressure in the constant flow model (Figure 2) and a reduced coronary flow in the constant pressure model (Figure 3). Smaller effects were observed at 200 nmol/L peptide (Online Figure II). Even in the constant flow model it would seem that oxygen supply to the heart was restricted by the presence of 2.5 μmol/L TAT-HK2 (but not 200 nmol/L) because RPP impairment correlated with a decrease in the PCr:ATP ratio (Figure. 2F). This drop in RPP was prevented when diethylamine NONOate was added to reverse the vasoconstriction (Figure 3).
Although we confirmed that perfusion with TAT-HK2 reduced the ability of IP to protect hearts from ischemia–reperfusion injury (Figure 2A, 2C, and 2E), this might be explained if TAT-HK2–induced vasoconstriction caused hypoxia and mitochondrial dysfunction on reperfusion. Consistent with this, 2.5 μmol/L TAT-HK2 exerted similar effects on infarct size, whether it was added before the IP protocol or during reperfusion (Figure 2C and 2E). At 200 nmol/L the damaging effects of TAT-HK2 were less but still observed. Hypercontracture was significantly increased (Online Figure IIIE), but the slight increase in infarct size of the IP group was not significant (Online Figure IIIF), unlike the increase in LDH used by Smeele et al9 as an indicator of necrotic damage. Even at 2.5 µmol/L TAT-HK2, the infarct size of the IP group peptide was significantly less than the non-IP hearts (Figure 2E), despite being associated with complete inhibition of hemodynamic function recovery (Figure 2C). These data show that in the presence of TAT-HK2 peptide there was a disparity between the reduction in infarct development and recovery of the hemodynamic function. We suggest that this is likely to be secondary to its effect on vasculature because the effects of 200 nmol/L TAT-HK2 on both parameters were abolished in the presence of the NO-donor diethylamine NONOate (Online Figure IIIB and IIIF). We do not know the mechanism by which the TAT-HK2 peptide causes vasoconstriction.
Limitations and Possible Explanations of Disparities Between Studies
A key difference between our studies and those of Smeele et al9 is that they used immunogold electron microscopy to demonstrate colocalization of HK2 with mitochondria rather than determining HK2 remaining bound to isolated mitochondria as we do here and had been done in previous studies.6 Another difference is that our studies were performed with rat hearts, whereas those of Smeele et al9 used mouse hearts. It is possible that rat and mouse hearts respond differently to the peptide, although the N-terminal sequences of mouse and rat HK2 are identical (MIASHMIACLFTELN), which makes this less likely. It should also be noted that the rodent sequence has 4 different residues (italic) to the human sequence (MIASHLLAYFFTELN) on which the TAT-HK2 peptide used in both studies is based. We cannot rule out that this difference might make the human TAT-HK2 peptide inefficient at displacing rat and mouse mitoHK2. Nor can we be certain that under the conditions used the peptide reaches the myocyte mitochondria at sufficient concentration and for sufficient time to dissociate mitoHK2. However, Smeele et al9 performed experiments with fluorescein isothiocyanate–labeled TAT-HK2 peptide to show its uniform distribution in the myocardium at concentrations from 200 nmol/L to 10 μmol/L, although it remains possible that the presence of the hydrophobic fluorescein moiety might enhance the permeability of peptide across the plasma membrane, enabling it to distribute more uniformly than the unlabeled peptide. Importantly, both studies suffer the same limitations in this regard.
In the perfused rodent heart, human TAT-HK2 should be used with considerable caution and careful attention to dosage because some of its effects may be mediated by vasoconstriction of the coronary vasculature rather than dissociation of HK2 from myocyte mitochondria.
Sources of Funding
This work was supported by a Program Grant from the British Heart Foundation (RG/08/001/24717).
In November 2012, the average time from submission to first decision for all original research papers submitted to Circulation Research was 15.8 days.
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.112.274233/-/DC1.
Non-standard Abbreviations and Acronyms
- ischemic preconditioning
- mitochondrial-bound hexokinase 2
- rate pressure product
- transactivating transcriptional factor of human immunodeficiency virus
- cell permeable peptide containing the hexokinase 2 mitochondrial binding motif
- Received May 23, 2012.
- Revision received September 20, 2012.
- Accepted September 26, 2012.
- © 2013 American Heart Association, Inc.
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