Reducing mitochondrial bound hexokinase II mediates transition from non-injurious into injurious ischemia/reperfusion of the intact heart

Abstract

Ischemia/reperfusion (I/R) of the heart becomes injurious when duration of the ischemic insult exceeds a certain threshold (approximately ≥20 min). Mitochondrial bound hexokinase II (mtHKII) protects against I/R injury, with the amount of mtHKII correlating with injury. Here, we examine whether mtHKII can induce the transition from non-injurious to injurious I/R, by detaching HKII from mitochondria during a non-injurious I/R interval. Additionally, we examine possible underlying mechanisms (increased reactive oxygen species (ROS), increased oxygen consumption (MVO₂) and decreased cardiac energetics) associated with this transition. Langendorff perfused rat hearts were treated for 20 min with saline, TAT-only or 200 nM TAT-HKII, a peptide that translocates HKII from mitochondria. Then, hearts were exposed to non-injurious 15-min ischemia, followed by 30-min reperfusion. I/R injury was determined by necrosis (LDH release) and cardiac mechanical recovery. ROS were measured by DHE fluorescence. Changes in cardiac respiratory activity (cardiac MVO₂ and efficiency and mitochondrial oxygen tension (mitoPO₂) using protoporphyrin IX) and cardiac energetics (ATP, PCr, ∆G_ATP) were determined following peptide treatment. When exposed to 15-min ischemia, control hearts had no necrosis and 85% recovery of function. Conversely, TAT-HKII treatment resulted in significant LDH release and reduced cardiac recovery (25%), indicating injurious I/R. This was associated with increased ROS during ischemia and reperfusion. TAT-HKII treatment reduced MVO₂ and improved energetics (increased PCr) before ischemia, without affecting MVO₂/RPP ratio or mitoPO₂. In conclusion, a reduction in mtHKII turns non-injurious I/R into injurious I/R. Loss of mtHKII was associated with increased ROS during ischemia and reperfusion, but not with increased MVO₂ or decreased cardiac energetics before damage occurs.

Keywords: Hexokinase; Ischemia/reperfusion injury; Oxygen consumption; Reactive oxygen species.

Fig. 1

Perfusion protocols used for peptide effects on ischemia development, I/R injury and cardiac oxygen consumption, mitochondrial PO₂ within the intact heart, ROS production, mitochondria-hexokinase binding and cardiac energetic parameters (PCr, ATP). Hearts were perfused with saline, TAT-only or TAT-HK peptide for 15–20 min after which some hearts were exposed to 15-min ischemia and 7- or 30-min reperfusion. I/R, cardiac energetics and hearts for hexokinase measurements were paced at 320 bpm during peptide treatment (dotted). NADH experiments were performed during 15-min peptide treatment. Cardiac energetics was measured at the end of peptide treatment and at 7-min reperfusion

Fig. 2

Low-dose TAT-HKII caused no ischemia, whereas higher-dose TAT-HKII caused modest ischemia. Hearts were treated for 15 min with saline, 2.5 μM TAT-only or 200 nM or 1 μM TAT-HKII. NADH fluorescence in the heart during the 15-min peptide treatment (a), lactate in effluent after 15-min peptide treatment (b) and perfusion pressure Pperf at the end of peptide treatment (c). ^### p < 0.001 vs 2.5 μM TAT-only; **p < 0.01 vs saline. Mean + SEM, n = 3–4 for a and b, n = 5–7 for c. Data for 2.5 μM TAT-only and 200 nM TAT-HKII in a and b have been published before [23] and are provided to facilitate direct comparisons with the 1 μM TAT-HKII peptide treatment

Fig. 3

TAT-HKII treatment decreased cardiac oxygen consumption but was without effect on myocardial economy before ischemia. Langendorff perfused rat hearts were exposed to 20 min saline, 1 μM TAT-only or 200 nM TAT-HKII treatment. Cardiac oxygen consumption (MVO₂) (a), cardiac function (rate pressure product (RPP)) (b) and myocardial economy (MVO₂/RPP) (c) during Langendorff perfusion. Mitochondrial oxygen tension (mitoPO₂) after 20-min peptide treatment as percentage of baseline (d). **p < 0.01 vs saline. Mean + SEM, n = 5–8 per group

Fig. 4

A reduction in mtHKII turned reversible I/R into irreversible I/R. Hearts were exposed to 20-min treatment with saline, 1 μM TAT-only or 200 nM TAT-HKII, followed by 15-min ischemia and 30-min reperfusion. Total lactate dehydrogenase (LDH) activity released during 30-min reperfusion (a), rate pressure product (RPP) (b), end-diastolic pressure (EDP) (c) and cardiac oxygen consumption (MVO₂)/RPP (d) determined at 30-min reperfusion. **p < 0.01, ***p < 0.001 vs saline. Mean + SEM, n = 5–7 per group

Fig. 5

TAT-HKII treatment increased ROS production during ischemia and reperfusion. ROS levels before ischemia (peptide treatment) and during the I/R intervention (a), and ROS levels at the end of peptide treatment, at the end of ischemia and at the peak during early reperfusion (b). *p < 0.05 vs TAT-only. Mean + SEM, n = 6 per group

Fig. 6

TAT-HKII treatment reduced mitochondrial bound hexokinase. Hearts were treated for 20 min with saline, 1 μM TAT-only or 200 nM TAT-HKII, exposed to 15-min ischemia and 7-min reperfusion. Representative images of the western blots (a), semi-quantitative amount of hexokinase II (HKII)/COX IV bound to the mitochondria as determined by western blot (b) and HK activity corrected for citrate synthase (CS) activity at 7-min reperfusion (c). *p < 0.05 vs saline. Mean + SEM, n = 6 per group

Fig. 7

TAT-HKII increases PCr before ischemia, but lowers PCr after ischemia. Hearts were treated for 20 min with 1 μM TAT-only or 200 nM TAT-HKII. Energetics were measured at the end of peptide treatment (baseline) or after 15-min ischemia and 7-min reperfusion. Phosphocreatine (PCr) (a, b), ATP (c, d) and ratio PCr/ATP (e, f) at the end of 20-min treatment (a, c, e) and after ischemia (b, d, f). *p < 0.05, **p < 0.01 vs TAT-only at the same time-point. Mean + SEM, n = 4–6 per group