The chlorinating activity of human myeloperoxidase: high initial activity at neutral pH value and activation by electron donors

The steady-state activity of myeloperoxidase in the chlorination of monochlorodimedone at neutral pH was investigated. Using a stopped-flow spectrophotometer we were able to show that the enzymic activity at pH 7.2 rapidly declined in time. During the first 50-100 ms after addition of H2O2 to the enzyme, a turnover number of about 320 s-1 per haem was observed. However, this activity decreased rapidly to a value of about 25s-1 after 1 s. This shows that in classical steady-state activity measurements, the real activity of the enzyme at neutral pH is grossly underestimated. By following the transient spectra of myeloperoxidase during turnover it was shown that the decrease in activity was probably caused by the formation of an enzymically inactive form of the enzyme, Compound II. As demonstrated before (Bolscher, B.G.J.M., Zoutberg, G.R., Cuperus, R.A. and Wever, R. (1984) Biochim. Biophys. Acta 784, 189-191) reductants such as ascorbic acid and ferrocyanide convert Compound II, which accumulates during turnover, into active myeloperoxidase. Activity measurements in the presence of ascorbic acid showed, indeed, that the moderate enzymic activity was higher than in the absence of ascorbic acid. With 5-aminosalicylic acid present, however, the myeloperoxidase activity remained at a much higher level, namely about 150 s-1 per haem during the time interval from 100 ms to 5 s after mixing. From combined stopped-flow/rapid-scan experiments during turnover it became clear that in the presence of 5-aminosalicylic acid the initially formed Compound II was rapidly converted back to native enzyme. Presteady-state experiments showed that 5-aminosalicylic acid reacted with Compound II with a K2 of 3.2 x 10(5) M-1.s-1, whereas for ascorbic acid a K2 of 1.5 x 10(4) M-1.s-1 was measured at pH 7.2. In the presence of 5-aminosalicylic acid during the time interval in which the myeloperoxidase activity remained constant, a Km for H2O2 at pH 7.2 was determined of about 30 microM at 200 mM chloride. In the absence of reductants the same value was found during the first 100 ms after addition of H2O2 to the enzyme. The physiological consequences of these findings are discussed.     

Cellular Resistance Against Troxacitabine in Human Cell Lines and Pediatric Patient Acute Myeloid Leukemia Blast Cells

Troxacitabine is a cytotoxic deoxycytidine analogue with an unnatural L-configuration, which is activated by deoxycytidine kinase (dCK). The configuration is responsible for differences in the uptake and metabolism of troxacitabine compared to other deoxynucleoside analogues. To determine whether troxacitabine has an advantage over other nucleoside analogues several cell lines resistant to cladribine and gemcitabine were exposed to troxacitabine, while blast cells from pediatric leukemia patients were tested for cross-resistance with other deoxynucleoside analogues. The gemcitabine resistant AG6000 (IC₅₀: >3000 nM), and the cladribine resistant CEM (IC₅₀: 150 nM) and HL-60 (IC₅₀: >3000 nM) cell lines, all with no or decreased dCK expression, were less sensitive to troxacitabine than their wild type counterparts (IC₅₀; A2780: 410, CEM: 71 and HL-60: 158 nM). dCK protein expression in CEM was higher than in HL-60, which, in turn, was higher than in A2780. Catalytically inactive p53 seems to increase the sensitivity to troxacitabine. The patient samples showed a large range of sensitivity to troxacitabine, similar to other deoxynucleoside analogues. Cross-resistance with all other deoxynucleoside analogues was observed.     

Ischemic preconditioning, insulin, and morphine all cause hexokinase redistribution

Association of hexokinase (HK) with mitochondria preserves mitochondrial integrity and is an important mechanism by which cancer cells are protected against hypoxic conditions. Maintenance of mitochondrial integrity also figures prominently as a major characteristic of many cardioprotective manipulations. In this study, we provide evidence that cardioprotective interventions may promote HK redistribution from the cytosol to the mitochondria in the heart. Isolated Langendorff-perfused rat hearts (n = 6/group) were subjected to normoxic perfusion (control, Con), three 5-min ischemia-reperfusion periods (ischemic preconditioning, IPC), 1 U/l insulin (Ins), or 1 microM morphine (Mor). Hearts were immediately homogenized and centrifuged to obtain whole cell, cytosolic, and mitochondrial fractions. HK, lactate dehydrogenase (LDH), and citrate synthase (CS) enzyme activities were determined. No change in LDH or CS present in the cytosol fraction relative to whole cell activity was observed with any of the cardioprotective interventions. By contrast, HK present in the cytosol fraction relative to whole cell activity decreased significantly (P < 0.05) with all cardioprotective interventions, from 0.58 +/- 0.03 (Con) to 0.46 +/- 0.04 (IPC), 0.41 +/- 0.01 (Ins), and 0.45 +/- 0.02 (Mor). In addition, HK relative to CS activity in the mitochondrial fraction increased significantly with cardioprotection, from 0.15 +/- 0.001 (Con) to 0.21 +/- 0.002 (IPC), 0.18 +/- 0.003 (Ins), and 0.21 +/- 0.005 (Mor). Our novel data suggest that well-known cardioprotective interventions share a common end-effector mechanism of cytosolic HK translocation. Association of HK with mitochondria may promote inhibition of the mitochondrial permeability transition pore and thereby reduce cell death and apoptosis.     

Reduced hexokinase II impairs muscle function 2 wk after ischemia-reperfusion through increased cell necrosis and fibrosis

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.     

Deletion of the Innate Immune NLRP3 Receptor Abolishes Cardiac Ischemic Preconditioning and Is Associated with Decreased Il-6/STAT3 Signaling

Objective

Recent studies indicate that the innate immune system is not only triggered by exogenous pathogens and pollutants, but also by endogenous danger signals released during ischemia and necrosis. As triggers for the innate immune NLRP3 inflammasome protein complex appear to overlap with those for cardiac ischemia-reperfusion (I/R) and ischemic preconditioning (IPC), we explored the possibility that the NLRP3 inflammasome is involved in IPC and acute I/R injury of the heart.

Principal Findings

Baseline cardiac performance and acute I/R injury were investigated in isolated, Langendorff-perfused hearts from wild-type (WT), ASC^−/− and NLRP3^−/− mice. Deletion of NLRP3 inflammasome components ASC^−/− or NLRP3^−/− did not affect baseline performance. The deletions exacerbated I/R-induced mechanical dysfunction, but were without effect on I/R-induced cell death. When subjected to IPC, WT and ASC^−/− hearts were protected against I/R injury (improved function and less cell death). However, IPC did not protect NLRP3^−/− hearts against I/R injury. NLRP3^−/− hearts had significantly decreased cardiac IL-6 levels with a trend towards lower IL-1β levels at end reperfusion, suggesting abrogation of IPC through diminished IL-6 and/or IL-1β signaling. Subsequent experiments showed that neutralising IL-6 using an antibody against IL-6 abrogated IPC in WT hearts. However, inhibition of the IL-1r receptor with the IL-1 receptor inhibitor Anakinra (100 mg/L) did not abrogate IPC in WT hearts. Analysis of survival kinases after IPC demonstrated decreased STAT3 expression in NLRP3^−/− hearts when compared to WT hearts.

Conclusions

The data suggest that the innate immune NLRP3 protein, in an NLRP3-inflammasome-independent fashion, is an integral component of IPC in the isolated heart, possibly through an IL-6/STAT3 dependent mechanism.     

The dynamic regulation of myocardial oxidative phosphorylation: Analysis of the response time of oxygen consumption

Although usually steady-state fluxes and metabolite levels are assessed for the study of metabolic regulation, much can be learned from studying the transient response during quick changes of an input to the system. To this end we study the transient response of O₂ consumption in the heart during steps in heart rate. The time course is characterized by the mean response time of O₂ consumption which is the first statistical moment of the impulse response function of the system (for mono-exponential responses equal to the time constant). The time course of O₂ uptake during quick changes is measured with O₂ electrodes in the arterial perfusate and venous effluent of the heart, but the venous signal is delayed with respect to O₂ consumption in the mitochondria due to O₂ diffusion and vascular transport. We correct for this transport delay by using the mass balance of O₂, with all terms (e.g. O₂ consumption and vascular O₂ transport) taken as function of time. Integration of this mass balance over the duration of the response yields a relation between the mean transit time for O₂ and changes in cardiac O₂ content. Experimental data on the response times of venous [O₂] during step changes in arterial [O₂] or in perfusion flow are used to calculate the transport time between mitochondria and the venous O₂ electrode. By subtracting the transport time from the response time measured in the venous outflow the mean response time of mitochondrial O₂ consumption (t_mito) to the step in heart rate is obtained.In isolated rabbit heart we found that t_mito to heart rate steps is 4-12 s at 37°C. This means that oxidative phosphorylation responds to changing ATP hydrolysis with some delay, so that the phosphocreatine levels in the heart must be decreased, at least in the early stages after an increase in cardiac ATP hydrolysis. Changes in ADP and inorganic phosphate (P_i) thus play a role in regulating the dynamic adaptation of oxidative phosphorylation, although most steady state NMR measurements in the heart had suggested that ADP and P_i do not change. Indeed, we found with ³¹P-NMR spectroscopy that phosphocreatine (PCr) and P_i change in the first seconds after a quick change in ATP hydrolysis, but remarkably they do this significantly faster (time constant ~2.5 s) than mitochondrial O₂ consumption (time constant 12 s). Although it is quite likely that other factors besides ADP and P_i regulate cardiac oxidative phosphorylation, a fascinating alternative explanation is that the first changes in PCr measured with NMR spectroscopy took exclusively place in or near the myofibrils, and that a metabolic wave must then travel with some delay to the mitochondria to stimulate oxidative phosphorylation. The t_mito slows with falling temperature, intracellular acidosis, and sometimes also during reperfusion following ischemia and with decreased mitochondrial aerobic capacity. In conclusion, the study of the dynamic adaptation of cardiac oxidative phosphorylation to demand using the mean response time of cardiac mitochondrial O₂ consumption is a very valuable tool to investigate the regulation of cardiac mitochondrial energy metabolism in health and disease.     

Mitochondrial hexokinase and cardioprotection of the intact heart

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.     

The effect of standard chow and reduced hexokinase II on growth, cardiac and skeletal muscle hexokinase and low-flow cardiac ischaemia-reperfusion injury

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(+/-) 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.     

In vivo mitochondrial oxygen tension measured by a delayed fluorescence lifetime technique

Mitochondrial oxygen tension (mitoPO₂) is a key parameter for cellular function, which is considered to be affected under various pathophysiological circumstances. Although many techniques for assessing in vivo oxygenation are available, no technique for measuring mitoPO₂ in vivo exists. Here we report in vivo measurement of mitoPO₂ and the recovery of mitoPO₂ histograms in rat liver by a novel optical technique under normal and pathological circumstances. The technique is based on oxygen-dependent quenching of the delayed fluorescence lifetime of protoporphyrin IX. Application of 5-aminolevulinic acid enhanced mitochondrial protoporphyrin IX levels and induced oxygen-dependent delayed fluorescence in various tissues, without affecting mitochondrial respiration. Using fluorescence microscopy, we demonstrate in isolated hepatocytes that the signal is of mitochondrial origin. The delayed fluorescence lifetime was calibrated in isolated hepatocytes and isolated perfused livers. Ultimately, the technique was applied to measure mitoPO₂ in rat liver in vivo. The results demonstrate mitoPO₂ values of ∼30-40 mmHg. mitoPO₂ was highly sensitive to small changes in inspired oxygen concentration around atmospheric oxygen level. Ischemia-reperfusion interventions showed altered mitoPO₂ distribution, which flattened overall compared to baseline conditions. The reported technology is scalable from microscopic to macroscopic applications, and its reliance on an endogenous compound greatly enhances its potential field of applications.