Skip Navigation

Cardiovascular Research 2004 62(1):145-153; doi:10.1016/j.cardiores.2004.01.010
© 2004 by European Society of Cardiology
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Zuurbier, C.J
Right arrow Articles by Ince, C
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zuurbier, C.J
Right arrow Articles by Ince, C
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2004, European Society of Cardiology

Inhibition of the pentose phosphate pathway decreases ischemia–reperfusion-induced creatine kinase release in the heart

C.J Zuurbier*,a, O Eerbeekb, P.T Goedhartb, E.A Struysc, N.M Verhoevenc, C Jakobsc and C Inceb

aDepartment of Anaesthesiology, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands
bDepartment of Physiology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
cMetabolic Unit, Department of Clinical Chemistry, VU University Medical Center, Amsterdam, The Netherlands

* Corresponding author. Tel.: +31-20-566-5259; fax: +31-20-697-9004. Email address: c.j.zuurbier{at}amc.uva.nl

Received 9 September 2003; revised 6 January 2004; accepted 8 January 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: The oxidative pentose phosphate pathway (oxPPP) produces NADPH, which can be used to maintain glutathione in its reduced state (anti-oxidant; beneficial effects) or to produce radicals or nitric oxide (NO) through NADPH oxidase/NO synthase (detrimental effects). Changes in cytosolic redox status have been implicated in ischemic preconditioning (PC). This study investigates whether (1) PC affects mitochondrial redox state, (2) the oxPPP plays a protective or detrimental role in ischemia (I)–reperfusion (R) injury in the intact heart and (3) PPP is altered with PC. Methods: Isolated rat hearts were subjected to 40-min global I and 30-min R (CO, control). Ischemia was either preceded by three 5-min I/R periods (PC) and/or oxPPP inhibition by 6-aminonicotinamide (6AN) or NADPH oxidase/NO synthase inhibition by diphenyleneiodonium (DPI). NADH videofluorometry was used to determine mitochondrial redox state. PPP intermediates were determined in CO and PC hearts using tandem mass spectrometry. Results: PC reduced ischemic damage (creatine kinase, CK, release from 337±64 to 147±41 U/R/gdw) and contracture (from 59±5 to 31±3 mm Hg) and increased recovery of contractility (from 48±10% to 88±8%), as compared to CO. PC was without effect on NADH fluorometry. Inhibition of the oxPPP reduced injury (CK release: 91±24 U/R/gdw) to similar levels as PC, without improving contractility. Inhibition of NADPH oxidase/NO synthase mimicked the effects of oxPPP inhibition on injury (CK release: 140±22 U/R/gdw). Although levels of ribose-5P and (ribulose-5P+xylulose-5P) rose several fold during ischemia with minor changes in sedoheptulose-7P, demonstrating an active PPP in the heart, PC did not affect these levels. Conclusions: (1) PC can attenuate cardiac reperfusion injury without alterations in mitochondrial redox state; (2) inhibition of the oxPPP protects the heart against I/R-induced CK release; and (3) PC does not result in altered activity of the PPP.

KEYWORDS Energy metabolism; Ischemia; Necrosis; Preconditioning; Reperfusion


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Ischemic preconditioning is a phenomenon in which transient nonlethal periods of ischemia protect the heart against a subsequent lethal ischemic period [1]. Although much research has been devoted to this phenomenon, the final end-effector or mechanism of protection remains elusive. One important trigger and/or mediator of the preconditioned state involves short periods of reactive oxygen species (ROS) generation [2–5], resulting in a pro-oxidant environment. Recently, it has been shown that diazoxide, which, at low concentrations, is a selective opener of the mito(K+ATP) channel, and which features prominently as a trigger of preconditioning [6], also results in increased ROS production [7]. It therefore seems likely that the pro-oxidant environment is an important cellular trigger that activates the cellular, protective mechanism of preconditioning. In contrast, the end-effector mechanism of preconditioning is related to reduced radical and NO production during the lethal ischemia–reperfusion period [8–11]. One regulatory pathway, which may result in both a pro-oxidant or anti-oxidant cellular environment, is the oxidative pentose phosphate pathway (oxPPP). This pathway produces NADPH which may feed either glutathione reductase for maintaining glutathione in its reduced state (anti-oxidant) or NADPH oxidase and NO synthase for producing radicals and NO, respectively (pro-oxidant; [12]). It is unknown whether a reduced flux through the oxPPP may partially explain the reduced radical/NO production observed in preconditioned tissue during the lethal ischemia–reperfusion period. Little or no attention has been paid to the role of the oxPPP in cardiac ischemia–reperfusion injury, let alone ischemic preconditioning. This is even more surprising when one realizes that the PPP is closely connected with glycolysis, a pathway that is strongly affected by preconditioning. A study by Turrens et al. [13] is the only study that has examined the relation between preconditioning and the oxPPP by determining the enzyme kinetics (Vmax) of this pathway in homogenized tissue samples. Although this study has provided important information that preconditioning is not related to changes in these variables, it is well accepted that Vmax does not necessarily dictate the in vivo flux of the pathway. In this study, we therefore focus on the role of the PPP in the setting of ischemia–reperfusion injury and ischemic preconditioning in the intact heart.

In order to investigate whether reduced flux through the oxPPP may mediate cardioprotection, we studied ischemia–reperfusion injury and preconditioning with and without the oxPPP inhibitor 6-aminonicotinamide [14–16] in the Langendorff-perfused rat heart. Although preconditioning is associated with changes in the redox state of thiol-containing compounds [5,7], controversy exists whether the mitochondrial redox state is affected by ischemia and preconditioning [17–19]. To this end, we used NADH videofluorometry of the surface of the left ventricle to monitor the mitochondrial redox state [20–22]. In addition, a newly developed method using tandem mass spectrometry for determining PPP intermediates was employed [23]. To our knowledge, this is the first study for the heart that reports on these intermediates and how they change upon ischemia and preconditioning.

The data indicate that although the pentose phosphate pathway is active during cardiac ischemia and inhibition reduces CK release during reperfusion, the protective actions of ischemic preconditioning are probably not mediated by changes in this pathway.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Heart perfusion
All procedures followed were in accordance with the Animal Ethical Commission of the University of Amsterdam and conform to NIH guidelines. Male Wistar rats (280–370 g) were anesthetized with pentobarbital (60 mg kg–1). Tracheotomy was performed and mechanical ventilation was initiated. Following the opening of the thorax and administration of intravenous heparin (150 IU), the aorta was cannulated in situ, and perfusion was started before excision of the heart. The hearts were perfused according to the method of Langendorff at 37 °C with Tyrode's solution containing (mmol l–1) NaCl 128.3, KCl 4.7, CaCl2 1.4, MgCl2 1.1, NaHCO3 20.2, NaH2PO4 0.4 and glucose 11.0, gassed with 95%O2/5%CO2. Thebesian venous effluent from the ventricular wall was drained from the left ventricular lumen by a cannula pierced through the apex. A water-filled latex balloon was inserted into the left ventricle through the left atrium and connected to a pressure transducer for assessment of contractile performance. End-diastolic pressure was set at 3–6 mm Hg by adjusting balloon volume. Hearts were perfused at a constant perfusion pressure of 80–85 mm Hg, measured just above the aortic cannula. Perfusion and left ventricular pressure were recorded and digitized with a sampling frequency of 100 Hz. At the end of the experiment, the hearts were stored for 2 days at 70 °C for determination of dry weight.

2.2. NADH surface fluorescence
The videofluoremeter was attached to a brace and positioned in front of the left ventricle of the heart as described elsewhere [24]. The fluorometer consisted of a 100-W mercury arc lamp, a fluorescence unit housing a dichroic mirror for separation of excitation and emission light and a Princeton camera (PP0304N, 25 mm S20 red enhanced photocathode). A UG-1 barrier filter in front of the Hg lamp allowed transmission of the 360-nm excitation light, whereas a band-pass filter in front of the camera allowed transmission of the NADH fluorescence light at 460±20 nm. An electronically controlled shutter in front of the Hg lamp permitted intermittent (120 ms) illumination of the left ventricular surface, preventing excessive bleaching of the endogenous NADH compound [24]. The exposure time of the camera was set at 10 ms. NADH fluorescence was determined at t=0, 9, 19, 29 and 34 min of baseline perfusion, at 3 and 39 min of ischemia, and at 5, 15 and 30 min of reperfusion (see Fig. 1).


Figure 1
View larger version (14K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Perfusion protocol with the different interventions for the experimental groups. Global ischemia is depicted by solid bars, and the vertical and reverse hatching depict the periods during which 500 µM 6-aminonicotinamide (6AN) and 2 µM diphenyleneiodonium (DPI) were administered. {dagger}: NADH image.

 
The fluorescence images were analyzed offline by image processing software (WinView32—Princeton Instruments). A constant, large area of the left ventricle was chosen (approximately 30–50 mm2 of heart surface) which was visible throughout the experiment. The averaged gray level of the selected area (approximately 60,000–80,000 pixels) was determined and corrected for fluctuations in excitation lamp intensities by normalization with the fluorescence intensity of a piece of uranyl positioned next to the heart [24]. Subsequently, the relative gray level was calculated by using the minimal and maximal fluorescence levels determined during the experiment. The maximal value was determined at 3 min of the global 40-min no-flow ischemia, resulting in a maximal level of NADH in the reduced state. The minimal value was determined at the end of the experiment following 5-min perfusion with the uncoupling agent CCCP (Sigma-Aldrich; final concentration 5 µM).

2.3. Experimental protocol
Following stabilization of left ventricular pressure, five different groups of hearts were studied (Fig. 1). Group 1 (CO, control; n=10) was subjected to 35 min of perfusion, followed by 40 min of global ischemia and 30 min of reperfusion. Switching a valve in the perfusion system, preventing perfusate to flow into the aortic cannula, imposed global ischemia. Group 2 (PC, preconditioned; n=8) received three 5-min periods of ischemia each followed by 5-min reperfusion, except that the last reperfusion lasted for 10 min, before the start of the 40-min ischemia and 30-min reperfusion. Group 3 (6AN group; n=6) was similar to group 1, except that after 5-min perfusion, the perfusate was switched to one containing 500 µM 6-aminonicotinamide (Sigma-Aldrich) for the remaining part of the protocol. In this way, the hearts were perfused for 30 min with 6AN before the onset of ischemia. This treatment to induce oxPPP inhibition by 6AN was based on studies in isolated kidneys and lungs, demonstrating inhibition after 15-min perfusion with 250–500 µM 6-aminonicotinamide [15,16]. In group 4 (6AN+preconditioning; n=6), the 6-aminonicotinamide and preconditioning treatment were combined, with similar timing as in groups 2 and 3. Finally, in group 5 (DPI; n=7), the irreversible flavoenzyme inhibitor diphenyleneiodonium (Sigma-Aldrich; 2 µM in perfusate) was administered for 5 min during 28–33 min of perfusion. In this way, the inhibitor was washed out during the last 2 min of perfusion before the 40-min ischemia. This group allowed us to compare direct inhibition by DPI of the "detrimental enzymes" for oxPPP-produced NADPH (NADPH oxidase and NO synthase) with the inhibition of oxPPP by 6AN.

During the 40-min global ischemia, the volume of the balloon was kept intact. All hearts were submerged in the experimental perfusate solution (37 °C) gassed with 95%N2/5%CO2, starting at 4-min ischemia up to 38 min of ischemia. This allowed NADH images to be recorded at 3 and 39 min of ischemia. The venous effluent was collected at fixed times throughout the protocol (at 1, 5, 10, 15, 20 and 30 min of reperfusion) for determination of venous creatine kinase leakage as index of cardiac injury.

To determine whether 6AN inhibit the oxPPP in the heart with our treatment protocol, myocardial NADPH levels were biochemically determined in a subset of hearts perfused for 30 min with (n=6) or without 500 µM 6AN (n=5). In addition, to compare the observed, relative, NADH changes during the experimental protocol using the videofluorometry technique with NADH levels from the whole heart, NADH was determined biochemically at 30 min of perfusion (same hearts as for the NADPH determinations) and following 40 min of ischemia and 10 min of reperfusion (n=5).

In a separate series of 36 hearts, the PPP intermediates ribose-5-phosphate, ribulose-5-phopshate, xylulose-5-phosphate and sedoheptulose-7-phosphate were determined at 30 min of perfusion (baseline), at 15 and 40 min of ischemia and at 10 min of reperfusion for control and preconditioned hearts. These measurements were also additionally determined for the 6AN group at baseline (n=4) and at 40-min ischemia (n=5). These intermediates are produced by both the oxidative and the nonoxidative PPP, with important connections to the glycolytic pathway (Fig. 2).


Figure 2
View larger version (14K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Schematic representation of glucose-6-phosphate metabolism by the oxidative pentose phosphate pathway (ox PPP), the nonoxidative pentose phosphate pathway (nonox PPP, dashed lines) and glycolysis. The interconversions aligned with the dashed ellipse are mediated by the transketolase and transaldolase enzymes; PRPP: phosphoribosyl pyrophosphate.

 
Four non-ischemic time control experiments were conducted to determine the extent of perfusion-dependent functional deterioration and creatine kinase release that occurred independent of ischemia–reperfusion injury.

2.4. Biochemical analysis
Creatine kinase was determined according to standard techniques [25], with albumin (bovine serum fatty acid free; Intergen) added to the collected effluent to recover most of the enzyme [26]. CK release was normalized to gram dry tissue.

For all myocardial metabolites, the hearts were freeze clamped with liquid N2-precooled Wollenberger tongs and subsequently freeze-dried overnight. Tissue was stored at –80 °C before metabolite extraction. NADPH and NADH were extracted from freeze-dried tissue according to Klingenberg [27] and determined fluorimetrically using an enzymatic technique [28] in a Perkin-Elmer 3000 Fluorescence Spectrometer.

Sugar phosphates were extracted with 50% acetonitrile (Merck, Sharpe & Dohme) from ~1 mg freeze-dried tissue (duplicate), containing the stable-isotope-labeled D-[13C6]glucose-6-phosphate internal standard according to Huck et al. [23]. The sugar phosphates in the extracts were analyzed by liquid chromatography-tandem mass spectrometry using an ion-pair-loaded C18 HPLC column (Waters Chromatography). We focused our analysis on the intermediates that are specific for the PPP, i.e. ribose-5P, ribulose-5P, xylulose-5P and sedoheptulose-7P. Ribulose-5P and xylulose-5P are presented together because they elute as one peak [23].

2.5. Statistical analysis
Data are represented as mean±S.E.M., unless indicated otherwise. Two-way ANOVA (by treatment group and by time) for repeated measurements were performed on NADH values throughout the experiment. One-way ANOVA (treatment group) on group means were performed for flow, heart rate and relative NADH fluorescence at baseline, for end-diastolic pressure and developed left ventricular pressure (DLVP) at baseline and end of reperfusion and for total CK release during reperfusion. The ANOVA analysis was followed by planned comparisons among group means (comparing intervention groups 2–5 with control group 1, using Bonferroni corrections) when a significant main effect (p<0.05) was found.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
One-way ANOVA demonstrated no significant differences between group means at baseline for the important physiological parameters. Consequently, all values across groups were averaged and amounted to flow (61.0±2.2 ml/min/gdw), end-diastolic pressure (EDP; 4.1±0.4 mm Hg), developed left ventricular pressure (DLVP; 102±5 mm Hg), heart rate (240±4 beats/min) and relative NADH fluorescence (30.4±1.2%).

The time control experiments showed a rather constant end-diastolic pressure (from 4.4±0.2 mm Hg at t=0 min to 4.1±0.5 mm Hg at 105-min perfusion), a 19% decrease in developed left ventricular pressure (at 105 min of perfusion, DLVP was 81±5% of the value at baseline) and a CK release of 4 U/105 min/gdw for the entire perfusion protocol or 0.4 U/30 min/gdw, when determined over the last 30 min of perfusion.

Thirty minutes of perfusing the hearts with 500 µM 6AN did result in decreased NADPH levels (958±116 vs. 616±61 nmol/gdw for control and 6AN hearts, respectively; p=0.02), indicating inhibition of the oxPPP.

Fig. 3 shows changes in relative NADH fluorescence of the left ventricular free wall for each of the five study groups before, during and after ischemia. Although the first 5-min I/R period resulted in a trend towards decreased NADH levels, indicative of a pro-oxidant mitochondrial environment with ischemic preconditioning, this decrease in relative NADH levels did not reach significance. Similarly, both preconditioning and inhibition of the oxPPP did not significantly change NADH levels at the end of the 40-min ischemic period as compared to control. Also, during reperfusion, no significant differences were observed in NADH fluorescence between the different groups. The data also indicate that NADH levels return to baseline values during reperfusion for most groups. The biochemically determined NADH levels at baseline (838±228 nmol/gdw) and 10-min reperfusion (1223±185 nmol/gdw) (p=0.20) also clearly indicate that ischemia–reperfusion in this heart model does not result in decreased levels of NADH.


Figure 3
View larger version (16K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 The relative intensity of endogenous NADH fluorescence as function of time for the different experimental groups. The solid bar denotes the 40-min global ischemia that all groups were subjected to.

 
No changes were observed in EDP (2.8±0.4 mm Hg) or %DLVP (94±3% of the value at t=0 min) between groups just before the 40-min ischemic period (t=35 min; see Fig. 1), demonstrating that ischemic preconditioning, 6AN or DPI did not affect mechanical performance before the lethal ischemic insult. The I/R intervention resulted in a 48±10% recovery of DLVP and 59±5 mm Hg contracture. Ischemic preconditioning improved DLVP recovery to 88±8%; however, no significant improvement in DLVP was observed with 6AN or DPI (Fig. 4). Inhibition of oxPPP with 6AN was not associated with significant attenuation of contracture development, whereas DPI significantly reduced contracture.


Figure 4
View larger version (14K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Developed left ventricular pressure at 30-min reperfusion relative to baseline (t=0 min) values (DLVP%), and diastolic pressure at 30-min reperfusion for the control (co), preconditioned (pc), control plus 6-aminonicotinamide (co+6AN), preconditioned plus 6-aminomicotinamide (pc+6AN) and the diphenyleneiodonium (DPI) group. *p<0.05 vs. control.

 
Forty minutes of global ischemia resulted in a large release of creatine kinase during the 30-min reperfusion (Fig. 5). Ischemic preconditioning decreased this release significantly with approximately 60%. Inhibition of the oxPPP reduced CK release to similar levels as ischemic preconditioning, with no further protection offered by ischemic preconditioning. DPI mimicked the effects of ischemic preconditioning and oxPPP inhibition on CK release.


Figure 5
View larger version (16K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 Creatine kinase release during the 30-min reperfusion period per gram heart dry weight (gdw) for the five5 different groups (for details, see Fig. 3). *p<0.05 vs. control.

 
Table 1 depicts the levels of sugar phosphates that are intermediates in both the oxidative and nonoxidative pentose pathway. During the 40-min ischemia, the ribose-5P and the (ribulose-5P+xylulose-5P) levels rose approximately 3.5 and 15 times, respectively, with minor changes in sedoheptulose-7P. These values were partially recovered at 10 min of reperfusion. However, at any one of the four time points determined, values were similar for preconditioned hearts as compared to control hearts. Although there seems to be a trend for decreased buildup of PPP intermediates at 40-min ischemia in the hearts with inhibition of the oxidative PPP, this trend did not reach significance (e.g. ribose-5P decreased from 59±6 nmol/gdw for control to 36±11 nmol/gdw in the presence of 6AN, p=0.11).


View this table:
[in this window]
[in a new window]

  		Table 1 Concentrations of PPP intermediates (nmol/gdw) for control (CO), preconditioned (PC) and 6-aminonicotinamide (6AN) hearts

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The major findings of this study can be summarized as follows: (1) ischemic preconditioning can attenuate cardiac ischemic damage without alterations in mitochondrial redox state; (2) inhibition of the oxidative pentose phosphate pathway protects the heart against ischemic damage; and (3) ischemic preconditioning does not result in altered activity of the pentose phosphate pathway.

4.1. Unaltered NADH levels with ischemic preconditioning
In our study, NADH levels determined from epifluorescence were not significantly higher during index ischemia or reperfusion for preconditioned hearts as compared to nonpreconditioned hearts. These data are in conflict with recently published data showing that preconditioning protected NADH levels during both ischemia and reperfusion [17]. In that same study, it was also shown that in the nonpreconditioned hearts, NADH epifluorescence decreased during reperfusion to levels far below baseline. However, in a previous study of that same group, no changes in NADH epifluorescence with ischemia–reperfusion had been observed [18]. It is possible that these conflicting results are due to optical artifacts and/or imaging of only the most outer surface of the heart, which are limitations inherent to the NADH epifluorescence technique. Therefore, we also performed global, biochemical determinations of NADH in nonpreconditioned hearts. These data showed no decreased NADH levels following ischemia–reperfusion, in accordance with observations made by others [29]. Our data suggest that the mitochondrial redox level of the pyridine nucleotides is not significantly affected by ischemic preconditioning, in contrast to the preconditioned-induced thiol redox changes [5]. These data are in concordance with a recent report that also showed an unaltered redox level of pyridine nucleotides with preconditioning in isolated mitochondria [19].

4.2. Inhibition of oxPPP confers cardioprotection
To our knowledge, this is the first study that has manipulated the oxidative pentose phosphate pathway in the setting of cardiac ischemia–reperfusion injury. That little attention has been paid to this pathway during ischemia probably stems from the reported low capacity of this pathway in the heart [30]. Although within the mitochondrial compartment NADPH is mostly produced by transhydrogenase and isocitrate dehydrogenase, recent data indicate that for the cytosolic compartment G6PD, and thus the oxPPP is a critical supplier of NADPH [31]. This cytosolically produced NADPH can be directed to maintain cytosolic glutathione in its reduced state [31]. Depending, however, on cellular signals (e.g. calcium) that may activate NADPH oxidase and NO synthases, this NADPH can also be used to support a pro-oxidant environment [12]. Our data indicate that during an acute cardiac ischemia–reperfusion assault, the oxPPP-produced NADPH is mostly directed to the NADPH oxidase and/or NO synthase. This is also supported by our observation that direct inhibition of NADPH oxidase and NO synthase by DPI resulted in a similar reduction in CK release as oxPPP inhibition.

That oxPPP inhibition only protected against CK release and not against mechanical dysfunction supports other studies that have observed this dichotomy between reduction in necrosis and improved functional recovery. It supports the contention that protection against ischemic damage or mechanical dysfunction does not follow completely identical paths [32,33]. Our data would suggest that the oxidant pathway figures more prominently in ischemic damage than in contractile impairment. However, it is also possible that identical paths are still involved, but that it is just a matter of intensity of the ischemic–reperfusion insult. All studies that have shown a dichotomy report that first infarct reduction takes place before increased functional recovery [32–35]. To what extent the reduction in infarct is due to the inhibition of a pathway not involved in reducing functional recovery or just to diminished activation of a pathway involved both in necrosis and dysfunction is presently unclear. Alternatively, it is also possible that functional recovery is reduced with oxPPP inhibition due to the reported observations that inhibition of glucose-6-phosphate dehydrogenase may impair intracellular calcium transport [31]. Although we observed no contractile impairment after 30-min oxPPP inhibition, we cannot exclude that prolonged inhibition does indeed result in decreased mechanical performance. Further studies are needed to resolve these issues.

4.3. Preconditioning not associated with change in pentose phosphate pathway
Since the first studies on preconditioning, it has been known from the examination of glycolytic intermediates that changes in glycolytic flux are a consistent finding in myocardium protected by ischemic preconditioning [1,36]. The mechanism by which this occurs is still elusive [37]. Because the pentose phosphate pathway derives its intermediates to a large extent from glycolysis, we hypothesised that PPP intermediates would also deviate between preconditioned and control myocardium. This is why we choose to examine the nature of the changes of these PPP intermediates. However, our data clearly show no difference in PPP intermediate levels following ischemic preconditioning. Both ribose-5P and (ribulose-5P+xylulose-5P) rose several-fold during ischemia. This increase is likely due to activation of G6PD by oxidative stress [31,38] and by substrate effects due to increased amounts of glucose-6P and fructose-6P during the initial period of ischemia [37]. The end-product of the irreversible oxPPP is ribulose-5P, which can be converted into ribose-5p and xylulose-5P by the nonoxPPP enzymes ribose-5P isomerase and ribulose-5P-epimerase. The other nonoxPPP enzymes transketolase (TK) and transaldolase (TA) interconvert the aforementioned pentoses with glycolytic intermediates (fructose-6P, glyceraldehyde 3P) and sedoheptulose-7P/erythrose-4P (see Fig. 2; [39]). The results with the inhibitor of the oxPPP, showing only a nonsignificant trend in a decreased buildup of PPP intermediates, indicate that at least during ischemia, the flux through the nonoxPPP is higher than that of the oxPPP in the heart. This is consistent with observations made by Goodwin et al. [40], although controversy over this issue exists for the heart in normoxic conditions [41]. Our data showing the similar changes in PPP intermediates between control and preconditioned hearts suggest that ischemic preconditioning is not associated with a major alteration in the regulation of the pentose phosphate pathway in the heart.

4.4. Methodological considerations
In this study, glucose was the only exogenous fuel for the isolated heart, a strategy chosen by most researchers examining preconditioning in isolated hearts. Although it is known that even under these conditions the heart still derives >50% of its energy from oxidation of endogenous stored fatty acids [42], our perfusion medium does not completely mimic the in vivo condition. Additional studies are needed to determine to what extent the results obtained are influenced by only providing glucose as substrate.

This is the first study that has used the drug 6-aminonicotinamide in the isolated heart. 6AN is a competitive inhibitor of the glucose-6-phosphate dehydrogenase (G6PD) of the oxPPP, by the production of a nonmetabolizable analogue of NADP [14]. Although it has been shown that, when intraperitoneally applied to the intact animal, 2 h are necessary before inhibition of the oxPPP can be measured, studies in isolated perfused organs (lung, kidney) and cells show inhibition within 30–60 min treatment with 6AN [15,16,43–45]. The reduction of NADPH levels following 30 min of 6AN perfusion observed in this study confirms also that in the heart, G6PD is partially inhibited.

CK enzyme release into the venous effluent was used as index of ischemic damage in this study. It may be argued that no enzyme release is possible from flow-obstructed areas of the post-ischemic heart, and therefore enzyme release is not a good indicator of ischemic damage. However, flow at the end of reperfusion is similar for all five groups (73±2.1 ml/min/gdw), with a tendency of being increased as compared to baseline levels (61.0±2.2 ml/min/gdw). In addition, flow-obstructed areas should be associated with increased NADH videofluorometry values, not observed in this study. It therefore seems likely that no flow-obstructed areas are present in our hearts, allowing enzyme release to be used as an index of damage. It is also known that CK does not originate exclusively from irreversible damaged cardiomyocytes, such that CK release is best viewed as a semiquantitative indicator of ischemic damage and not necrosis per se. That CK release may be used as an indicator of cardiac injury is corroborated by other studies that have shown a good correlation between enzyme release and other indices of cardiac injury in isolated hearts subjected to global ischemia–reperfusion protocols [32,46–48].

In summary, our study indicates that the protective actions of ischemic preconditioning are not mediated by changes in the pentose phosphate pathway. In addition, our studies reveal that during ischemia–reperfusion, the pentose phosphate pathway is active in the heart and that inhibition of the oxidative part of this pathway during an acute ischemia–reperfusion assault represents a novel mechanism through which cardiac injury, as indexed by enzyme release, may be reduced.


   Notes
 
Time for primary review 21 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

  1. Murry C.E., Jennings R.B., Reimer K.A. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation (1986) 74:1124–1136.[Abstract/Free Full Text]
  2. Baines C.P., Goto M., Downey J.M. Oxygen radicals released during ischemic preconditioning contribute to cardioprotection in the rabbit myocardium. J. Mol. Cell. Cardiol. (1997) 29:207–216.[CrossRef][ISI][Medline]
  3. Tritto I., D'Andrea D., Eramo N., et al. Oxygen radicals can induce preconditioning in rabbit hearts. Circ. Res. (1997) 80:743–748.[Abstract/Free Full Text]
  4. Van den Hoek T.L., Becker L.B., Shao Z., et al. Reactive oxygen species released from mitochondria during brief hypoxia induce preconditioning in cardiomyocytes. J. Biol. Chem. (1998) 273:18092–18098.[Abstract/Free Full Text]
  5. Chen W., Gabel S., Steenbergen C., et al. A redox-based mechanism for cardioprotection induced by ischemic preconditioning in perfused rat heart. Circ. Res. (1995) 77:424–429.[Abstract/Free Full Text]
  6. Garlid K.D., Paucek P., Yarov-Yarovoy V., et al. Cardioprotective effect of diazoxide and its interaction with mitochondrial ATP-sensitive K+ channels: possible mechanism of cardioprotection. Circ. Res. (1997) 81:1072–1082.[Abstract/Free Full Text]
  7. Forbes R.A., Steenbergen C., Murphy E. Diazoxide-induced cardioprotection requires signaling through a redox-sensitive mechanism. Circ. Res. (2001) 88:802–809.[Abstract/Free Full Text]
  8. Crestanello J.A., Lingle D.M., Kamelgard J., et al. Ischemic preconditioning decreases oxidative stress during reperfusion: a chemiluminescence study. J. Surg. Res. (1996) 65:53–58.[CrossRef][ISI][Medline]
  9. Tosaki A., Cordis G.A., Szerdahelyi H., et al. Effect of preconditioning on reperfusion arrhythmias, myocardial function, formation of free radicals, and ion shifts in isolated ischemic/reperfused rat hearts. J. Cardiovasc. Pharmacol. (1994) 23:365–373.[ISI][Medline]
  10. Kevin L.G., Camara A.K.S., Riess M.L., et al. Ischemic preconditioning alters real-time measure of O2 radicals in intact hearts with ischemia and reperfusion. Am. J. Physiol. Heart Circ. Physiol. (2003) 284:H566–H574.[Abstract/Free Full Text]
  11. Csonka C., Szilvassy Z., Fulop F., et al. Classic preconditioning decreases the harmful accumulation of nitric oxide during ischemia and reperfusion in rat hearts. Circulation (1999) 100:2260–2266.[Abstract/Free Full Text]
  12. Spolarics Z. Endotoxemia, pentose cycle, and the oxidant/antioxidant balance in the hepatic sinusoid. J. Leukoc. Biol. (1998) 63:534–541.[Abstract]
  13. Turrens J.F., Thornton J., Barnard M.L., et al. Protection from reperfusion injury by preconditioning hearts does not involve increased antioxidant defenses. Am. J. Physiol. Heart Circ. Physiol. (1992) 262:H585–H589.[Abstract/Free Full Text]
  14. Köhler E., Barrach H.-J., Neubert D. Inhibition of NAD dependent oxidoreductases by the 6-aminonicotidamine analogue of NADP. FEBS Lett. (1970) 6:225–228.[CrossRef][ISI][Medline]
  15. Gupte S.A., Li K.-X., Okada T., et al. Inhibitors of pentose phosphate pathway cause vasodilation: involvement of voltage-gated potassium channels. J. Pharmacol. Exp. Ther. (2002) 301:299–305.[Abstract/Free Full Text]
  16. Rostand S.G., Work J. Effect of 6-aminonicotinamide on renin release in isolated rat kidney: possible role for the pentose pathway. Am. J. Physiol. Renal Fluid Electrolyte Physiol. (1985) 249:F213–F219.[Abstract/Free Full Text]
  17. Riess M.L., Camara A.K.S., Chen Q., et al. Altered NADH and improved function by anesthetic and ischemic preconditioning in guinea pig intact hearts. Am. J. Physiol. Heart Circ. Physiol. (2002) 283:H53–H60.[Abstract/Free Full Text]
  18. Varadarajan S.G., An J., Novalija E., et al. Changes in [Na(+)](i), compartmental [Ca(2+)], and NADH with dysfunction after global ischemia in intact hearts. Am. J. Physiol. Heart Circ. Physiol. (2001) 280:H280–H293.[Abstract/Free Full Text]
  19. Da Silva M.M., Sartori A., Belisle E., et al. Ischemic preconditioning inhibits mitochondrial respiration, increases H2O2 release, and enhances K+ transport. Am. J. Physiol. Heart Circ. Physiol. (2003) 285:H154–H162.[Abstract/Free Full Text]
  20. Barlow C.H., Chance B. Ischemic areas in perfused rat hearts: measurements by NADH fluorescence photography. Science (1976) 193:909–910.[Abstract/Free Full Text]
  21. Zuurbier C.J., Kruyver B., Eerbeek O., et al. Commonly used numbers of microspheres affect cardiac vascular resistance. J. Cardiovasc. Pharmacol. (2003) 41:223–232.[CrossRef][ISI][Medline]
  22. Zuurbier C.J., van Iterson M., Ince C. Functional heterogeneity of oxygen supply–consumption ratio in the heart. Cardiovasc. Res. (1999) 44:488–497.[Abstract/Free Full Text]
  23. Huck J.H.J., Struys E.A., Verhoeven N.M., et al. Profiling of pentose phosphate pathway intermediates in blood spots by tandem mass spectrometry: application to transaldolase deficiency. Clin. Chem. (2003) 49:1375–1380.[Abstract/Free Full Text]
  24. Ashruf J.F., Coremans J.M.C.C., Bruining H.A., et al. Increase of cardiac work is associated with decrease of mitochondrial NADH. Am. J. Physiol. Heart Circ. Physiol. (1995) 269:H856–H862.[Abstract/Free Full Text]
  25. Szasz G., Gruber W., Bernt E. Creatine kinase in serum: 1. Determination of optimum reaction conditions. Clin. Chem. (1976) 22:650–656.[Abstract/Free Full Text]
  26. Dunphy G., Ely D. Decreased storage stability of creatine kinase in a cardiac reperfusion solution. Clin. Chem. (1990) 36:778–780.[Abstract/Free Full Text]
  27. Klingenberg M. Methods of enzymatic Analysis, 2nd ed. Bergmeyer H.U., ed. (1974) New York: Academic Press. 2045–2057.
  28. Zhang Z., Yu J., Stanton R.C. A method for determination of pyridine nucleotides using a single extract. Anal. Biochem. (2000) 285:163–167.[CrossRef][ISI][Medline]
  29. Ceconi C., Bernocchi P., Boraso A., et al. New insights on myocardial pyridine nucleotides and thiol redox state in ischemia and reperfusion damage. Cardiovasc. Res. (2000) 47:586–594.[Abstract/Free Full Text]
  30. Zimmer H.G. The oxidative pentose phosphate pathway in the heart: regulation, physiological significance, and clinical implications. Basic Res. Cardiol. (1992) 87:303–316.[CrossRef][ISI][Medline]
  31. Jain M., Brenner D.A., Cui L., et al. Glucose-6-phosphate dehydrogenase modulates cytosolic redox status and contractile phenotype in adult cardiomyocytes. Circ. Res. (2003) 93:e9–e16.[Abstract/Free Full Text]
  32. Peart J., Headrick J.P. Adenosine-mediated early preconditioning in mouse: protective signaling and concentration dependent effects. Cardiovasc. Res. (2003) 58:589–601.[Abstract/Free Full Text]
  33. Toyoda Y., Friehs I., Parker R.A., et al. Differential role of sarcolemmal and mitochondrial KATP channels in adenosine-enhanced ischemic preconditioning. Am. J. Physiol. Heart Circ. Physiol. (2000) 279:H2694–H2703.[Abstract/Free Full Text]
  34. Xi L., Hess M.L., Kukreja R.C. Ischemic preconditioning in isolated perfused mouse heart: reduction in infarct size without improvement of post-ischemic ventricular function. Mol. Cell. Biochem. (1998) 186:69–77.[CrossRef][ISI][Medline]
  35. Saurin A.T., Pennington D.J., Raat N.J.H., et al. Targeted disruption of the protein kinase C epsilon gene abolishes the infarct size reduction that follows ischaemic preconditioning of isolated buffer-perfused mouse hearts. Cardiovasc. Res. (2002) 55:672–680.[Abstract/Free Full Text]
  36. Zuurbier C.J., Ince C. Postischemic changes in the response time of oxygen consumption to demand in the isolated rat heart are mediated partly by calcium and glycolysis. Pflügers Arch. (2002) 443:908–916.[CrossRef][ISI][Medline]
  37. Vogt A.M., Poolman M., Ackermann C., et al. Regulation of glycolytic flux in ischemic preconditioning. J. Biol. Chem. (2002) 277:24411–24419.[Abstract/Free Full Text]
  38. Zimmer H.-G., Bünger R., Koshine H., et al. Rapid stimulation of the hexose monophosphate shunt in the isolated perfused rat heart: possible involvement of oxidized glutathione. J. Mol. Cell. Cardiol. (1981) 13:531–535.[CrossRef][ISI][Medline]
  39. Casazza J.P., Veech R.L. The interdependence of glycolytic and pentose cycle intermediates in ad libitum fed rats. J. Biol. Chem. (1986) 261:690–698.[Abstract/Free Full Text]
  40. Goodwin G.W., Cohen D.M., Taegtmeyer H. [5-3H]glucose overestimates glycolytic flux in isolated working rat heart: role of the pentose phosphate pathway. Am. J. Physiol. Endocrinol. Metab. (2001) 280:E502–E508.[Abstract/Free Full Text]
  41. Leong H.S., Grist M., Parsons H., et al. Accelerated rates of glycolysis in the hypertrophied heart: are they a methodological artifact? Am. J. Physiol. Endocrinol. Metab. (2002) 282:E1039–E1045.[Abstract/Free Full Text]
  42. Saddik M., Lopaschuk G.D. Myocardial triglyceride turnover and contributor to energy substrate utilization in isolated working rat hearts. J. Biol. Chem. (1991) 266:8162–8170.[Abstract/Free Full Text]
  43. Bender J.G., van Epps D.E. Inhibition of human neutrophil function by 6-aminonicotinamide: the role of the hexose monophosphate shunt in cell activation. Immunopharmacology (1985) 10:191–199.[CrossRef][ISI][Medline]
  44. Olsen L.F., Kummer U., Kindzelskii A.L., et al. A model of the oscillatory metabolism of activated neutrophils. Biophys. J. (2003) 84:69–81.[Abstract/Free Full Text]
  45. Gupte S.A., Arshad M., Viola S., et al. Pentose phosphate pathway coordinates multiple redox-controlled relaxing mechanisms in bovine coronary arteries. Am. J. Physiol. Heart Circ. Physiol. (2003) 285:H2316–H2326.[Abstract/Free Full Text]
  46. Gustafsson A.B., Sayen M.R., Williams S.D., et al. TAT protein transduction into isolated perfused hearts. Circulation (2003) 106:735–739.
  47. Humphrey S.M., Thomson R.W., Gavin J.B. The influence of the no-reflow phenomenon on reperfusion and reoxygenation damage and enzyme release from anoxic and ischaemic isolated rat hearts. J. Mol. Cell. Cardiol. (1984) 16:915–930.[CrossRef][ISI][Medline]
  48. Kanno S., Lee P.C., Zhang Y., et al. Attenuation of myocardial ischemia/reperfusion injury by superinduction of inducible nitric oxide synthase. Circulation (2000) 101:2742–2748.[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Zuurbier, C.J
Right arrow Articles by Ince, C
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zuurbier, C.J
Right arrow Articles by Ince, C
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?