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Involvement of energetic metabolism in the effects of ischemic postconditioning on the ischemic-reperfused heart of fed and fasted rats

The Journal of Physiological Sciences volume 61, pages 303–312 (2011)Cite this article

An Erratum to this article was published on 30 April 2014

Abstract

The effects of ischemic-postconditioning (IPOC) on functional recovery and cell viability of ischemic-reperfused hearts from fed and fasted rats were studied in relation to triacylglycerol and glycogen mobilization, ATP content, glucose-6-phosphate dehydrogenase activity and reduced/oxidized glutathione (GSH/GSSG). Oxidative damage was estimated by measuring thiobarbituric acid reactive substances (TBARS). IPOC improved contractile recovery and cell viability in the fed but attenuated them in the fasted hearts. In both groups ischemia lowered glycogen. IPOC further reduced it. Triacylglycerol remained unchanged during ischemia-reperfusion in both groups, but triacylglycerol mobilization was activated by IPOC in the fasted group. ATP was increased by IPOC in the fed hearts, but lowered in the fasted ones, which appeared to be associated with the rates of ATP synthesis in isolated mitochondria. In the fed hearts IPOC raised glucose-6-phosphate dehydrogenase activity and GSH/GSSG, and lowered TBARS. These results suggest that IPOC effects are associated with changes in the ATP supply, mobilization of energy sources and glutathione antioxidant ratio.

Introduction

After exposure to ischemia, reperfusion of the myocardium is the prerequisite for any potential recovery of contractile function. However, reperfusion by itself can cause additional damage, also known as reperfusion injury [1]. Reperfusion injury could be attenuated by interventions applied either before the onset of ischemia or during reperfusion. Among these procedures, ischemic postconditioning (IPOC), defined as rapid intermittent interruptions of blood flow in the early phase of reperfusion, has been shown to attenuate myocardial injury in many experimental studies as well as in clinical settings [2, 3]. Interestingly, the reduction of infarct size by postconditioning persists for up to 72 h [4], suggesting that IPOC effects the long-term reduction in reperfusion damage and therefore not merely the delay of an inevitable injury. This procedure seems to activate multiple intracellular molecular pathways that attenuate reperfusion injury, but their respective contribution to the amelioration of necrosis, apoptosis, endothelial injury and/or microvascular and macrovascular injury is not yet fully understood [3, 4].

On the other hand, it has been found that previous fasting protects the heart against ischemia-reperfusion injury by improving functional recovery [5, 6], increasing the ratio of reduced to oxidized glutathione (GSH/GSSG) and decreasing oxidative damage in the Langendorff perfused rat hearts submitted to no-flow global ischemia-reperfusion [6]. These changes appear to be a consequence of the increased hydrolysis of endogenous triacylglycerol (TAG) and the upregulation of glucose-6-phosphate dehydrogenase (G6PDH), the first and rate-limiting enzyme of the pentose phosphate pathway, that occurs in the fasted rat heart perfused under aerobic conditions before ischemia-reperfusion [6]. It is well known that glucose-6-phosphate, which is derived either from glycogenolysis or from glucose taken up by the cell, is metabolized mainly via glycolysis. However, a small amount enters the pentose phosphate pathway in which G6PDH is the first and rate-limiting enzyme. This pathway provides reducing equivalents in the form of NADPH, which is required by the heart in order to maintain tissue levels of GSH that are essential for detoxification of reactive oxygen species. Furthermore, it has been shown that specific adaptive regulation of G6PDH, which is exerted both before and after transcription, is an important component of the integrated response to hormones, nutrients and oxidative stress [7].

Until now no study has examined the modulatory role of the nutritional condition in IPOC-mediated cardioprotection. The aim of the present work was therefore to assess the functional and metabolic response to no-flow ischemia in ischemic-postconditioned hearts from fed and fasted rats and compare this response to non-postconditioned hearts. TAG and glycogen mobilization were measured. Since fasting results in increases in G6PDH activity and the GSH/GSSG ratio, it seemed appropriate to investigate whether the protection afforded by IPOC is associated with changes in G6PDH activity and the GSH/GSSG ratio in the tissue. In order to assess the oxidative damage to the tissue, the concentration of compounds that react with thiobarbituric acid (TBARS) was determined.

Materials and methods

Experimental protocol

This study conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication no. 85–23, revised 1996; http://acu.od.nih.gov/regs/guide.pdf) and Argentine Law No. 14346 concerning animal protection.

Female Wistar rats, weighing 250–350 g, maintained on a 12-h dark-light cycle, fed ad libitum or fasted for 24 h, were used in the study. Rats were anaesthetized with diethylether, and then heparin (250 IU) was injected into the jugular vein. Hearts were excised quickly and cooled in ice-cold saline until contractions stopped. Hearts were then mounted on a modified Langendorff apparatus (Hugo Sachs Elektronik, March-Hugstetten, Germany) and perfused at a constant pressure of 70 mmHg with a non-recirculating Krebs-Ringer bicarbonate solution of the following composition (mM): NaCl 120; NaHCO3 25; KCl 4.8; MgSO4 1.33; KH2PO4 1.2; CaCl2 1.6; Na2EDTA 0.02; glucose 10. The perfusate was gassed with 95% O2 and 5% CO2 (pH 7.4), and kept at a constant temperature of 37°C. In the conventional Langendorff preparation, oxygen is provided by gassing the perfusion solution with a sintered glass bubbling device with high concentrations of oxygen because of the low oxygen carrying capacity of crystalloid buffers. Typically a mixture of 95% oxygen and 5% carbon dioxide is used to ensure adequate O2 delivery to the cells.

After a 25-min equilibration period, hearts were subjected to 25 min of global ischemia, followed by 30 min of reperfusion (RP). Ischemia was started by shutting off the flow of perfusate. IPOC was induced by six cycles of 10-s reperfusion interspersed by 10-s no-flow ischemia immediately after sustained ischemia.

Only hearts with left ventricular developed pressure (LVDP) >60 mmHg and heart rate (HR) >200 beats/min at the end of the equilibration period were included in the study.

It is worth noting that Langendorff perfused rat hearts subjected to 25 min of total global ischemia followed by 30 min reperfusion have been extensively used for the evaluation of cardioprotective interventions on necrosis, functional recovery and the study of metabolic pathways [6, 8, 9].

Measurement of heart function

The left atrium was removed, and a latex balloon connected to a pressure transducer was inserted into the left ventricle through the mitral valve in order to measure left ventricular pressures. The volume of the balloon was adjusted to obtain an initial left ventricular end diastolic pressure (LVEDP) of 10 mmHg. This allowed continuous measurement of end diastolic and systolic pressure changes during ischemia and reperfusion. Values for LVDP, peak rate of contraction (+dP/dt) and peak rate of relaxation (−dP/dt) were obtained using a digital data acquisition system. Heart rate was measured by means of a counter that was triggered by the LVDP pulse. Rate-pressure product (RPP) was determined by multiplying HR by LVDP.

Measurement of cell viability

At the end of the RP period the hearts were removed, frozen and cut into six to eight slices of approximately 0.8 up to 1 mm of thickness. Following defrosting the slices were incubated at room temperature with 1% triphenyltetrazolium chloride in phosphate buffer (100 mM, pH 7.4) for 90 min and fixed in 10% formaldehyde solution to distinguish clearly stained viable tissue and unstained necrotic tissue. The areas of viable tissue were determined by computer morphometry (Scion Image B 4). The risk area was the sum of total ventricular area minus cavities. The cellular viability was calculated as percentage of risk area.

TBARS, G6PDH and GSH/GSSG assay

TBARS, G6PDH and GSH/GSSG were measured from parallel experiments in separate hearts treated according to the above protocols. Frozen heart tissue was homogenized in 5 ml of 50 mM cold phosphate buffer (pH 7.4). An aliquot was taken for measurement of TBARS as a marker of lipid peroxidation. The rest of the homogenate was centrifuged at 10,000 rpm for 10 min at 0°C, and the supernatant separated and used for measurement of G6PDH and GSH/GSSG.

G6PDH activity was measured using spectrophotometry [10]. Enzyme activity was expressed as units/g protein. The amount of protein was determined by the method of Lowry et al. [11], with bovine serum albumin (BSA) as the standard.

GSH/GSSG was determined using a commercially available kit (Calbiochem, La Jolla, CA). The technique is based on the enzymatic recycling method described by Tietze [12].

Levels of TBARS were determined using a commercially available kit (Cayman Chemical Co., Ann Arbor, MI) based on the spectrophotometric method described by Yagi [13].

ATP, TAG and glycogen assay

Tissue ATP, glycogen and TAG were measured from parallel experiments in separate hearts treated according to the above protocols. A sample of approximately 60 mg of wet tissue was used to determine the dry-to-wet ratio and to calculate the total dry weight (g) of the heart.

Tissue levels of ATP were determined by luciferin-luciferase luminometry (Sigma bioluminescent assay kit) in ~200 mg neutralized HClO4 extracts of frozen ventricular tissue according to a standard technique [14].

For measurement of TAG content, ~200 mg frozen heart tissue was homogenized in chloroform:methanol (2:1). Following overnight extraction at 4°C, the homogenate was filtered, mixed with an equal volume of cold saline and centrifuged at 0°C. The lower phase was then transferred to a fresh tube, and the phospholipids were adsorbed by addition of activated silicic and shaking for 90 s using a vortex mixer. The silicic acid was packed by centrifugation at 20°C for 10 min, and an aliquot was transferred to a fresh tube and reduced to dryness. TAG was saponified with ethanolic KOH at 60°C for 15 min. The amount of glycerol was measured by spectrophotometry [15].

Glycogen was determined in ~200 mg samples of frozen ventricular tissue according to the method of Walaas and Walaas [16] with the use of amyloglucosidase.

Measurement of mitochondrial ATP synthesis

At the end of ischemia-reperfusion, hearts were removed from the Langendorff apparatus, and mitochondria were isolated by differential centrifugation after tissue homogenization in ice-cold sucrose buffer solution (300 mM sucrose, 10 mM Tris-Cl, 2 mM EGTA, 5 mg/ml BSA, pH 7.4). The mitochondrial pellet was then washed three times in sucrose isolation buffer solution lacking BSA. Cardiac mitochondria prepared with this procedure have been shown to be metabolically active with respiratory control ratios of 3.5–5.0 with succinate and 8.0–10.0 with glutamate/malate, and corresponding ADP/O ratios of 1.5–1.7 and 2.5–2.7, respectively [17]. Since it is well documented that complex I of the respiratory chain is the most sensitive to reperfusion injury [18], mitochondrial ATP synthesis was measured in the presence of the complex I substrates pyruvate and malate. Mitochondria (1 mg protein/mL) were incubated for 10 min in a 25°C metabolic shaker in a medium containing (mM): KCl 125, Mops 20, Tris 10, EGTA 0.5, KH2PO4 2.4, MgCl2 2.5, malate 2.5 and pyruvate 2.5, pH 7.4. After 2 min, ATP synthesis was initiated by addition of ADP 2.5 mM. ADP 2.5 mM was used because it corresponds to a physiological concentration found in myocytes. Aliquots were taken from the incubation mixture at 3-min intervals for 10 min, and reactions were stopped by adding perchloric acid. The neutralized supernatant was assayed for ATP by luciferin-luciferase luminometry (Sigma bioluminescent assay kit). Mitochondrial protein concentration was determined by the method of Lowry using BSA as a standard, and the rate of mitochondrial ATP synthesis was calculated and expressed as nmol per minute per milligram of mitochondrial protein.

Statistical analysis

All data are presented as mean ± SEM. Changes in the ventricular contractile function were statistically compared using a three-factor ANOVA for repeated measurements in one factor, followed by Tukey’s test. Differences between the same biochemical measurements at different times or between variables in control and postconditioned fed and fasted hearts were assessed using factorial ANOVA followed by Tukey’s test. Statistical significance was set at p < 0.05.

Results

At the end of the equilibration period, fasting did not exhibit any effect on baseline values of HR, RPP, +dP/dt and −dP/dt (data not shown).

The exposure to 25 min of global ischemia led to complete cessation of spontaneous contractions, and during the 30 min of reperfusion the HR gradually returned to pre-ischemic values, which were similar in both fed and fasted control hearts (15-min RP: control fed 190.33 ± 27.35 and control fasted 196.12 ± 16.54; 30-min RP: control fed 222.63 ± 12.69 and control fasted 218.25 ± 8.22; expressed as beats/min.). In addition, there was no significant difference in HR between control and postconditioned hearts during reperfusion, nor between fed and fasted postconditioned hearts (15-min RP: postconditioned fed 207.00 ± 14.21 and postconditioned fasted 191.00 ± 17.9; 30-min RP: postconditioned fed 231.24 ± 5.40 and postconditioned fasted 210.63 ± 5.90 beats/min). Upon reperfusion, hearts from fasted rats exhibited faster recovery of RPP, +dP/dt and −dP/dt compared with hearts from fed rats (Fig. 1a–c). The rate of recovery of RPP, +dP/dt and −dP/dt in fed rat hearts was improved by IPOC, but markedly reduced in hearts from fasted rats. During reperfusion and concurrently with the faster rate of recovery of contractile function, hearts from fasted rats exhibited a smaller amplitude of LVEDP than hearts from fed animals (Fig. 1d). The amplitude of LVEDP was significantly reduced by IPOC in the fed hearts, but increased in the fasted hearts when compared with the control groups. Hence, the difference between hearts from fed and fasted rats disappeared in postconditioned hearts.

Fig. 1
figure 1

Changes in: A rate-pressure product (RPP); B peak rate of contraction (+dP/dt); C peak rate of relaxation (−dP/dt) and D left ventricular end-diastolic pressure (LVEDP) due to ischemia-reperfusion in control and postconditioned hearts from fed and fasted rats. Values are expressed as percentages of the respective basal values at the end of the 25-min equilibration period. Open symbols fed hearts. Filled symbols fasted hearts. Squares control hearts. Triangles postconditioned hearts. Values are mean ± SEM (n = 8) A, B, C: a, p < 0.01 versus control fed, b, p < 0.05 versus postconditioned fasted, c, p < 0.05 versus postconditioned fed, d, p < 0.01 versus postconditioned fasted, e, p < 0.01 versus postconditioned fed. D a, p < 0.05 versus control fasted, b, p < 0.05 versus postconditioned fed., c, p < 0.05 versus postconditioned fasted

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In accordance with previous data [6] the percentage of viable myocytes was similar in both nutritional conditions (fed 41.2 ± 1.1% and fasted 40.1 ± 0.5%; n = 8). Compared with hearts from control animals, cell viability was increased in the postconditioned fed hearts (61.0 ± 0.7%, p < 0.05 vs. control fed; n = 8), whereas it was decreased in the postconditioned fasted hearts (33.5 ± 1.2%, p < 0.05 vs. control fasted; n = 8).

Glycogen content measured in vivo (n = 8) was higher in the fasted group than in the fed group (362.15 ± 8.12 vs. 229.21 ± 6.11 μg/100 mg dry weight; p < 0.01) and decreased only in the fasted hearts during the pre-ischemic stabilization period (fasted: 321.35 ± 7.23 μg/100 mg dry weight, p < 0.05 vs. in vivo; fed: 247 ± 4.11 μg/100 mg dry weight, n.s. vs. in vivo). In spite of this, the fasted group exhibited a higher glycogen content before the onset of ischemia compared with the fed group (Fig. 2). During ischemia, glycogen content fell to similar values in both nutritional groups, and no further decrease occurred during reperfusion. These changes in glycogen content resulted in a marked difference in glycogen breakdown, which was about 25% higher in the fasted than in the fed group. Interestingly, IPOC reduced glycogen content close to exhaustion during reperfusion in both nutritional conditions (Fig. 2).

Fig. 2
figure 2

Glycogen content of hearts subjected to ischemia-reperfusion. Values are mean ± SEM (n = 8) and are expressed as μg glycogen/100 mg dry weight. Open bars fed hearts, filled bars fasted hearts. Pre-ischemic refers to the end of the 25-min pre-ischemic period. Ischemic refers to the end of the 25-min ischemic period. Reperfusion refers to the end of the 30-min reperfusion period. C control hearts. IPOC postconditioned hearts. a, p < 0.01 versus pre-ischemic in the same nutritional condition; b, p < 0.01 versus fed pre-ischemic; c, p < 0.01 vs. C reperfusion in the same nutritional condition

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TAG content measured immediately after excision of the hearts (in vivo, n = 8), was higher in the fasted group than in the fed group (19.52 ± 1.77 vs. 8.66 ± 1.88 μmol/g dry weight; p < 0.01). No change in TAG content was seen throughout the experiment in the hearts from fed rats (Fig. 3). In contrast, during the pre-ischemic period, the TAG content of the hearts from the fasted group decreased compared with that in hearts immediately after their removal from rats to 16.29 ± 1.24 μmol/g dry weight (p < 0.05). This indicates that the consumption of TAG increased in fasted rat hearts. However, at the end of the ischemic-RP period, there was no further change in TAG levels in fasted rats, indicating that, in this group, the utilization of endogenous TAG did not persist during the post-ischemic period (Fig. 3). TAG content at the end of the 30-min reperfusion period was not affected by IPOC in the fed group, but was markedly lowered in the fasted group (Fig. 3). Oxfenicine (2 mM), a carnitine palmitoyltransferase I inhibitor, added during the 30-min RP period abolished TAG breakdown in the postconditioned fasted hearts (postconditioned fasted plus oxfenicine: 12.90 ± 1.31 vs. postconditioned fasted: 7.77 ± 1.21 μmol/g dry weight; p < 0,01) and the noxious effects of IPOC on functional recovery in the fasted hearts (Table 1), but did not affect the recovery in the postconditioned fed ones.

Fig. 3
figure 3

TAG content of hearts subjected to ischemia-reperfusion. Values are mean ± SEM (n = 8) and are expressed as μmol/g dry weight. Open bars fed hearts, filled bars fasted hearts. Pre-ischemic refers to the end of the 25-min pre-ischemic period. Ischemic refers to the end of the 25-min ischemic period. Reperfusion refers to the end of the 30-min reperfusion period. C control hearts. IPOC postconditioned hearts. a, p < 0.01 versus fed hearts, same treatment; b, p < 0.01 versus IPOC reperfusion fasted heart and versus pre-ischemic fasted hearts

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Table 1 Effects of oxfenicine on contractile recovery of postconditioned fasted hearts
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Immediately before the beginning of ischemia, the myocardial ATP level was similar in both nutritional groups (Fig. 4). At the end of ischemia, ATP levels declined to the same extent in both groups, but at the end of reperfusion they were higher in the fasted group (Fig. 4). Accordingly, the rate of ATP synthesis in mitochondria isolated from hearts exposed to ischemia-reperfusion was higher in the fasted than in the fed group (Fig. 5). At the end of reperfusion, IPOC markedly raised myocardial ATP content in the fed condition, whereas this intervention decreased myocardial ATP content in the hearts from fasted rats (Fig. 4) compared with the control ones. Furthermore, the rate of ATP synthesis in the isolated mitochondria of fed rat hearts was increased by IPOC, but reduced in the fasted group (Fig. 5). It is worth noting that the effects of IPOC on functional recovery and the extent of cell viability in the fed and fasted hearts appeared to be associated with the changes in the in vitro rates of ATP production, suggesting that the mitochondrial ATP synthesis capacity observed in vitro could also occur in situ.

Fig. 4
figure 4

ATP content of hearts subjected to ischemia-repefusion. Values are mean ± SEM (n = 8) and are expressed as μmol/g dry weight. Open bars fed hearts; filled bars fasted hearts. Pre-ischemic refers to the end of the 25-min pre-ischemic period. Reperfusion refers to the end of the 30-min reperfusion period. C control hearts. IPOC postconditioned hearts. a, p < 0.05 versus C reperfusion in the same nutritional condition. b, p < 0.05 versus pre-ischemic in the same nutritional condition. c, p < 0.05 versus C reperfusion fed and versus ischemic fasted

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Fig. 5
figure 5

ADP-stimulated ATP production in isolated mitochondria from hearts subjected to ischemia-reperfusion. Open bars fed hearts; filled bars fasted hearts. Values are mean ± SEM (n = 10) and are expressed as nmol/min/mg mitochondrial protein. a, p < 0.05 versus control fed and versus postconditioned fasted; b, p < 0.01 versus control fasted

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As indicated in Table 2, fasting increased G6PDH activity 1.44 times, increased the GSH/GSSG ratio by a factor of 3.63 and decreased TBARS levels by 44% in the pre-ischemic hearts. Ischemia-reperfusion did not affect G6PDH activity, but decreased the GSH/GSSG ratio by approximately 50% in both the fed and fasted groups. Thus, fasted hearts at the end of reperfusion exhibited a markedly higher GSH/GSSG ratio than fed ones. Along with the changes in glutathione status, tissue levels of TBARS were increased during ischemia-reperfusion in both nutritional conditions (Table 2). Despite these increases, TBARS levels remained lower in the fasted group compared with the fed one (Table 2). In the reperfused fed hearts, IPOC significantly raised G6PDH activity and the GSH/GSSG ratio, and reduced TBARS content (Table 2). However, in the fasted group IPOC did not cause changes in any of these variables. Accordingly, the differences between the fed and fasted rat hearts were abolished in the IPOC-treated hearts.

Table 2 Glucose-6-phosphate dehydrogenase (G6PDH) activity, reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio and thiobarbituric acid-reactive substances (TBARS) levels in control and postconditioned hearts from fed and fasted rats
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Discussion

In agreement with previous reports, present data indicate that IPOC accelerated functional recovery of isolated hearts from fed rats [2–4]. Fasting status per se exerted protective effects on the contractile function [5, 6], which were attenuated by IPOC, suggesting that the latter might interfere with some of the “beneficial” biochemical pathways activated during fasting. These findings of greater or lesser rates of functional recovery—specially during early reperfusion—in the fed and fasted postconditioned hearts were supported by concordant measurement of cell viability. Thus, the internal consistency of these data strongly supports the protective or deleterious effects of this intervention.

Consistent with earlier observations, the hearts from fasted rats had higher in vivo TAG and glycogen levels than the fed hearts [6, 19]. Fasting is a condition in which serum fatty acids and myocardial fatty acids uptake are elevated, and a shift in substrate preference from glucose to fatty acids occurs. The enhanced oxidation of fatty acids increases glycogen content through the inhibition of the glycolytic pathway. In addition, in this state, myocardial TAG also increases because fatty acids and acyl-CoA that are not used for β-oxidation are sequestered into TAG. In the absence of exogenous fatty acids, TAG levels decreased during the pre-ischemic period only in the fasted hearts, but remained higher with regard to the fed hearts. It seems reasonable to suggest that oxidation of endogenous fatty acids is responsible for TAG breakdown. On the other hand, the breakdown of glycogen during the pre-ischemic period, observed only in hearts from fasted rats, could be due to oxidation of glucose through the pentose phosphate pathway.

As has been previously shown [6], ischemia evoked a significant decrease in glycogen content in both nutritional groups, but since the pre-ischemic glycogen level was higher in the fasted group, the extent of glycogen breakdown was higher in the fasted condition. However, although pre-ischemic TAG content was higher in the fasted group, no lipolysis occurred after ischemia-reperfusion in either group. Therefore, during ischemia, the energy supply was greater in the fasted than in the fed condition. However, since IPOC evoked a further decrease in glycogen content in both nutritional conditions, it evidently improved the glucose supply during reperfusion. Furthermore, IPOC increased lipolytic activity during reperfusion, but only in the fasted group, which suggests that it might have evoked a catabolic shift towards lipid oxidation through the increase in the supply of endogenous fatty acids. Thus, it is plausible to speculate that the differences between the two groups might be related to the kind of endogenous substrates supplied during reperfusion.

On the basis of these findings, it is possible that glycogen breakdown may give rise to a pyruvate pool that is more tightly coupled with oxidation than pyruvate derived from extracellular glucose [20]. Therefore, increased glycogen breakdown elicited by IPOC in the fed rat hearts may contribute to the increased rate of mitochondrial ATP production and ATP content, consequently accelerating functional recovery. In very broad terms, the present findings agree with those of Correa et al. [21], who have reported that glucose uptake and consumption were significantly increased during the early RP phase in postconditioned rat hearts, suggesting that IPOC enhances the contribution of glucose catabolism to the total energy expenditure during reperfusion.

The protective effects of fasting have been ascribed to metabolic changes occurring during ischemia, such as reduced glycolysis [6]. The latter would provide protection, at least in part, by decreasing the exchange of Na+ for H+ which in turn reduces calcium overload [6, 22]. These changes probably arise as a consequence of the increased catabolism of fatty acids derived from endogenous TAG that occurs in hearts from fasted rats during the aerobic pre-ischemic period [6]. Interestingly, according to present and previous data, TAG mobilization does not persist during reperfusion [6]. It has been reported that reperfusion of ischemic hearts results in a rapid recovery of the ability to oxidize fatty acids [23, 24], and an over-reliance on fatty acid oxidation has a detrimental effect on functional recovery [25]. Since endogenous TAG, through lipolysis, is a potential source of fatty acids for oxidative metabolism [5, 15, 26], it is plausible to infer that IPOC may accelerate fatty acid β-oxidation in fasted rat hearts. Hence, the noxious effects of IPOC in this nutritional condition might be the consequence of increased lipolysis during reperfusion. Supporting this hypothesis, oxfenicine, a carnitine palmitoyltransferase I inhibitor, added immediately upon RP abolished TAG breakdown and the noxious effects of IPOC on functional recovery in the fasted hearts, but did not affect the recovery in the postconditioned fed ones. Therefore, while endogenous pre-ischemic TAG breakdown is beneficial to the ischemic-reperfused heart, it becomes harmful when it occurs during reperfusion. In regard to the latter finding, it is worthwhile to recall that an increased rate of fatty acid β-oxidation inhibits glucose utilization, with glucose oxidation being inhibited to the largest extent, followed by a smaller effect on glycolysis and glucose uptake [27–29]. Since glycogenolysis is stimulated by IPOC, the concomitant stimulation of TAG breakdown may lead to an increase in H+ production during the critical period in which the heart is attempting to recover from the acidosis that occurred during ischemia [24, 30, 31]. Consequently, calcium overload might be enhanced [32], leading to contracture [33] and having adverse effects on mitochondrial function and contractile recovery [34]. Calcium overload, especially when this is accompanied by oxidative stress and adenine nucleotide depletion, conditions that are exactly those that the heart experiences during post-ischemic reperfusion, provides a powerful stimulus to mitochondrial permeability transition pore (MPTP) opening. When it opens, the MPTP allows free passage of any molecule <1,500 Da across the mitochondrial inner membrane, including protons. Under such conditions the mitochondria are unable to synthesize ATP by oxidative phosphorylation; indeed by reversing the proton-translocating ATPase such uncoupled mitochondria actively hydrolyse ATP synthesized by glycolysis and remaining healthy mitochondria [26, 34].

AMP-activated protein kinase (AMPK), which plays an important role in regulating both fatty acid and glucose metabolism by switching on catabolic pathways that generate ATP [31, 35, 36], has been shown to be upregulated in the setting of IPOC [36]. The present data show that IPOC caused an increase in TAG breakdown in the fasted heart, where TAG was elevated, increasing the chances that AMPK mediates that process. As a consequence, it is possible that these changes lead to a glucose-sparing effect concomitant with an “over-dependence” on endogenous fatty acids as the energy source for the reperfused fasted rat hearts, which may be linked to the effects of IPOC on functional performance, ATP synthesis and tissue damage. However, assessment of the role of AMPK in IPOC was beyond the scope of this study.

This study also demonstrates that protection induced by IPOC in the fed hearts is associated with an increase in G6PDH activity and a resultant increase in the glutathione antioxidant ratio, which in turn reduces oxidative damage. It has been traditionally thought that G6PDH, the rate limiting enzyme of the pentose phosphate pathway, is a typical “housekeeping” enzyme that provides the cytosolic NADPH required to regenerate reduced glutathione, essential for detoxification of reactive oxygen species [37, 38]. The present findings support the hypothesis that G6PDH activity is associated with the preservation of the GSH/GSSG ratio and subsequent protection against oxidative damage [39]. Furthermore, IPOC increased the ATP content and mitochondrial rate of ATP synthesis in the fed group. It should also be noted that IPOC did not, however, affect the fasting-induced upregulation of G6PDH activity, the GSH/GSSG ratio or TBARS levels, yet it decreased cell viability and delayed functional recovery. Since IPOC decreased ATP synthesis and ATP content in the fasted group, it is likely that these noxious effects of IPOC are mainly linked to the decrease in ATP supply and not to the oxidative status.

In summary, the present results are consistent with previously reported protective effects of IPOC and provide evidence that this protection occurs together with stimulation of glycogen breakdown and the pentose phosphate pathway, an increase in ATP content and synthesis and an increase in the GSH/GSSG ratio in fed rat hearts. On the other hand, IPOC partially attenuates the protection afforded by previous fasting, although in this nutritional condition it increases both glycogen and TAG breakdown without changing either the oxidative status of glutathione or the pentose pathway activity.

References

  1. Yellon DM, Hausenloy DJ (2007) Myocardial reperfusion injury. N Engl J Med 357(11):1121–1135

    Article  PubMed  CAS  Google Scholar 

  2. Zhao ZQ, Vinten-Johansen J (2006) Postconditioning: reduction of reperfusion-induced injury. Cardiovasc Res 70(2):200–211

    Article  PubMed  CAS  Google Scholar 

  3. Darling CE, Solari PB, Smith CS, Furman MI, Przyklenk K (2007) Postconditioning the human heart: multiple balloon inflations during primary angioplasty may confer cardioprotection. Basic Res Cardiol 102(3):274–278

    Article  PubMed  Google Scholar 

  4. Argaud L, Gareau-Roesch O, Raisky O, Loufouat J, Robert D, Ovize M (2005) Postconditioning inhibits mitochondrial permeability transition. Circulation 111:194–197

    Article  PubMed  CAS  Google Scholar 

  5. Montessuit C, Papageorgiou I, Tardy-Cantalupi I, Rosenblatt-Velin N, Lerch R (2000) Postischemic recovery of heart metabolism and function: role of mitochondrial fatty acid transfer. J Appl Physiol 89:111–119

    PubMed  CAS  Google Scholar 

  6. Marina Prendes MG, González MS, Torresin ME, Hermann R, Pascale NG, Jaitovich MM et al (2009) Involvement of mitochondrial permeability transition, glutathione status, pentose phosphate pathway and oxidative damage in the protective effect of fasting on the ischaemic-reperfused rat heart. Clin Exp Pharmacol Physiol 36(7):637–642

    Article  PubMed  Google Scholar 

  7. Zimmer HG (1992) The oxidative pentose phosphate pathway in the heart: regulation, physiological significance, and clinical implications. Basic Res Cardiol 87:303–316

    Article  PubMed  CAS  Google Scholar 

  8. Schaefer S, Ramasamy R (1997) Glycogen utilization and ischemic injury in the isolated rat heart. Cardiovasc Res 35:90–98

    Article  PubMed  CAS  Google Scholar 

  9. Wang L, Cherednichenko G, Hernandez L, Halow J, Camacho SA, Figueredo V et al (2001) Preconditioning limits mitochondrial Ca 2+ during ischemia in rat hearts: role of KATP channels. Am J Physiol Heart Circ Physiol 280:H2321–H2328

    PubMed  CAS  Google Scholar 

  10. Lohr GW, Waller HD (1974) Glucose-6-phosphate dehydrogenase. In: Bergmeyer HU (ed) Methods of enzymatic analysis, 3rd edn. Verlag Chemie, Wehnheim, pp 636–643

    Google Scholar 

  11. Lowry OH, Rosebrough NJ, Farr AL, Randal RI (1951) Protein measurement with Folin phenol reagent. J Biol Chem 193:265–275

    PubMed  CAS  Google Scholar 

  12. Tietze F (1969) Enzymatic method for quantitative determination of nanogram amounts of total and oxidized glutathione. Applications to mammalian blood and other tissues. Anal Biochem 27:502–522

    Article  PubMed  CAS  Google Scholar 

  13. Yagi K (1998) Simple assay for the level of total lipid peroxides in serum or plasma. Methods Mol Biol 108:101–106

    PubMed  CAS  Google Scholar 

  14. Strehler BL (1974) Adenosine-5′-triphosphate and creatine phosphate: determination with luciferase. In: Bergmeyer HV (ed) Methods of enzymatic analysis, vol 4, 2nd edn. Academic Press, New York, pp 2112–2115

    Google Scholar 

  15. Varela A, Savino EA (1988) Endogenous triacylglycerol utilization by isolated rat atria. Rev Esp Fisiol 44(1):87–92

    PubMed  CAS  Google Scholar 

  16. Walaas O, Walaas E (1950) Effect of epinephrine on rat diaphragm. J Biol Chem 187:769–776

    PubMed  CAS  Google Scholar 

  17. Solem L, Wallace K (1993) Selective activation of the sodium-independent, cyclosporin A sensitive calcium pore of cardiac mitochondria by doxorubicin. Toxicol Appl Pharmacol 121(1):150–157

    Article  Google Scholar 

  18. Solaini G, Harris DA (2005) Biochemical dysfunction in heart mitochondria exposed to ischaemia and reperfusion. Biochem J 390(2):377–394

    Article  PubMed  CAS  Google Scholar 

  19. Doenst T, Guthrie PH, Chemnitius J-M, Zech R, Taegtmeyer H (1996) Fasting, lactate, and insulin improve ischemia tolerance in rat heart: a comparison with ischemic preconditioning. Am J Physiol Heart Circ Physiol 270:H1607–H1615

    CAS  Google Scholar 

  20. Goodwin GW, Taylor CS, Taegtmeyer H (1998) Regulation of energy metabolism of the heart during acute increase in heart work. J Biol Chem 273:29530–29539

    Article  PubMed  CAS  Google Scholar 

  21. Correa F, García N, Gallardo-Pérez JC, Carreño Fuentes L, Rodríguez-Enríquez S, Marín-Hernández A et al (2008) Post-conditioning preserves glycolytic ATP during early reperfusion: a survival mechanism of the reperfused heart. Cell Physiol Biochem 22(5):635–644

    Article  PubMed  CAS  Google Scholar 

  22. Ramasamy R, Liu H, Cherednichenko G, Schaefer S (2001) Fasting limits the increase in intracellular calcium during ischemia in isolated rat hearts. Basic Res Cardiol 96(5):463–470

    Article  PubMed  CAS  Google Scholar 

  23. Lopaschuk GD, Spafford MA, Davies NJ, Wall SR (1990) Glucose and palmitate oxidation in isolated working rat hearts reperfused after a period of transient global ischemia. Circ Res 66:546–553

    PubMed  CAS  Google Scholar 

  24. Kantor PF, Dyck JR, Lopaschuk G (1999) Fatty acid oxidation in the reperfused ischemic heart. Am J Med Sci 318(1):3–14

    Article  PubMed  CAS  Google Scholar 

  25. Lopaschuk GD, Barr R, Thomas PD, Dyck JRB (2003) Beneficial effects of trimetazidine in ex vivo working ischemic hearts are due to a stimulation of glucose oxidation secondary to inhibition of long-chain 3-ketoacyl coenzyme A thiolase. Circ Res 93:e33–e37

    Article  PubMed  CAS  Google Scholar 

  26. Saddik M, Lopaschuk GD (1991) Myocardial triglyceride turnover and contribution to energy substrate utilization in isolated working rat hearts. J Biol Chem 266:8162–8170

    PubMed  CAS  Google Scholar 

  27. Sugden MC, Holness MJ (1994) Interactive regulation of the pyruvate dehydrogenase complex and the carnitine palmitoyltransferase system. Faseb J 8:54–61

    PubMed  CAS  Google Scholar 

  28. Taegtmeyer H, Wilson CR, Razeghi P, Sharma S (2005) Metabolic energetic and genetics in the heart. Ann N Y Acad Sci 1047:208–218

    Article  PubMed  CAS  Google Scholar 

  29. Jaswal JS, Lopaschuk GD (2007) Partial inhibition of fatty acid β-oxidation with trimetazidine a novel approach to the treatment of ischemic heart disease. Arch Med Sci 3(3A):S1–S9

    CAS  Google Scholar 

  30. Kudo N, Barr AJ, Barr RL, Desai S, Lopaschuk GD (1995) High rates of fatty acid oxidation during reperfusion of ischemic hearts are associated with a decrease in malonyl-CoA levels due to an increase in 5′-AMP-activated protein kinase inhibition of acetyl-CoA carboxylase. J Biol Chem 270(29):17513–17520

    Article  PubMed  CAS  Google Scholar 

  31. Dyck JRP, Lopaschuk GD (2006) AMPK alterations in cardiac physiology and pathology: enemy or ally? J Physiol 574:95–112

    Article  PubMed  CAS  Google Scholar 

  32. Tani M, Neely JR (1989) Role of intracellular Na+ in Ca2+ overload and depressed recovery of ventricular function of reperfused ischemic rat hearts. Possible involvement of H+–Na+ and Na+-Ca2+ exchange. Circ Res 65(4):1045–1056

    PubMed  CAS  Google Scholar 

  33. Piper HM, Abdallah Y, Schäfer C (2004) The first minutes of reperfusion: a window of opportunity for cardioprotection. Cardiovasc Res 61(3):365–371

    Article  PubMed  CAS  Google Scholar 

  34. García-Rivas GJ, Guerrero-Hernández A, Guerrero-Serna G, Rodríguez-Zavala JS, Zazueta C (2005) Inhibition of the mitochondrial calcium uniporter by the oxo-bridged dinuclear ruthenium amine complex (Ru360) prevents from irreversible injury in postischemic rat heart. FEBS J 272(13):3477–3488

    Article  Google Scholar 

  35. Towler MC, Hardie DG (2007) AMP-activated protein kinase in metabolic control and insulin signaling. Circ Res 100:328–341

    Article  PubMed  CAS  Google Scholar 

  36. Bouhidel O, Pons S, Souktani R, Zini R, Berdeaux A, Bijan Ghaleh B (2008) Myocardial ischemic postconditioning against ischemia-reperfusion is impaired in ob/ob mice. Am J Physiol; 295(4):H1580–H1586

    CAS  Google Scholar 

  37. Burns AH, Reddy WJ (1977) Hexose monophosphate shunt in isolated cardiac myocytes from normal rats. Am J Physiol 232(6):E570–E573

    PubMed  CAS  Google Scholar 

  38. Kletzien RF, Harris PKW, Van Noorden CJF (1994) Glucose-6-phosphate dehydrogenase: a “housekeeping” enzyme subject to tissue-specific regulation by hormones, nutrients, and oxidant stress. Faseb J 8(2):174–181

    PubMed  CAS  Google Scholar 

  39. Jain M, Cui L, Brenner DA, Wang B, Handy DE, Leopold JA et al (2004) Increased myocardial dysfunction after ischemia-reperfusion in mice lacking glucose-6-phosphate dehydrogenase. Circulation 109(7):898–903

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

The authors thank Norma Gladys Infante for technical assistance and Ariel Jaitovich for proofreading, grammar review and stylistic improvement. This research was supported, in part, by grants from the Universidad de Buenos Aires, PIP-CONICET 5976, ANPCyT BID contract 1728/OC-AR project 26026 and IQUIMEFA-CONICET.

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Authors and Affiliations

  1. Physiology Unit, Department of Biological Sciences, School of Pharmacy and Biochemistry, University of Buenos Aires and IQUIMEFA-CONICET, Buenos Aires, Argentina

    M. G. Marina Prendes, R. Hermann, M. E. Torresin, E. A. Savino & A. Varela

  2. Physiopathology Unit, Department of Biological Sciences, School of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, Argentina

    P. Souto & S. Tallis

  3. Cátedra de Fisiología, Facultad de Farmacia y Bioquímica, University of Buenos Aires, Junín 956, 1113, Buenos Aires, Argentina

    A. Varela

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  1. M. G. Marina Prendes
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  2. R. Hermann
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  4. P. Souto
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Correspondence to A. Varela.

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An erratum to this article is available at http://dx.doi.org/10.1007/s12576-014-0307-x.

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Marina Prendes, M.G., Hermann, R., Torresin, M.E. et al. Involvement of energetic metabolism in the effects of ischemic postconditioning on the ischemic-reperfused heart of fed and fasted rats. J Physiol Sci 61, 303–312 (2011). https://doi.org/10.1007/s12576-011-0152-0

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The Journal of Physiological Sciences

ISSN: 1880-6562

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