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Cardiovascular Research 1999 41(3):594-602; doi:10.1016/S0008-6363(98)00256-9
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Copyright © 1999, European Society of Cardiology

Loss of protection by hypoxic preconditioning in aging Fischer 344 rat hearts related to myocardial glycogen content and Na+ imbalance

Masato Tani*, Yukako Honma, Michiyo Takayama, Hiroshi Hasegawa, Ken Shinmura, Yoshinori Ebihara and Kayoko Tamaki

Department of Geriatrics, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160, Japan

* Corresponding author. Tel.: +81-3-3353-1211, ext. 2915; Fax: +81-3-5269-2468.

Received 18 March 1998; accepted 27 July 1998


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objectives: The objective of this study was to determine whether hypoxic preconditioning (HP) could lessen the myocardial increase in [Na+]i, thus protecting the aging myocardium against ischemia. Background: A decrease in ischemic tolerance with aging is associated with an accelerated increase in [Na+]i during ischemia. Ischemic preconditioning fails to protect the middle-aged and senescent myocardium against ischemia. Methods: Isolated hearts of young adult (12-week-old), middle-aged (50-week-old) and senescent (100-week-old) Fischer 344 rats were subjected to 25 min of ischemia with or without HP followed by 30 min of reperfusion. Left ventricular (LV) function, myocardial energy metabolites and [Na+]i were measured. Results: In the older groups, the recovery of LV function and high-energy phosphates (HEPs) was lower with an increased release of creatine kinase (CK) during reperfusion than in the young group. The increased [Na+]i at the end of ischemia was greater in the former groups than in the young group. HP decreased myocardial glycogen and lessened the increased [Na+]i in the young group, resulting in an improved recovery of LV function and HEPs, as well as decreased CK release. However, the levels of glycogen before HP in the older groups were higher than in the young group and its levels after HP were similar to that before HP in the young group. HP did not affect the [Na+]i, exacerbated CK release and inhibited the recovery of LV function and HEPs in the older groups. Conclusions: HP failed to lessen the increased [Na+]i or to protect the aging hearts, probably due to the preexistence of increased glycogen level.

KEYWORDS ATP, adenosine triphosphate; [Ca2+]i, intracellular Ca2+ content; CK, creatine kinase; dP/dt, the first derivative of left ventricular pressure; HP, hypoxic preconditioning; IP, ischemic preconditioning; LV, left ventricular; LVDP, left ventricular developed pressure; LVEDP, left ventricular end-diastolic pressure; [Na+]i, intracellular Na+ content


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Several clinical studies have reported that the morbidity and mortality rates following an acute myocardial infarction are higher in the elderly [1–4]. The hearts of senescent animals are less tolerant than those of young adult animals to ischemia–reperfusion injury [5–7]. The loss of a number of myocytes, the development of hypertrophy and the increase in the volume of interstitial tissue [8]may hinder the delivery of oxygen to myocytes subjected to ischemia, while a weakening in the defense against oxidative stress [9]and in the aerobic metabolism of the postischemic myocardium [10]have been reported in aged animals, although the causes of such decreased ischemic tolerance with aging have not been established.

Ataka et al. [11]reported that the increase in cytosolic [Ca2+] is accelerated in the senescent myocardium during ischemia. This increase in cytosolic [Ca2+] may progress to a lethal cellular injury, and is thought to result in part from the excessive influx of Ca2+ via a Na+–Ca2+ exchange mechanism [12, 13]. We previously reported that the myocardial content of Na+ ([Na+]i) was increased in the middle-aged and senescent myocardium of Fischer 344 rats before ischemia, and that the increased [Na+]i was further exacerbated at the end of ischemia compared with that seen in the young adult myocardium [14].

Ischemic preconditioning (IP) and hypoxic preconditioning (HP) both similarly protect the hearts of the young animals against myocardial ischemia–reperfusion injury. However, this protective effect of IP is lost in myocardium that is vulnerable to ischemia with aging [15, 16]. It remains unclear whether or not the mechanisms that work during IP and HP procedures are identical. However, we previously reported that HP depletes myocardial glycogen partially and reduces the increased lactate and [Na+]i at the end of ischemia in young Sprague-Dawley rats, resulting in a lessening of the myocardial Ca2+ overload and an improved recovery of myocardial function [13, 17]. The objective of the present study was to determine whether HP improves the myocardial ionic imbalance and ischemia–reperfusion-induced injury in the myocardium of aging rats.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
We studied male Fischer 344 rats (Charles River Japan), a strain that has been extensively used as a model of aging [18]. This strain does not exhibit significant coronary, vascular or valvular abnormalities with aging and the survival curve shows almost no decline until rats reach 70- to 80 weeks of age [18, 19].

2.1 Preparation and instrumentation
Sixty young adult (12-week-old; weighing 180–230 g), 60 middle- aged (50-week-old; weighing 380–420 g), and 60 senescent (100-week-old; weighing 410–450 g) rats were anesthetized by an intraperitoneal injection of sodium pentobarbital (40 mg/kg). The heart was removed and perfused at 37°C using a modified Krebs-Henseleit buffer that was gassed with 95% O2–5% CO2 and containing (mmol/l) NaCl 118, NaHCO3 25, KCl 4.7, MgSO4 1.2, KH2PO4 1.2, CaCl2 1.75, EDTA 0.5, glucose 11, and pyruvate 5, according to the Langendorff technique (coronary perfusion pressure=70 mmHg) [12]. We confirmed that there were no significant changes in pH or electrolyte concentrations in the perfusate throughout the experiments. A plastic catheter with a latex balloon tip was inserted into the ventricle for left ventricular (LV) pressure measurement. Platinum electrodes were attached to the right atrium and the aorta, to pace the heart during pressure measurement. An epicardial electrocardiogram was recorded using three platinum electrodes that were attached to the left atrium, the right ventricle and the apex of the LV, respectively. All of the procedures in this investigation conformed with the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1985).

2.2 Measurement of LV function
Eight hearts from each age group were subjected to 20 min of oxygenated recirculating perfusion [HP(–)] (Fig. 1A). Another eight hearts from each age group were subjected to 20 min of oxygenated and 10 min of hypoxic (gassed with 95% N2–5% CO2) perfusion [HP(+)]. During the last 5 min of the 20 min of oxygenated perfusion, the hearts were electrically paced at 5 Hz for 5 min and the LV end-diastolic pressure (LVEDP) was adjusted to 10 mmHg by filling the balloon inserted into the LV with fluid. LV function (LV systolic pressure, LVSP; LV developed pressure, LVDP=LVSP–LVEDP; and the maximal and minimal values of the rate of LV pressure change, i.e., LV peak positive dP/dt and LV peak negative dP/dt, respectively) was measured before inducing the period of sustained global ischemia in order to record pre-ischemic control values. During the hypoxic perfusion in HP(+), the coronary perfusion pressure (i.e., aortic pressure) was adjusted to maintain the rate of coronary flow at the same rate as during oxygenated perfusion (Table 1). The hearts, either with or without HP, were then subjected to 25 min of sustained global ischemia followed by 30 min of reperfusion (Fig. 1A). The electrical pacing was discontinued during hypoxic perfusion and sustained global ischemia because such pacing throughout the sustained ischemia increased the rate of ventricular tachyarrhythmias during reperfusion [20]. The epicardial electrocardiograms were analyzed to determine the incidence of ventricular tachycardia (VT) and/or ventricular fibrillation (VF) during the first 25 min of reperfusion, according to the criteria of the Lambeth Conventions [21]. In hearts with VT or VF after 25 min of reperfusion, electrical or mechanical conversion was performed. After 25 min of reperfusion, the electrical pacing was reinstated and the postischemic LV function was recorded. The LVEDP was then readjusted to 10 mmHg in order to calculate the percentage recovery of LV function after reperfusion. The hearts from these three groups, either with or without HP, were then frozen with Wollenberger clamps after 30 min of reperfusion and were used for assay of energy metabolites.


Figure 1
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  		Fig. 1 Perfusion protocols. (A) Perfusion protocols for LV pressure measurement. These hearts were also used for measurement of myocardial metabolites after 30 min of reperfusion. Eight hearts of each age group were used for each protocol. Arrows indicate time points of left ventricular functional measurement. (B) Perfusion protocols for analysis of myocardial metabolites before ischemia (n=5 for each age group), after hypoxic perfusion (n=5 for each age group) and at the end of 25 min ischemia with or without HP (n=5 for each age group). (C) Perfusion protocols for measurement of [Na+]i and [Ca2+]i before ischemia (n=8 for each age group) and at the end of ischemia with or without HP (n=8 for each age group); {square}, oxygenated non-recirculating washout perfusion; {image}, oxygenated recirculating perfusion; {image}, hypoxic recirculating perfusion; {blacksquare}, total global ischemia; blk14, washout with 20 ml of ice-cold 0.35 mol/l sucrose–5 mmol/l histidine (pH 7.4).

 

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  		Table 1 Coronary flow rate during oxygenated and hypoxic perfusion

 
2.3 Analysis of myocardial energy metabolites
Another 20 hearts from each age group were perfused and were frozen at each time point as described in Fig. 1B. These frozen hearts and those frozen after 30 min of reperfusion (described in Fig. 1A) were pulverized with a mortar and pestle that had been cooled in liquid nitrogen. An aliquot of the frozen tissue powder was then extracted into ice-cold perchloric acid (6%, w/v). The neutralized perchloric acid extracts were assayed for ATP, creatine phosphate and lactate using a standard enzymatic procedure [12]. To determine the myocardial glycogen content, a portion of frozen tissue powder (150–200 mg) was mixed with 0.3 ml of 30% KOH and boiled for 30 min, resuspended with 0.2 ml of 2% Na2SO4 plus 2 ml of absolute ethanol, and placed in a freezer at –4°C overnight. The resuspension was then centrifuged at 4000 rpm (2300 g) for 10 min and the resultant pellet was resuspended in 2 ml of 66% ethanol and recentrifuged at 4000 rpm for 10 min. The pellet was then digested with 1 ml of 2 N H2SO4 for 3 h in a boiling bath. The resultant solution was neutralized with 1 ml of 0.5 M MOPS and 30% KOH and used to assay for glucose as a degradation product of glycogen. Myocardial energy metabolites were expressed as mol/g dry weight of tissue (mol/g dry).

2.4 Analysis of release of creatine kinase (CK)
In the hearts used for analysis of LV function in Fig. 1A, the recirculating coronary perfusate (50 ml/heart) used during the 20 min of oxygenated pre-ischemic perfusion or the 30 min of reperfusion were assayed for CK activity released from the myocytes (n=8 for each age group). CK activity was measured by the adenosine diphosphate-dependent dephosphorylation method that uses creatine phosphate as the substrate [22]. CK release was presented as U/g dry weight of tissue (U/g dry).

2.5 Analysis of [Na+]i and [Ca2+]i
The other 24 hearts from each age group were perfused with the buffer containing [14C]-labeled sorbitol (5 Ci/50 ml), as described in Fig. 1C. The coronary artery was washed out with 20 ml of ice-cold 0.35 M sucrose/5 mM histidine (pH=7.4) before or at the end of 25 min of ischemia (n=8 for each age group), as described previously [23]. The ventricles were then frozen with Wollenberger clamps and pulverized. The frozen tissue powder was digested in concentrated nitric acid at 60°C for 48 h and its ionic content was measured with an atomic absorption spectrometer (Hitachi, Tokyo, Japan). The amounts of [14C]-labeled sorbitol in the solution and the perfusate were determined with a liquid scintillation spectrophotometer. The volume of perfusate in the solution was calculated by dividing the residual radioactivity due to the [14C]-labeled sorbitol by the sorbitol radioactivity in the perfusate. The intracellular [Na+]i or [Ca2+]i was calculated by subtracting the extracellular ionic content from the total ionic content. Intracellular ionic contents were expressed as mol/g dry.

2.6 Statistical analysis
Data are expressed as mean±SEM. Comparisons among groups or among time points in each group were performed using two-way analysis of variance (ANOVA) followed by Tukey’s test. A chi-square test followed by Fisher’s Exact test was used to evaluate the differences in the incidence of VT or VF during reperfusion. A level of p<0.05 was accepted as being statistically significant.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
The heart weight/body weight ratio was 0.31±0.03 (12-week-old rats), 0.30±0.04 (50-week-old rats) and 0.34±0.05 (100-week-old rats). There was no significant difference in the ratio among groups.

3.1 Recovery of LV function
The three age groups did not differ significantly in their indices of LV function before ischemia (Table 2). LVEDP increased more in the 50- and 100-week-old rats than in the 12-week-old rats at the end of ischemia (Table 3). Electrical or mechanical conversion was needed for one, one or one heart of 12-, 50- or 100-week-old group without HP and zero, two or one heart of 12-, 50- or 100-week-old group with HP. In the hearts without HP, the percentage recoveries of the LVSP, the LVDP, the LV peak positive dP/dt and the LV peak negative dP/dt were significantly lower in the 50- and 100-week-old rats than in the 12-week-old rats regardless of the readjustment of LVEDP (Tables 3 and 4Go). When HP was performed in all three groups, the recovery of LV function was improved only in the 12-week-old rats (Tables 3 and 4Go).


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  		Table 2 Comparison of LV function before ischemia

 

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  		Table 3 Effects of hypoxic preconditioning (HP) on LV function after reperfusion. LV function after 25 min of reperfusion before readjustment of LVEDP

 

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  		Table 4 Effects of hypoxic preconditioning (HP) on LV function after reperfusion. Percentage recovery of LV function after readjustment of LVEDP to 10 mmHg

 
3.2 Myocardial energy metabolite levels
The content of myocardial ATP, creatine phosphate or lactate did not differ among the three groups before ischemia (Table 5). In the hearts without HP, the myocardial levels of glycogen before ischemia in the older groups were 1.5 times higher than the level found in the 12-week-old rats (Table 5). At the end of 25 min of ischemia, the cardiac glycogen level decreased in all three groups, but the levels were still significantly higher in the older groups (Table 6). In the hearts without HP, the levels of the high-energy phosphates at the end of ischemia did not differ significantly among the three groups (Table 6). The recovery of the level of creatine phosphate during the 30 min of reperfusion after ischemia in the hearts without HP was greater in the 12-week-old rats than in the older groups (Table 7). The lactate levels before ischemia, at the end of ischemia, and after 30 min of reperfusion did not differ among the three groups.


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  		Table 5 Comparison of metabolites before ischemia

 

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  		Table 6 Comparison of metabolites at the end of ischemia

 

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  		Table 7 Effects of hypoxic preconditioning (HP) on myocardial metabolites after reperfusion

 
HP reduced the myocardial glycogen content in all three groups; again the level of glycogen was higher in the older groups than in the 12-week-old rats (Table 5), although the value was equivalent to that before HP in the 12-week-old rats. The glycogen level at the end of ischemia was lower in the preconditioned hearts than in the non-preconditioned hearts in the older age groups (Table 6). When HP was performed in the 12-week-old rats, the levels of high-energy phosphates were recovered during 30 min of reperfusion in an accelerated fashion (Table 7). However, in the older groups, HP did not improve the recovery of ATP during reperfusion.

3.3 The release of CK
The release of CK into the coronary effluent did not differ significantly among the three groups during control normoxic perfusion. The release of CK during 30 min of reperfusion increased in the three groups; the increase was significantly higher in the older groups (Fig. 2). When HP was performed in the 12-week-old rats, a reduction in the increase in CK release was associated with improved recovery of LV function and myocardial metabolites (Tables 3 and 7GoFig. 2). In the other groups, however, HP did not reduce the amount of CK released.


Figure 2
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  		Fig. 2 Comparison of levels (solid bars) of creatine kinase (CK) released into the coronary effluent during 20 min of pre-ischemic oxygenated perfusion with those during 30 min of reperfusion, and with (open bars) or without (hatched bars) hypoxic preconditioning (HP). Data are displayed as the mean values±SEM from eight hearts. *p<0.05 vs. the corresponding values in the 12-week-old rats. #p<0.05 vs. before ischemia in the same age group. $ p<0.05 vs. HP(–) in the same age group.

 
3.4 Reperfusion-induced VT and VF
The incidence of reperfusion-induced VF and VT during post-ischemic reperfusion did not differ significantly among the three groups, if they had not undergone HP (Fig. 3). HP increased the incidence of VF in the older groups, although statistical significance was observed only between the 50-week-old and 12-week-old rats.


Figure 3
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  		Fig. 3 Incidence of reperfusion-induced ventricular tachycardia (solid bars) and ventricular fibrillation (open bars). Data were obtained from eight hearts. *p<0.05 vs. the corresponding values in the 12-week-old rats.

 
3.5 Intracellular ion levels before and at the end of ischemia
Before ischemia, the myocardial [Na+]i was greater in the older groups than in the 12-week-old rats (Fig. 4) whereas the [Ca2+]i values did not differ (12-week-old, 4.7±0.6; 50-week-old, 4.4±0.5; 100-week-old, 3.8±0.3 mol/g dry). In the hearts without HP, myocardial [Na+]i increased but [Ca2+]i did not change during ischemia in any of the groups (12-week-old, 3.5±0.5; 50-week-old, 4.0±0.6; 100-week-old, 4.2±0.3 mol/g dry). However, the change in the myocardial [Na+]i was greater in the older groups than in the 12-week-old rats (Fig. 4). HP reduced the increased levels of myocardial [Na+]i in the 12-week-old rats, but did not change the levels of myocardial [Na+]i in the older groups. Again, HP had no effect on myocardial [Ca2+]i during ischemia in all age groups (12-week-old, 4.4±0.2; 50-week-old, 4.8±0.7; 100-week-old, 4.6±0.3 mol/g dry).


Figure 4
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  		Fig. 4 Comparison of pre-ischemic levels of myocardial [Na+]i (solid bars) with those at the end of ischemia, and with (open bars) or without (hatched bars) hypoxic preconditioning (HP). Data are displayed as the mean values±SEM from eight hearts. *p<0.05 vs. the corresponding values in the 12-week-old rats. #p<0.05 vs. before ischemia in the same age group. $ p<0.05 vs. HP(–) in the same age group.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
The present study showed that HP was of no benefit to the hearts of aging rats. Such hearts are less tolerant of ischemia–reperfusion injury, although HP reduced the damage to myocardium of the young adult rats. The loss of efficacy of HP in the aging hearts was associated with the levels of glycogen before ischemia, accumulation of lactate and an impaired [Na+]i during ischemic insult.

4.1 Changes in intracellular ion homeostasis in the aging myocardium
The increase in [Na+]i that occurs during ischemia contributes to myocardial ischemia–reperfusion injury through an overloading of cellular Ca2+ via Na+–Ca2+ exchange [24]. We previously showed that interventions that increase [Na+]i during ischemia exacerbate reperfusion injury [13, 25, 26]. Lustyik [27]showed that the concentrations of intracellular Na+, K+ and Cl– were elevated in the non-ischemic aged myocardium. That author concluded that the elevated concentrations of intracellular ions were due mainly to the decrease in intracellular water content that occurs with aging. However, the difference in intracellular water content between the young adult and aged rats in that study was only 6%, which does not explain the two-fold increase in the concentration of intracellular Na+ or the 50% increase in that of intracellular K+ in the aged rats. The increased levels of [Na+]i in the present study were expressed as µmol/g dry and also suggested a net increase in these ion levels with aging, although Ruch et al. [28]and Abete et al. [29]reported no significant changes in the free Na+ activities of the left atrial or papillary muscles with aging, when measured by an ion-selective microelectrode.

Results of previous studies suggest that there is a decrease in the number, activity and functional capacity of Na+–K+ pumps of the ventricular and atrial muscles with aging [15, 30, 31]. Since the main mechanism of Na+ extrusion from myocardial cells is thought to be performed by the Na+–K+ pump, the increase in [Na+]i in the aging myocardium perfused under normoxic conditions may be attributed to a decreased functional activity of the Na+–K+ pump.

The increased [Na+]i at the end of ischemia was exacerbated in the aging myocardium compared with that in the young adult. This may be attributed in part to the decrease in Na+–K+ pump activity in the former. The Na+–K+ pump is mainly driven by glycolytic ATP [32]. In the young adult myocardium, the [Na+]i does not increase when the substrate for glycolysis is present during hypoxia, but does increase when the substrate is omitted from the perfusate [26]. The activity of some glycolytic enzymes, including phosphorylase, is reportedly increased in the aged myocardium [33]. This suggests that a possible increase in glycolytic flux is required to compensate for the decrease in the functional activity of the Na+–K+ pump, even under normoxic conditions. It, thus, seems likely that [Na+]i regulation is more susceptible to changes in glycolytic flux in the aged myocardium. The occurrence of an accelerated increase in [Na+]i is very plausible under either sustained ischemic or substrate-free hypoxic conditions, wherein the Na+–K+ pump would be expected to be inhibited by the decrease in glycolytic ATP production, myocardial acidosis and the accumulation of inorganic phosphate. Recently, van Emous et al. [34]have reported that pre-administration of ouabain did not accelerate the rate of increase in [Na+]i during ischemia in isolated rat hearts significantly, which indicates that Na+–K+ ATPase may be inhibited during ischemia, although the enzyme is relatively insensitive to ouabain in this species. While we have proposed the Na+–H+ exchanger as another important route of Na+ influx in the ischemic–reperfused myocardium [13, 24], others have reported age-related changes in the voltage-gated Na+ channels. Butwell et al. [35]showed that lidocaine, a voltage-gated Na+-channel blocker, reduced ischemic contracture and the increase in [Na+]i in rat hearts. Na+–HCO3– symport may also contribute to Na+ influx, especially, during reperfusion [36]. However, Imanishi et al. [37]reported an increase in [Na+]i during ischemia in rat hearts that was of the same extent as that reported by van Emous et al. [34], although the former group used a bicarbonate-free buffer while the latter group employed a modified Krebs-Henseleit buffer. These observations suggest that the contribution of Na+–HCO3– symport to changes in [Na+]i during this particular period is minor. In addition, Imanishi et al. [37]reported that inhibition of Na+–H+ exchange during ischemia resulted in reduction of the increase in [Na+]i by ~70%.

4.2 Loss of beneficial effects of HP in the aging myocardium
We [13]and others [38]have demonstrated that the reduced [Na+]i accumulation during sustained ischemia that is produced by HP or IP is important in myocardial protection. The reduced [Na+]i accumulation we observed during ischemia was attributed to a diminished activation of Na+–H+ exchange, which, in turn, was due to a lessened accumulation of H+ secondary to depletion of glycogen and a reduction in glycolysis. Thus, the reduction of excess Na+ influx caused by HP or IP is obtained at the risk of excessive inhibition of Na+ extrusion by the Na+–K+ pump. To the contrary, Langestrom et al. [39]found that glycogen depletion before ischemia was detrimental in rabbit hearts. However, the initial glycogen content of their control hearts was ~50 µmol/g dry and was much lower than the values in the previous and present studies [17, 40]. King and Opie [41]showed that pre-ischemic depletion of glycogen from 16.3 to 10.8 µmol/g wet weight of tissue (µmol/g wet) by acetate perfusion ameliorated the beneficial effects of IP, which further decreased glycogen levels to 5.9 µmol/g wet before ischemia. These values of glycogen were calculated as 102, 63 and 38 mol/g dry when tissue dry/wet ratio was assumed to be ~16%. King and Opie [41]also reported that the beneficial effect of glycogen depletion by IP was relatively diminished because of excess accumulation of the metabolites of glycogenolysis and glycolysis when the pre-ischemic glycogen level was increased to 21.4 µmol/g wet (~135 µmol/g dry) by the addition of insulin with glucose in the perfusate, although insulin has lots of action on myocardium and may affect ischemic tolerance via mechanisms other than glycogen accumulation. In the present study, the pre-ischemic glycogen level was much higher in the older groups and the level of glycogen after HP in these groups was still relatively high and was equivalent to that before HP in the 12-week-old rats. Therefore, a moderate cellular level of glycogen is thought to be optimal, not only for the purpose of preventing excess glycolytic product accumulation, but also for allowing minimal ATP production to maintain energy-dependent cellular processes, including the ion pumps [40, 41]. In addition, the optimal level of glycogen may vary with experimental conditions, including species differences, and may also be age-dependent, since the rate of glycolytic flux and the regulation of intracellular ion homeostasis change with aging.

HP improved the recovery of LV function and high-energy phosphates, and reduced CK release during reperfusion in the 12-week-old rats, which is consistent with our previous results [13]. However, HP failed to show beneficial effects in the older groups. Surprisingly, the release of CK during reperfusion was not increased much by HP in the 100-week-old rats, compared with the loss of the recovery of LV function. These observations suggest that HP not only accelerates irreversible myocardial damage but the functional and metabolic stunning of the aged myocardium. A decreased recovery of high energy phosphates may cause the accelerated functional impairment in ischemic myocardium, although the metabolic efficacy of contraction during reperfusion is decreased to the same extent in the adult and the senescent myocardium [10].

Time for primary review 21 days.


   Acknowledgements
 
This study was supported in part by grants from the Keio Health Consulting Center and the Ministry of Education, Culture and Science, Japan.


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

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