Cardiovascular Research

Cardiovascular Research 1998 40(1):74-87; doi:10.1016/S0008-6363(98)00123-0
© 1998 by European Society of Cardiology

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Copyright © 1998, European Society of Cardiology

Extracellular adenosine levels and cellular energy metabolism in ischemically preconditioned rat heart

Glenn J. Harrison^*, Roger J. Willis and John P. Headrick

Rotary Centre for Cardiovascular Research, School of Health Sciences, Griffith University Gold Coast Campus, QLD 4217, Australia

^* Corresponding author. Tel.: +61 7 5594 8519; Fax: +61 7 5594 8908; E-mail: g.harrison@gu.edu.au

Received 1 September 1997; accepted 27 March 1998

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusions References

 
Objective: Microdialysis and ³¹P-NMR spectroscopy were used to test opposing hypotheses that ischemic preconditioning inhibits adenine nucleotide degradation and purine efflux, or that preconditioning activates cardiovascular adenosine formation to provide enhanced cardioprotection. Methods: ³¹P-NMR spectra and matching interstitial fluid (ISF) or venous effluent samples were obtained from Langendorff perfused rat hearts. Control hearts (n=9) underwent 30 min of global normothermic ischemia and 30 min reperfusion. Preconditioned hearts (n=6) were subjected to a 5 min ischemic episode and 10 min reflow prior to 30 min ischemia and 30 min reperfusion. Effects of repetitive ischemia–reperfusion (3x5 min ischemic episodes) on adenosine levels and energy metabolism were also assessed (n=8). Results: Preconditioning improved post-ischemic recovery of heart rate x left ventricular developed pressure (71±5 vs 43±8%, P<0.05) and end-diastolic pressure (14±3 vs 29±4 mmHg, P<0.05) compared with control hearts, respectively. Preconditioning did not alter intracellular ATP, phosphocreatine (PCr), inorganic phosphate (P_i), H⁺ or free Mg²⁺ during global ischemia, but improved recoveries of PCr, P_i, and G_ATP on reperfusion. ISF adenosine increased more than 20-fold during 30 min ischemia. The 5 min preconditioning episode increased ISF adenosine 3-fold, and reduced ISF adenosine and inosine during subsequent prolonged ischemia by up to 75%. Venous purine levels during reperfusion were also reduced by preconditioning. Accumulation of adenosine in ISF and venous effluent during repetitive ischemia was progressively reduced despite comparable changes in substrate for adenosine formation via 5'-nucleotidase (5'-AMP), and in allosteric modulators of this enzyme (Mg²⁺, H⁺, P_i, ADP, ATP). Conclusions: (i) Ischemic preconditioning reduces interstitial and vascular adenosine levels during ischemia–reperfusion, (ii) reduced ISF adenosine during ischemia is not due to reduced ischemic depletion of adenine nucleotides in preconditioned rat hearts, (iii) preconditioning may inhibit adenosine formation via 5'-nucleotidase in ischemic rat hearts, and (iv) improved functional recovery with preconditioning is unrelated to metabolic/bioenergetic changes during the ischemic insult, but may be related to improved post-ischemic recovery of [P_i] and G_ATP in this model.

KEYWORDS Adenosine; Preconditioning; Energy metabolism; Microdialysis; ³¹P-NMR; Rat

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusions References

 
Induction of a brief period of ischemia renders the heart more tolerant to subsequent and more sustained ischemic insult. This potent cardioprotective phenomenon, termed ‘myocardial preconditioning’, was first described by Murry et al. in 1986 [1]. Since that time it has been shown to effectively reduce cardiovascular injury and improve myocardial metabolic state during ischemia–reperfusion in a number of species [2, 3]. Whether experimental findings of preconditioning in animals translate to its natural occurrence in man or provides potential clinical applications remains speculative [4]. The therapeutic potential of ischemic preconditioning will depend on identification and pharmacological manipulation of the endogenous effector(s) of the phenomenon. Although the precise mechanism of preconditioning is unknown, activation of adenosine A₁ and/or A₃ receptors has been shown to play a key role in rabbit [5–8], pig [9], dog [2, 10], and rat heart [11–13]. Interestingly, the effect of preconditioning on adenosine levels, and therefore adenosine receptor activation, remains controversial. Some investigators have documented reductions in accumulation of interstitial adenosine during prolonged ischemia [14–17], consistent with the hypothesis that preconditioning slows adenine nucleotide degradation during ischemic insult [1, 18]. In contrast, there is evidence of increased adenosine formation with preconditioning, and it has been proposed that amplified adenosine formation contributes to cardioprotection via increased activation of cardiovascular adenosine receptors [10, 19, 20].

Although it is suggested that reductions in adenosine levels following preconditioning are secondary to reduced adenine nucleotide depletion [16–18], or that enhanced adenosine release with preconditioning (when observed) is due to activation of 5'-AMP hydrolysis by 5'-nucleotidase [10, 19, 20], no studies have examined the relationship between extracellular purine levels and cellular metabolite levels, or substrate for the enzyme 5'-nucleotidase. The purpose of the present study was to employ simultaneous cardiac microdialysis and ³¹P-NMR spectroscopy to assess the relationships between ISF and vascular adenosine levels, intracellular metabolic state, substrate levels for adenosine formation ([5'-AMP]), and functional recovery from ischemia in control and preconditioned hearts.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusions References

 
2.1 Isolated heart preparation
All experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996) and the work was approved by the institutional animal experimental ethics committee. Male Sprague-Dawley rats (300–350 g), fed ad libitum prior to experimentation, were anaesthetised with sodium pentobarbitone (60 mg/kg administered intraperitoneally). Hearts were rapidly excised into ice-cold perfusion fluid. Following aortic cannulation, hearts were perfused in a non-recirculating Langendorff mode at a coronary perfusion pressure of 100 mmHg and were permitted to beat at intrinsic heart rate throughout experiments. Perfusion fluid was a modified Krebs–Henseleit buffer (KHB) containing (in mM): NaCl 119, NaHCO₃ 25, KCl 4.7, CaCl₂ 1.75, MgCl₂ 1.2, EDTA 0.5, and glucose 11. The buffer was equilibrated with 95% O₂: 5% CO₂ at 37°C, giving a pH of 7.4. The left ventricle was vented to remove Thebesian drainage and a water-filled latex balloon inserted via the left atrium. This balloon was connected to a P23XL pressure transducer (Viggo Spectramed, Oxnard, CA), and left ventricular pressures, heart rate, and ±dP/dt were measured and stored using a MacLab data acquisition system (ADInstruments, Castle Hill, Australia). Coronary flow rate was obtained by timed collection into a graduated cylinder.

2.2 Cardiac microdialysis
To sample interstitial purine metabolites during experiments, the technique of cardiac microdialysis was utilised [11, 15, 21]. Probes were constructed from 300 µm diameter dialysis fibre (5000 Dalton cut-off) joined to fine silica tubing to form an 8 mm window for exchange of myocardial interstitial solutes with dialysate [11, 21]. The silica outflow tube of each probe was extended to permit collection of dialysate from outside the NMR magnet. A single microdialysis probe was implanted in the left ventricular free wall, approximately mid-way between base and apex. Placement of probes within the myocardial wall was verified at the end of experiments by dissection and visual inspection, and only data from hearts in which probes were located fully within the ventricular wall are reported [11, 21], since incorrect placement may artificially lower the interstitial concentrations. We note that close agreement between basal ISF adenosine levels in this study and previous studies of perfused rat myocardium [11, 22]suggest this is unlikely. Probes were connected to a 1 ml gas-tight syringe and perfused at 1 µl/min with KHB degassed with argon to minimise oxygen delivery to the tissue during ischemia. Dialysate was collected as 5 or 10 min aliquots and stored at –80°C for later analysis for adenosine and inosine levels via HPLC. To determine the recovery rate for adenosine and inosine, the 8 mm probes were perfused at 1 µl/min whilst immersed in 1 µM solutions of adenosine and inosine in KHB at 37°C. The relative recovery rates for the purine metabolites (dialysate concentration/external concentrationx100%) were determined for each probe. These values were then used to convert dialysate concentrations to estimated external (ISF) concentrations [11, 15, 21, 22]. Collection of dialysate samples was timed to match acquisition of ³¹P-NMR spectra during the ischemia–reperfusion protocols.

2.3 HPLC analysis of purine metabolites
Thawed 5 and 10 µl dialysate samples were diluted with 100 µl of HPLC grade water and analysed for adenosine and inosine using a Waters automated HPLC system. Diluted samples (100 µl) were injected onto a 30 cm C-18 column and eluted at a flow of 1 ml/min with an initial 10 min isocratic period (100 mM KH₂PO₄, pH 5.0) followed by a 10 min gradient from 0% to 25% methanol in 100 mM KH₂PO₄. Eluent absorbance was continuously monitored at 254 nm and peaks were identified and quantified by comparison of retention times and peak areas with known external standards run with the samples. Interstitial purine concentrations were estimated from dialysate concentrations by correction for percentage recovery of microdialysis catheters determined in vitro.

2.4 ³¹P-NMR spectroscopy
Following cannulation and microdialysis probe insertion, hearts were located within the centre of a 25 mm double-tuned NMR coil and introduced into the centre of an 18.3 cm bore Oxford 7.05 T magnet. Perfusate temperature was maintained at 37°C in the magnet, and the hearts were additionally superfused with KHB maintained at 37±0.2°C by a thermostatically regulated heater. Temperature within the heart chamber was verified prior to each experiment. The myocardial and perfusate proton signal was maximised using an Oxford Instruments 15-channel shim supply. ³¹P-NMR spectra were then acquired at 121.47 MHz using 90° RF pulses with an interpulse delay of 2 seconds. Spectra consisted of either 148 free induction decays (FID) acquired over 5 min periods, or 297 FIDs over 10 min periods. Spectra were multiplied by a 20 Hz line broadening factor to improve signal-to-noise ratios. Spectral intensities for β-ATP, phosphocreatine (PCr), and inorganic phosphate (P_i) were determined by computer integration using the resident SISCO software. Spectral intensities were corrected for partial saturation using spin-lattice relaxation (T₁) values of 2.17 s, 0.94 s and 1.75 s for PCr, β-ATP, and P_i, respectively [23].

2.5 Metabolic calculations
Measurement of myocardial phosphorus metabolites, pH, [Mg²⁺] and G_ATP has been described in detail by us previously [11, 23, 24]. Briefly, saturation-corrected spectral intensities were converted to intracellular concentrations by calibration against myocardial ATP determined in a group of freeze-clamped control hearts stabilised for 60 min (n=6). Powdered frozen tissue was extracted with perchloric acid and samples analysed for tissue ATP, PCr and creatine (Cr) using HPLC as outlined previously [11, 23]. Tissue contents assessed by HPLC (µmol/g wet weight) were converted to mM intracellular concentrations using a cytosolic volume of 0.48 ml/g [25]. This analysis yielded total tissue ATP and PCr+Cr concentrations of 10.5±0.9 mM and 27.0±2.4 mM, respectively, in normoxic hearts. This tissue ATP concentration was then assigned to the saturation corrected β-ATP intensity in baseline spectra, and all other saturation corrected intensities were normalised against the baseline [ATP]/β-ATP intensity ratio [11, 23]. Intracellular pH (pH_i) was calculated from the chemical shift of P_i relative to PCr, and free intracellular [Mg²⁺] ([Mg²⁺]_i) was calculated from the chemical shifts of the -P and β-P resonances in ³¹P-spectra, as previously described in detail [11, 23, 24]. Free cytosolic [5'-AMP] was determined from the adenylate kinase equilibrium:

where K'_ak is the apparent adenylate kinase equilibrium adjusted for measured pH_i and [Mg²⁺]_i [11, 23]. Free cytosolic [ADP] is estimated from the creatine kinase equilibrium:

where the creatine concentration ([Cr]) was determined by subtraction of PCr from the total tissue creatine content (PCr+Cr), assuming that myocardial PCr+Cr is reasonably conserved for the duration of the experiment [11, 23, 24], and K'_ck is the apparent creatine kinase equilibrium adjusted for measured pH_i and [Mg²⁺]_i [11, 23]. The free energy of ATP hydrolysis (G_ATP) was calculated as:

where R is the ideal gas constant (8.31 J/K/mole), T is temperature in Kelvin (310 K), and the standard free energy of ATP hydrolysis (G_ATP^°) is calculated as –RT.ln K'_ATP where K'_ATP is the apparent ATPase equilibrium adjusted for measured pH_i and [Mg²⁺]_i [11, 23].

2.6 Creatine kinase and lactate dehydrogenase assays
Venous effluent samples were assayed for creatine kinase (CK) and lactate dehydrogenase (LDH) as markers of cellular damage. CK was assayed spectrophotometrically by coupling the CK reaction to the hexokinase and glucose-6-P dehydrogenase reactions using a commercially available diagnostic kit from Sigma Chemicals (St. Louis, MO). LDH activity in effluent samples was determined spectrophotometrically by monitoring loss of NADH (at 340 nm) as pyruvate is converted to lactate. The LDH activity was measured by addition of effluent or tissue homogenate samples to 0.1 M imidazole buffer (pH 7.0) containing 150 µM NADH and 1 mM Na-pyruvate. A unit of LDH is defined as the amount necessary to catalyze 1 µmol NADH per min. Total myocardial CK and LDH activities were assayed in a group of normoxic hearts (n=8) perfused for 5 min. After 5 min of perfusion the hearts were removed from the perfusion apparatus, gently blotted, weighed and then homogenized in 10 volumes of ice cold isotonic buffer containing: 250 mM sucrose, 50 mM imidazole acetate, 10 mM magnesium acetate, 4 mM KH₂PO₄, 2 mM EDTA, and 50 µM N-acetylcysteine. Approximately half of the homogenate was set aside for assay of CK while half was used for assay of LDH. To inhibit adenylate kinase in homogenate samples used for subsequent CK analysis 12.5 µM sulfur in 0.8% ethanol was added. Homogenates were centrifuged at 2,000 g for 5 min, and the supernatant re-centrifuged for 10 min at 20,000 g. The final supernatant was used for enzymatic analysis, and tissue activities are expressed as U/g wet weight.

2.7 Experimental protocol
All hearts were stabilised for 60 min after dialysis probe insertion. Hearts were then randomly assigned to one of two experimental groups. Control hearts (n=9) underwent 30 min of global normothermic ischemia followed by 30 min of reperfusion. Hemodynamic parameters, NMR spectra and dialysate samples were gathered every 10 min throughout the experimental protocol. To precondition hearts in the second group (n=6), a single 5 min period of global ischemia was instigated, followed by 10 min of reflow prior to the sustained 30 min ischemic insult and 30 min of reperfusion. To measure changes in energy metabolism and adenosine concentration during the preconditioning episode, a 5 min dialysate sample and NMR spectrum were obtained. All other measurements were obtained over 10 min cycles corresponding to control hearts.

A parallel study was undertaken with hearts perfused outside of the NMR magnet to assess effects of preconditioning on vascular purine levels and extracellular markers of cellular damage (CK and LDH). Hearts were perfused as described above and the pulmonary artery was cannulated with polyethylene tubing to permit collection of venous effluent. Venous effluent was collected throughout the equilibration and experimental periods for subsequent analysis of cumulative CK and LDH efflux, with timed 500 µl aliquots acquired for analysis by HPLC for adenosine, inosine, hypoxanthine, xanthine and uric acid [11, 23]. All samples were stored at –80°C until analysis. Hearts were subjected to the same ischemia–reperfusion protocols outlined above (n=8 per group). Venous effluent samples were obtained every 5 min prior to prolonged ischemia, every 2 min for the initial 10 min of reperfusion following prolonged ischemia, followed by every 5 min for the remainder of the reperfusion period. For clarity, only every second value obtained is shown for the reperfusion period in Fig. 5. At the end of the experiments hearts were freeze-clamped and assayed for total tissue ATP, PCr and Cr as described above. The metabolite analysis was also performed on groups of control and preconditioned hearts freeze-clamped at the end of the ischemic period (n=7 per group).

View larger version (47K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Changes in venous adenosine and inosine concentrations, and venous purine efflux (adenosine+inosine+hypoxanthine+xanthine+uric acid) during 30 min of global ischemia and 30 min reperfusion in control (n=8) and preconditioned (Precon, n=8) hearts. Preconditioned hearts were subjected to a single 5 min ischemic episode (PC) followed by 10 min reperfusion prior to the prolonged ischemia. Values are means±SEM. *P<0.05, **P<0.01 versus control hearts; P<0.05 versus pre-ischemia.

 
Finally, in order to further assess the mechanism of impaired adenosine formation during repeated ischemia, a group of hearts (n=8) were stabilised in the NMR magnet as described above with microdialysis fibers implanted in the left ventricle. After acquisition of baseline measurements the hearts were subjected to 3 periods of global ischemia of 5 min duration each separated by 10 min reperfusion periods. Parallel ³¹P-spectral data, microdialysate samples and venous effluent samples were acquired over consecutive 5 min periods throughout these experiments.

2.8 Data analysis
All results are expressed as mean±SEM. Data points in figures are displayed at the midpoint of the 5 or 10 min periods over which ³¹P-NMR data or microdialysate samples were acquired. Statistical comparisons between control and preconditioned hearts were made using a repeated measures ANOVA with Scheffe's post-hoc test for individual comparisons. A value of P<0.05 was considered indicative of statistical significance.

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusions References

 
3.1 Effects of preconditioning on contractile function in the isolated rat heart
Table 1 displays the hemodynamic parameters measured in control and preconditioned hearts after stabilisation, and at the end of the reperfusion period subsequent to 30 min of ischemia. There were no differences in baseline values between control and preconditioned hearts for the measured contractile indices. A 30 min period of global ischemia is commonly used in perfused rat heart as it causes severe post-ischemic systolic and diastolic dysfunction. Recovery of the rate-pressure product after 30 min reperfusion was only 43% of the pre-ischemic value. End-diastolic pressure remained 4-fold higher than pre-ischemic values. A single preconditioning episode of 5 min significantly improved post-ischemic contractile recovery after 30 min ischemia. Recovery of the rate-pressure product in preconditioned hearts was 71% of the pre-ischemic value, and end-diastolic pressure only remained elevated at 2-fold higher than pre-ischemia. Recovery of coronary blood flow was not significantly altered by preconditioning in this model.

View this table: [in this window] [in a new window]   Table 1 Summary of functional data from control (n=9) and preconditioned (n=6) hearts

 
3.2 Effect of preconditioning on interstitial purine concentrations
Cardiac microdialysis was employed to continuously monitor ISF purine concentrations during ischemia and reperfusion in control and preconditioned hearts (Fig. 1). Baseline levels of ISF adenosine were 0.4–0.5 µM in both groups. During prolonged ischemia in control hearts ISF adenosine rose >20-fold, reaching a maximum of 7–8 µM in the final 10 min of ischemia. Adenosine levels recovered to pre-ischemic values after 30 min of reperfusion. The 5 min preconditioning episode caused a transient 3-fold increase in ISF adenosine concentration, which returned to baseline during reflow. Preconditioned hearts displayed significantly lower adenosine levels throughout the prolonged ischemia and during the first 10 min of reperfusion compared to control hearts.

View larger version (56K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Changes in interstitial fluid adenosine and inosine concentrations during 30 min of global ischemia and 30 min reperfusion in control (n=9) and preconditioned (Precon, n=6) hearts. Preconditioned hearts were subjected to a single 5 min ischemic episode (PC) followed by 10 min reperfusion prior to the prolonged ischemia. Interstitial fluid concentrations were estimated from dialysate concentrations following correction for partial recoveries determined in vitro (see Methods). Values are means±SEM. *P<0.05, **P<0.01 versus control hearts; P<0.05 versus pre-ischemia.

 
A similar pattern was observed for ISF inosine levels (Fig. 1, lower panel). Quantitatively, the increase in ISF inosine in control hearts was more pronounced than that for adenosine: ISF inosine increased >30-fold from its baseline concentration during the 30 min ischemic period. Inosine levels in preconditioned hearts were elevated 5-fold during the preconditioning episode, and were significantly lower than values in control hearts throughout the prolonged ischemic period (Fig. 1, lower panel). No difference was observed in ISF inosine between the two experimental groups during reperfusion.

3.3 Effect of preconditioning on metabolic state and markers of tissue injury during ischemia-reperfusion
High energy phosphate metabolism during 30 min ischemia and 30 min reperfusion in control and preconditioned hearts was assessed by ³¹P-NMR spectroscopy, and the results are presented in Figs. 2–4. As expected, prolonged ischemia led to rapid reductions in [ATP] and [PCr] with concomitant increases in [P_i] (Fig. 2). Although preconditioning did not alter the changes in [ATP], [PCr] and [P_i] during the ischemic insult, post-ischemic recovery of [PCr] and [P_i] was significantly improved in preconditioned hearts. Recovery of [ATP] was not significantly altered by preconditioning.

View larger version (47K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Changes in ATP, PCr and Pi during 30 min of global ischemia and 30 min reperfusion in control (n=9) and preconditioned (Precon, n=6) hearts. Preconditioned hearts were subjected to a single 5 min ischemic episode (PC) followed by 10 min reperfusion prior to the prolonged ischemia. Values are means±SEM. *P<0.05, **P<0.01 versus control hearts; P<0.05 versus pre-ischemia.

 

View larger version (46K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Changes in [5'-AMP], [Mg2+]i and pHi during 30 min of global ischemia and 30 min reperfusion in control (n=9) and preconditioned (Precon, n=6) hearts. Preconditioned hearts were subjected to a single 5 min ischemic episode (PC) followed by 10 min reperfusion prior to the prolonged ischemia. Values are means±SEM. **P<0.01 versus control hearts; P<0.05 versus pre-ischemia.

 

View larger version (49K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Changes in myocardial GATP during 30 min of global ischemia and 30 min reperfusion in control (n=9) and preconditioned (Precon, n=6) hearts. Preconditioned hearts were subjected to a single 5 min ischemic episode (PC) followed by 10 min reperfusion prior to the prolonged ischemia. Values are means±SEM. *P<0.05, **P<0.01 versus control hearts; P<0.05 versus pre-ischemia.

 
Changes in [5'-AMP] during the experimental protocol are shown in the top panel of Fig. 3. In preconditioned hearts [5'-AMP] increased ~10-fold during the preconditioning episode, recovering to baseline values prior to prolonged ischemia. There was no difference in the sharp increase in [5'-AMP] during prolonged ischemia in control and preconditioned hearts, although [5'-AMP] could only be accurately estimated during the initial 10 min period owing to rapid loss of PCr. No differences were evident for [5'-AMP] between the two groups during the reperfusion period (Fig. 3).

Impaired ion homeostasis was also indicated by marked elevations in [Mg²⁺]_i during ischemia in both groups (see centre panel, Fig. 3), consistent with previous studies of free [Mg²⁺]_i changes during global ischemia [11,24,26 and references therein]. There was an apparent surge in [Mg²⁺]_i during the final 10 min in control hearts. [Mg²⁺]_i in preconditioned hearts recovered to values which were insignificantly lower than those in controls. Myocardial pH_i fell rapidly to ~6.0 during ischemia, and recovered to pre-ischemic values during reperfusion in both groups. Although pH_i tended to be higher in preconditioned hearts throughout the prolonged ischemic period, this was not statistically significant. The free energy of ATP hydrolysis (G_ATP) fell rapidly and comparably during ischemia in the two experimental groups (Fig. 4). Myocardial G_ATP recovered to pre-ischemic levels in preconditioned hearts but remained significantly depressed in control hearts throughout reperfusion.

Total myocardial CK and LDH contents were 791±78 U/g and 342±31 U/g, respectively, in normoxic hearts. Myocardial efflux of CK and LDH was minimal during normoxic perfusion, and markedly enhanced by 30 min global ischemia (Table 2). Interestingly, efflux of LDH appears to be more sensitive indicator of cellular injury than CK, since in control hearts only 1–2% of myocardial CK was lost following 30 min ischemia whereas ~10% of myocardial LDH content was lost. Ischemic preconditioning significantly reduced the loss of both CK and LDH (Table 2). Efflux of both CK and LDH was reduced by ~50% in preconditioned compared with control hearts. Nevertheless, enzyme leakage was still significantly enhanced following ischemia in the preconditioned group.

View this table: [in this window] [in a new window]   Table 2 Myocardial efflux of creatine kinase and lactate dehydrogenase, two markers of cellular injury, from control (n=8) and preconditioned hearts (n=8) prior to and during ischemia-reperfusion

 
3.4 Effect of preconditioning on vascular purine concentrations and myocardial purine efflux
Effects of preconditioning on vascular purine levels were assessed in a separate series of hearts subjected to the same protocol as those located within the NMR magnet. Functional recoveries in these hearts were comparable to those perfused in the NMR magnet: preconditioning improved post-ischemic recovery of heart rate x left ventricular developed pressure (75±6 vs 42±9%, P<0.05) and end-diastolic pressure (17±4 vs 35±5 mmHg, P<0.05) compared with control hearts, respectively. Baseline venous adenosine was ~0.1 µM in both groups (Fig. 5). Reperfusion following 30 min of ischemia resulted in considerable washout of purine metabolites (Fig. 5). Adenosine levels were ~10 µM during early reperfusion, falling rapidly to less than pre-ischemic levels at the end of the reperfusion period. The 5 min preconditioning episode caused a transient increase in vascular adenosine levels during the initial reperfusion period, and the venous concentration returned to baseline prior to the prolonged ischemic episode. Compared with control hearts preconditioned hearts displayed significantly lower adenosine levels throughout the initial 10 min of reperfusion after 30 min of ischemia (Fig. 5). A similar pattern was observed for venous inosine levels and total purine efflux (Fig. 5). Total purine efflux during the 30 min reperfusion period was calculated to be 2.45±0.30 µmol/g in control hearts, and was significantly reduced to 1.58±0.18 µmol/g in preconditioned hearts. These purine efflux values equate well with overall loss of ATP measured in control hearts (2.5 µmol/g) and preconditioned hearts (1.8 µmol/g) (Fig. 2, Table 3).

View this table: [in this window] [in a new window]   Table 3 Myocardial ATP, PCr and Cr contents determined by chemical analysis of freeze-clamped hearts at the end of the stabilization period (Pre-Ischemia), after 75 min of normoxic perfusion following stabilisation (75 min Normoxia), at the end of 30 min global ischemia in control and preconditioned hearts, and after 30 min reperfusion following prolonged ischemia in control and preconditioned hearts

 
3.5 Relationship between cardiovascular adenosine levels and adenine nucleotides during repetitive ischemia–reperfusion
To further examine the mechanism of reduced extracellular adenosine and purine levels in preconditioned hearts we studied effects of repetitive ischemia–reperfusion. The metabolic effects of repeated 5 min periods of global ischemia, separated by 10 min of reperfusion, are depicted in Fig. 6. The metabolic response to each ischemic episode was comparable. Ischemic [ATP], [ADP] and [5'-AMP] values were similar during the three ischemic periods, as were the levels of allosteric modulators of 5'-nucleotidase (pH, Mg²⁺, P_i). However, there was a significant trend towards lower ADP and 5'-AMP levels in the intervening reperfusion periods. Despite the similar metabolic responses to each ischemic episode, accumulation of adenosine in the interstitial compartment declined significantly with each subsequent ischemic episode, and venous adenosine efflux also decreased (Fig. 7). A less pronounced decline in total purine efflux was also observed.

View larger version (28K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 Metabolic responses to 3x5 min periods of global normothermic ischemia separated by 10 min reperfusion periods (n=8). Values are means±SEM. *P<0.05 versus pre-ischemia.

 

View larger version (19K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 7 Changes in interstitial adenosine levels and venous adenosine and total purine efflux (adenosine+inosine+hypoxanthine+xanthine+uric acid) in hearts subjected to 3x5 min periods of global normothermic ischemia separated by 10 min reperfusion (n=8). Values are means±SEM. *P<0.05 versus pre-ischemia; P<0.05 versus values during the first ischemic episode.

 

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusions References

 
The primary aim of this study was to test opposing hypotheses that ischemic preconditioning inhibits adenosine formation and release into interstitial and vascular compartments of the heart as a result of reduced adenine nucleotide depletion during ischemia [1, 18], and that preconditioning actually enhances adenosine formation and release, thereby providing receptor-mediated cardioprotection [10, 19, 20]. The present data support inhibition of adenosine release into interstitial and vascular compartments in preconditioned hearts, but indicate that this reduction is unrelated to ischemic ATP depletion. The data suggest that preconditioning may inhibit myocardial adenosine formation via 5'-nucleotidase. Our data also suggest that protective effects of preconditioning are unrelated to metabolic changes (eg. ATP depletion, acidosis) during ischemia, in contrast to previous studies [19, 27–29, 31–33]. We obtain evidence that effects of preconditioning are related to differences in post-ischemic metabolic state.

4.1 Extracellular purine levels in preconditioned hearts
Adenosine exerts cardioprotective effects in ischemic myocardium [5–7, 11, 28, 34–36], and is a key mediator of preconditioning in several species [6, 7, 10–12, 32]. Recent studies suggest that preconditioning reduces interstitial adenosine during ischemia [14–17], consistent with the hypothesis that preconditioning protects the heart through reduced ischemic adenine nucleotide catabolism [1, 18]. Unfortunately, no study has assessed whether extracellular accumulation of adenosine does reflect ischemic adenine nucleotide degradation in control and preconditioned hearts. In the present study we used cardiac microdialysis and ³¹P-NMR spectroscopy to document relationships between ISF and venous adenosine levels, intracellular adenine nucleotide metabolism, and functional recovery from ischemia in control and preconditioned hearts. Microdialysis offers the advantages of monitoring ISF purine levels during zero-flow ischemia, and permitting acquisition of samples whilst hearts are in the NMR magnet. Baseline ISF adenosine levels obtained in the present study agree with previous values for perfused rat heart [11, 22].

We found that preconditioning transiently increased ISF adenosine and inosine levels (Fig. 1) and also increased vascular adenosine (Fig. 5), consistent with previous data [11]and the notion that transient adenosine receptor activation is involved in preconditioning. The rise in ISF adenosine is sufficient to activate A₁ and possibly A₃ adenosine receptors, assuming dissociation constants for adenosine in the µM range. A₁ receptor activation can mimic anti-infarction effects of preconditioning [5, 7, 14, 37], reduces post-ischemic contractile dysfunction [9, 11, 13, 34], and endogenous adenosine is involved in preconditioning in different models [eg. 6,7,11,12]. The precise mechanism by which transient receptor activation protects from subsequent prolonged ischemia is not known, but involves G_i dependent activation and translocation of protein kinase C [2, 8], and modification of ischemic and post-ischemic energy metabolism [11].

Preconditioning reduced adenosine in the interstitial compartment during ischemia and early reperfusion (Fig. 1), and in the vascular compartment during reperfusion (Fig. 5). Reduced ISF adenosine during prolonged ischemia has been observed in preconditioned rabbit [14, 16], dog [15]and pig heart [38], but not in rat. It is proposed that reduced ISF adenosine reflects reduced ATP depletion during ischemia [1, 18]. However, we obtain evidence that preconditioning depresses ISF adenosine during ischemia (Fig. 1) despite comparable rates and extent of ATP depletion (Fig. 2). Net myocardial ATP depletion during ischemia was ~0.2 mM per min in both groups. Additionally, rates of depletion of PCr and the increase in P_i during ischemia were similar in both groups (Fig. 2).

Adenosine formation occurs predominantly via hydrolysis of 5'-AMP, and under normoxic and hypoxic conditions this is predominantly intra- rather than extracellular 5'-AMP [eg. 39,40]. The rate of 5'-AMP hydrolysis is substrate limited since the K_M for 5'-AMP far exceeds intracellular substrate concentrations. Thus, changes in [5'-AMP] produce parallel changes in adenosine formation. The enzyme is also activated by ADP and Mg²⁺ and inhibited by ATP, H⁺, and P_i [40–42]. We observed 2- to 4-fold higher accumulation of ISF adenosine and inosine during ischemia in control versus preconditioned hearts despite comparable changes in all of these metabolic parameters. Indeed, at the time of the greatest difference between ISF adenosine (ie. the initial 10 min of ischemia) there are no differences in [5'-AMP], [ATP], [Mg²⁺], [H⁺], [P_i], or [ADP]. Since changes in interstitial adenosine levels parallel changes in net myocyte adenosine formation, these data indicate that preconditioning down-regulates adenosine formation without modifying substrate levels or known inhibitors/activators of myocardial 5'-nucleotidase. This is supported by similar reductions in other indicators of myocardial adenosine formation (coronary venous adenosine and net purine efflux) during reperfusion (Figs. 5 and 7). We eliminate a stimulatory effect of preconditioning on adenosine catabolism as an explanation since the breakdown product inosine was reduced by preconditioning and transient ischemia in interstitial and vascular compartments (Figs. 1 and 5 and 7), and adenosine transport and catabolism are likely to be saturated by high extracellular purine levels [43–45]. Furthermore, adenosine transport occurs via facilitated diffusion. Thus we eliminate increased adenosine uptake into myocytes, against the concentration gradient, as an explanation for reduced extracellular levels.

Inhibition of adenosine formation is supported by observations in repetitive ischemia (Figs. 6 and 7). Despite similar changes in 5'-AMP, ATP, ADP, P_i, H⁺ and Mg²⁺, interstitial adenosine accumulation and adenosine washout into the vascular compartment were progressively inhibited by prior ischemia. This is in agreement with recent studies of repetitive ischemia in rat heart [30]. Collectively, our data are consistent with inhibition rather than activation of adenosine formation by preconditioning. This may preserve the purine nucleotide pool in the face of supra-maximal adenosine receptor activation. Indeed, different purine efflux after 30 min global ischemia (2.45 µmol/g in control and 1.58 µmol/g in preconditioned hearts) matches the ultimate decline in myocardial [ATP] (i.e. 2.5 µmol/g in control and 1.8 µmol/g in preconditioned hearts) (Table 3). An alternate explanation for the present data is distinct compartmentation of myocardial adenine nucleotides, with preferential depletion of a specific pool during transient or repeated ischemia, as suggested by Goto et al. [16].

It is interesting to note that the adenosine concentration is comparable in both interstitial and vascular compartments during early reperfusion. Thus, adenosine receptor activation at this time (and presumably during ischemia) will be comparable in both compartments. Previous studies have examined relationships between vascular and interstitial adenosine, primarily in isolated hearts [39, 43, 44]. These studies employed differing indices of interstitial adenosine, but all verify significant concentration gradients from interstitium to vascular compartment under baseline conditions, as observed here (Figs. 1 and 5 and 7). The gradient reflects the highly active metabolic barrier represented by endothelial and smooth muscle cells interposed between interstitial and vascular compartments [39, 43–45]. Interventions including hypoxia [39, 43]and ischemia [44, 45]significantly reduce this gradient, probably as a result of saturation of endothelial and smooth muscle transport and catabolism by high extracellular purine levels [39, 43–45].

4.2 Enhanced adenosine release as a mediator of preconditioning?
Kitakaze et al. hypothesise that preconditioning activates 5'-nucleotidase, increasing adenosine formation and receptor activation to reduce injury [10, 19, 20]. Our data directly addresses this hypothesis since we measured interstitial and vascular adenosine levels in control and preconditioned hearts, and assessed changes in precursor for adenosine (5'-AMP) within the myocyte. It is the adenosine concentration (and resultant receptor activation) rather than 5'-nucleotidase activity itself which is protective in this scheme [19]. Our results are inconsistent with the model of Kitakaze et al. [10, 19, 20], but are in agreement with observations in rabbit, dog and pig hearts [14–17, 38]. The discrepancy between the data of Kitakaze and colleagues and depressed ISF adenosine in isolated and in situ hearts [14–17, 38]is not easily explained. As suggested by Van Wylen et al. [15]and by Kitakaze et al. [20], the discrepancy may involve spatial differences in adenosine concentrations between vascular lumen and interstitium, with interposed endothelial cells functioning as diffusion barrier, catabolic sink, and potential source of adenosine. Changes in adenosine in the vascular compartment may not be reflected in the interstitial compartment, and protective actions of adenosine in the vasculature and in myocytes are mechanistically distinct. Although vascular ecto-5'-nucleotidases may be activated by preconditioning [19, 20], ecto-5'-nucleotidase does not appear in significant quantities in endothelium of canine or rabbit hearts [46], ecto-5'-nucleotidase plays only a minor role in adenosine formation [39, 40], and our data show that net adenosine accumulation in both vascular and interstitial compartments is depressed by preconditioning (Figs. 5 and 7). Other recent studies report depression of vascular adenosine levels following transient ischemic stimuli [16, 30], and Przyklenk et al. have presented recent evidence that 5'-nucleotidase activation is not involved in preconditioning canine myocardium [47, 48].

4.3 Does preconditioning protect the adenine nucleotide pool and pH during ischemia?
Circumstantial evidence has led to the controversial hypothesis [16]that protection of the adenine nucleotide pool, and reduced purine efflux, is involved in cardioprotection afforded by preconditioning [1, 14, 15, 18]. Our data show that cardioprotection occurs without alterations in rate of ATP or PCr depletion, P_i accumulation, or acidosis during prolonged ischemia (Figs. 2–4). These observations are contrary to proposed roles for reduced ischemic ATP depletion [19, 27, 31, 32]and ischemic acidosis [27–29, 49], but agree with our previous observations [11]and an earlier study by Bradamante et al. [26]. Additionally, Kolocassides et al. recently demonstrated that preconditioning may actually accelerate ischemic ATP hydrolysis yet still improve functional recovery [50], and Bradamante et al. [30]report that ATP depletion is uninvolved in preconditioning, presenting evidence that enhanced bioenergetic state rather than absolute nucleotide concentrations, is involved. Given disparate effects of preconditioning on ischemic ATP depletion in various models [11, 18, 30, 50], and since earlier studies implicating ATP depletion failed to demonstrate a causal link between depletion and functional recovery [1, 18, 27, 31, 32], we conclude that ischemic ATP depletion itself is not crucial to the beneficial effects of preconditioning. In relation to our observation that preconditioning fails to modify acidosis during ischemia (Fig. 3), Cave et al. [51]showed that attenuation of acidosis is not a prerequisite for preconditioning, and Cross et al. recently showed that functional recovery from ischemia may actually be greater in hearts displaying greater degrees of acidosis [52]. These observations collectively indicate that attenuation of acidosis is not involved in preconditioning in rat heart.

4.4 Post-ischemic changes in cellular energy metabolism in preconditioned hearts
In contrast to metabolic changes during ischemia [18, 27, 31, 32], the present data and our previous findings [11]implicate differences in metabolic recovery during reperfusion in improved functional recovery with preconditioning (Figs. 2–4). Recoveries of G_ATP, PCr and P_i were all greater in preconditioned versus control hearts (Figs. 2 and 4). As with previous studies examining potential involvement of metabolic changes in myocardial preconditioning, eg. [1, 18, 26–32, 47–49], our data do not directly demonstrate causality. However, based on known sensitivity of rat and human myofibrils to P_i, a post-ischemic elevation in P_i to 12–14 mM in control hearts versus only 4 mM in preconditioned hearts should depress Ca²⁺ activated myofibrillar force by up to 40% in control versus preconditioned hearts [53]. Elevated P_i will also directly inhibit creatine kinase activity [54], potentially contributing to reduced bioenergetic recovery in control hearts (Fig. 4). Finally, the sustained decline in G_ATP from –60 kJ/mol to –56 kJ/mol may depress free-energy dependent processes including sarcoplasmic reticular Ca²⁺ ATPase activity in control compared with preconditioned hearts.

4.5 Study limitations: stunning versus necrosis
Studies examining myocardial metabolism and contractility in post-ischemic hearts often do not consider to what extent any changes may be due to necrosis rather than impaired metabolism or contractility within viable cells [e.g. 26,28,30,32,41,49]. While preconditioning has been shown to attenuate necrosis or infarction [1–3, 5, 17, 18], there is controversy regarding the ability of preconditioning to counteract stunning [55]. Mechanisms of inhibition of infarction versus stunning may differ [56, 57]. While it is difficult in perfused heart studies to verify the role of necrosis [56], we measured indicators of cellular injury (CK and LDH). Efflux of CK and LDH indicates that there is some tissue damage in both control and preconditioned hearts (Table 2). However, in control hearts loss of CK during reperfusion only amounts to 1% of total myocardial activity, and loss of LDH accounts for only 10% of total activity. Enzyme loss was approximately halved in preconditioned hearts. Whether 10% loss of LDH during reperfusion period reflects up to 10% tissue necrosis is unclear since necrotic cells may retain significant quantities of cytosolic enzyme after reperfusion. However, the low levels of efflux suggest a low degree of injury, and indicate that preconditioning substantially reduces this injury.

In addition to enzymatic markers, metabolic changes during ischemia–reperfusion also provide insight into degrees of necrosis. The creatine pool (PCr+Cr) is well preserved in both groups, declining by less than 10–15% in control and preconditioned hearts after ischemia–reperfusion (Table 3). This suggests a low degree of necrosis and cellular disruption. Secondly, based on post-ischemic [PCr] (Fig. 2) and PCr+Cr (Table 3) it can be calculated that cell necrosis (if any) must be less than 20% in control hearts and less than 10% in preconditioned hearts, assuming a negligible phosphorylation potential and ability to generate PCr in necrotic cells, and based on the thermodynamic improbability of PCr/Cr ratios exceeding 20–100 in viable cells. Finally, the total decline in ATP is inconsistent with significant necrosis. ATP declined by only 30–40% in control and preconditioned hearts (Fig. 2). Such changes (20–40% reductions) are characteristic of reversible injury in viable tissue subjected to quite moderate insults [58, 59]. Even assuming a decline in ATP of only 20–30% in viable cells, and negligible detectable ATP in necrotic tissue, it can be calculated that extent of necrosis must fall below 15% of myocardial mass in control hearts, and lower in preconditioned hearts. Were this upper limit of necrosis to occur, it still remains insufficient to account for 50% and 30% reductions in contractile function in control and preconditioned hearts, respectively. It follows that metabolic and contractile function are impaired in viable cells, and that while preconditioning may reduce necrosis, it also appears to attenuate post-ischemic abnormalities in contractile and metabolic function in viable cells in the rat heart model.

We can also consider to what extent necrosis may contribute to post-ischemic elevations in [P_i]. If necrosis was the sole explanation for elevated [P_i], we would predict 35% tissue necrosis in control hearts. Free [P_i] in necrotic cells might reach a maximal value equivalent to pre-ischemic [P_i]+[PCr]+3[ATP] (i.e. 50 mM) minus transsarcolemmal P_i efflux during reperfusion (up to ~0.15 µmol/g/min with significant transsarcolemmal [P_i] gradients, [60]). Thus, [P_i] might achieve 40 mM in necrotic tissue. At this concentration approximately 35% necrosis is necessary to solely account for the elevation in [P_i] to 14 mM. This far exceeds predicted levels of necrosis based upon changes in [ATP], [PCr] and PCr+Cr, as noted above.

	5 Conclusions

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusions References

 
In conclusion, this study demonstrates that elevations in interstitial and vascular adenosine during ischemia–reperfusion are markedly reduced by preconditioning. As opposed to current hypotheses [1, 18], the decline in extracellular adenosine is not due to reduced adenine nucleotide depletion during the ischemic insult in this model. Our data indicate that preconditioning reduces ischemic adenosine formation through inhibition of 5'-nucleotidase, since 4- to 5-fold differences in ISF and vascular [adenosine] occurred after preconditioning and repetitive ischemia despite similar intracellular substrate levels ([5'-AMP]), ATP levels, and changes in known modulators of 5'-nucleotidase activity ([ATP], [PCr], [P_i], [H⁺], and [Mg²⁺]_i). The present data show that preconditioning does not improve post-ischemic contractile function through limiting ATP depletion or acidosis during ischemia. Alternatively, enhanced functional recovery is associated with reduced post-ischemic [P_i] and elevated post-ischemic G_ATP. Thus, metabolic changes during reperfusion rather than ischemia itself may be important in beneficial effects of preconditioning.

Time for primary review 18 days

	Acknowledgements

 
The authors would like to thank Tilley Pain for technical assistance with HPLC analysis, and John Williams for constructing NMR coils used in this study. This work was supported in part by grants from the National Heart Foundation of Australia and from the National Health and Medical Research Council of Australia. A portion of this work appeared in abstract form in J Mol Cell Cardiol 1996;28:A274.

	References

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusions References

 

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