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Department of Biochemistry and Molecular Biology I, Faculties of 1 Biology2 Chemistry, Complutense University, 28040 Madrid, Spain
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Abstract |
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(Received 4 January 2005;
accepted after revision 21 February 2005; first published online 8 March 2005)
Corresponding author A. Megías: Departamento de Bioquimica y Biologia Molecular I, Facultad de Biología, Universidad Complutense, 28040 Madrid, España. Email: amegias{at}bbm1.ucm.es
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Introduction |
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The hypothesis that HSP72 plays an important role in myocardial protection against I/R injury is supported by recent investigations where expression levels of HSP72 were manipulated (Sakaguchi et al. 2000; Jayakumar et al. 2001; Grünenfelder et al. 2001; Suzuki et al. 2002; Paroo et al. 2002), although, the mechanism through which HSP72 protects cardiac function has not been established. Rat hearts transfected with HSP72 gene and thus overexpressing HSP72 protein show improved cardioprotection against I/R, and activation of ecto-5'-nucleotidase (ecto-5'-NT) has been reported to explain this phenomenon (Sakaguchi et al. 2000). Ecto-5'-NT is the main enzyme responsible for adenosine formation during ischaemic episodes, and adenosine is known to play a protective role against ischaemia through its vasodilator effect (Mubagwa & Flameng, 2001; Obata, 2002) and also possibly through modifying superoxide release from neutrophils and via inhibition of platelet aggregation (Ely & Berne, 1992). Although the aforementioned findings point to the possible involvement of ecto-5'-NT in cardioprotection, no study has been specifically conducted to assess whether training-induced improved tolerance to I/R is mediated by an increase in ecto-5'-NT activity.
Duration of exercise training may be a critical factor to evoke the adaptive response of the heart. Previous research from our laboratory supports this idea (Morán et al. 2003, 2004). However, most studies about cardioprotection linked to exercise use training programmes no longer than 12 weeks. Therefore, the purpose of the present work was to study the effects of a prolonged training protocol (24 weeks) on the cardiac antioxidant defence systems and their response to I/R and to examine the potential involvement of ecto-5'-NT in the training-induced cardioprotection.
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Methods |
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Ninety-five male Wistar rats (initial body mass, 146 ± 9 g) were obtained from Harlan (Barcelona, Spain). They were housed in an animal room at 22–24°C and given free access to commercial rat chow and tap water. The animals were adapted to an inverse 12–12 h light–dark cycle (dark period: 08.00–20.00 h) for 1 week before the beginning of the exercise programme. Rat care and handling and all the experimental procedures employed were in accordance with internationally accepted principles concerning the care and use of laboratory animals. The local ethical committee approved the study.
Rats were randomly divided into a sedentary (n = 63) and an exercise-trained group (n = 32). The rats in the trained group ran on a rodent motor-driven treadmill (Columbus Instruments, Columbus, OH, USA) and performed five sessions (Monday to Friday) per week for a total 24-week period. Mild electrical stimulation was used to encourage the rats to run (grid shock, < 1 mA). From the beginning of the training protocol and until the 4th week, the exercise load of trained rats was progressively increased in intensity and duration, so that after the fifth week the rats ran at 25 m min–1 for 45 min. Before every training session, the animals performed a warm-up period of 5 min at 20 m min–1. Sedentary rats performed a single exercise session once a week for 5 min at 15 m min–1 to familiarize themselves with treadmill exercise and handling.
Two weeks before the end of the training period, all animals performed a treadmill endurance exercise test. During the test, animals of both groups ran at 25 m min–1 with a 5% slope until fatigue occurred, that is until they could no longer maintain the required running pace. Total exercise duration was recorded. Immediately after the endurance exercise test, heart rate and mean arterial blood pressure were measured using an indirect blood pressure monitor (LE 5001, Letica Scientific Instruments).
Isolated perfused rat heart preparation and experimental protocols
At the conclusion of the training programme (48 h after the last exercise training session, in order to avoid the acute effects of exercise), all the rats were weighed, anaesthetized with sodium pentobarbital (50 mg (kg body weight)–1 I.P.) and injected with sodium heparin (50 mg (kg body weight)–1 I.P.). After thoracotomy, the heart was quickly exposed, arrested with ice-cold saline, and the aorta cannulated in situ. The hearts were excised and retrogradely perfused in vitro via the aorta, according to the Langendorff method, at a constant pressure of 70 mmHg at 37°C. The perfusion medium was modified Krebs–Henseleit buffer containing: (mM): NaCl 118, KCl 4.7, CaCl2 2.52, NaHCO3 24.88, KH2PO4 1.18, MgSO4 1.66 and glucose 11; pH 7.40. The perfusion medium was continuously bubbled with 95% O2–5% CO2. Each heart was housed in a controlled heart chamber that was maintained at 37°C.
To assess the validity of our perfusion model, hearts from sedentary rats exposed to 0, 5, 10, 20 and 30 min of normoxic perfusion (n = 5 for all groups) were studied. In addition, the hearts of a group of sedentary (n = 5) and a group of trained rats (n = 5) were excised after thoracotomy and freeze-clamping without being perfused (control hearts, C). The remaining hearts were subjected to the I/R protocol. After 30 min of normoxic perfusion (stabilization period), subgroups of sedentary and trained hearts were either freeze-clamped (perfused control hearts, P), or subjected to 20 min of global ischaemia (20 min ischaemia hearts, 20i), or exposed to 20 min of global ischaemia followed by 15 min of normoxic reperfusion (reperfused hearts, 20i–15r). Global normothermic ischaemia was induced by clamping the aortic inflow. During the ischaemic period, hearts remained immersed in perfusion buffer in the water-jacketed chamber maintained at 37°C. After experimental protocol the hearts were freeze-clamped and stored at –80°C until use. The hearts that did not recover sinusal rhythm within the first minute of stabilization or perfusion period were not used for the study (n = 12 for sedentary animals and n = 6 for trained rats). All I/R subgroups consisted of n = 7 hearts. Experimental protocols are depicted in Fig. 1.
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The soleus muscles were also removed, trimmed of connective tissue, weighed, fast frozen and stored at –80°C. To prepare cardiac homogenates, the atria and great blood vessels were removed from the ventricles. Cardiac and soleus homogenates were prepared as previously described (Delgado et al. 1999). The protein concentration of the homogenates was determined by the method of Lowry et al. (1951). Coronary effluent samples were collected at selected times during the normoxic perfusion and the I/R protocol.
To determine metabolites in heart and coronary effluents, samples were prepared as follows. A portion of the frozen ventricles was cut, weighed while frozen in liquid nitrogen (wet weight), and freeze-dried. After lyophilization, the dry tissue weight was determined and 10 mg of the dried ventricles was ground to powder and homogenized in 600 µl ice-cold 0.6 M HClO4 with a Polytron PT-10 (Kinematica, Switzerland). Aliquots of coronary effluent (200 µl) were also treated with 400 µl of 0.6 M HClO4, at 4°C for 10 min. The perchloric acid (HClO4) extracts were centrifuged for 10 min at 4°C and 17 000 x g. The supernatants were collected and aliquots of 500 µl were neutralized with 150 µl of 2.5 M KHCO3. After 10 min on ice they were centrifuged at 17 000 x g for 5 min at 4°C. The neutralized perchloric acid extracts were assayed to determine metabolites.
Determinations of lactate and high energy phosphates
Lactate was determined according to a standard enzymatic procedure (Noll, 1985) employing 200 µl perchloric acid extracts for coronary effluents and 50–100 µl for heart extracts. ATP and creatine phosphate (CrP) assays were measured in the heart perchloric acid extracts (100–200 µl) according to standard enzymatic procedures (Tratschold et al. 1985; Heinz & Weiber, 1985).
Enzyme activities in homogenates
In order to assess training efficacy, citrate synthase (CS) activity was measured in soleus muscle homogenates at 37°C in the presence of 0.2% Triton X-100 as previously described (Delgado et al. 1999) using aliquots of homogenates, to which defatted bovine serum albumin (final concentration 5 mg ml–1) was added before being frozen.
Antioxidant enzymatic activities were measured in heart homogenates. Superoxide dismutase (SOD) activity was determined spectrophotometrically by the method of Marklund & Marklund (1974). To assay the mitochondrial SOD (mtSOD) activity, the cytosolic SOD activity was inhibited by 1 mM KCN. Catalase (CAT) activity was determined as previously described by Aebi (1984). Glutathione reductase (GR) activity was measured as described by Carlberg & Mannervik (1985). Glutathione peroxidase (GPX) activity was assayed by the method of Flohé & Gunzler (1984).
Ecto-5'-NT activity in cardiac homogenates was determined as previously described (Delgado et al. 1999). The method involves two assays, the first one measures total 5'-NT activity (cytosolic plus ecto-5'-NT), and the second assay measures the cytosolic activity by inhibition of ecto-5'-NT with 1 mM
,ß-methyleneadenosine 5'-diphosphate. The ecto-5'-NT activity was calculated as the difference between both assays.
Cardiac damage markers
Creatine kinase (CK) activity was measured by a spectrophotometric assay employing a commercial kit (Boehringer Mannheim) immediately after collecting samples of coronary effluent. Cardiac troponin I (cTnI) concentration in coronary effluent aliquots was determined employing an immunoenzymometric assay kit for human cTnI (Sanofi Pasteur Diagnostics).
Oxidative stress markers
To assess the level of myocardial lipid peroxidation, thiobarbituric acid-reactive substances (TBARS) were assayed by the method of Ohkawa et al. (1979). To minimize artificial oxidation during assay, 0.05% butylated hydroxytoluene was added to the tissue homogenates.
Thiol group content was determined spectro-photometrically using the 5, 5'-dithiobis 2-nitrobenzoic acid-based technique as described by Jocelyn (1987). Total thiol content was measured after treating the homogenate samples with SDS (1% final concentration) to denature proteins, while non-protein thiols were assayed after acid precipitation of proteins with one volume of cold 0.33 M HClO4.
Quantification of HSP72
Quantification of HSP72 was performed as previously described by Hernando & Manso (1997). In brief, myocardial homogenate aliquots (80 µg tissue wet weight) were subjected to SDS-PAGE on 12.5% acrylamide gels. Resolved proteins were transferred to a nitrocellulose membrane and incubated with the monoclonal antibody against HSP-72 (1: 250, SPA-810, StressGen, Victoria, BC, Canada). Immunodetection of the primary antibody was carried out with peroxidase-labelled goat anti-mouse antibody (1: 5000, Bio-Rad). The blot was washed, incubated with enhanced chemiluminescence (ECL) (Amersham Pharmacia Biotech) and exposed to X-ray films. Quantification of immunoreactive bands was performed using computerized densitometry with commercially available software (Sigmagel 1.0). Standard curves were constructed during preliminary experiments to ensure linearity. Also, we determined the myosin content of the myocardium of sedentary and trained rats by electrophoresis on SDS-PAGE, followed by staining with Coomasie blue R-250. The myosin content in both groups did not differ, suggesting that changes in HSP72 may be attributed to those in cardiomyocytes per se. To allow comparison between blots, a standard sample was loaded on each gel along with the samples of the treatment groups.
Data analysis
All determinations were performed at least in duplicate. Means ± S.D. of n = 5–7 independent preparations are presented. A Student's t test for unpaired data was used to compare the means of the measured variables in sedentary and trained groups. Analysis of the data of normoxic perfusion protocol was performed with one-way analysis of variance (ANOVA) for myocardial metabolite content, and one-way ANOVA with repeated measures for coronary effluent determinations. In I/R groups, myocardial biochemical parameters were analysed by a two-way ANOVA to test the two main effects (training state and I/R protocol) and the interaction between them. Coronary effluent data were analysed by two-way ANOVA with repeated measures. When a significant P-value was obtained, the Scheffe test was employed post hoc to determine the differences between groups. A level of P < 0.05 was selected to indicate statistical significance.
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Results |
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After 24 weeks of treadmill training, body mass was significantly lower in trained compared with sedentary rats (P < 0.01) (Table 1). In addition, end-exercise heart rate was significantly lower in trained animals (P < 0.01), and time to fatigue in the endurance test (P < 0.01) and CS activity in soleus muscle homogenate (P < 0.05) were greater in the training group reflecting the effectiveness of the training programme. Training did not affect ventricular wet mass or tibia bone length.
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The results obtained for determinations in coronary effluents from sedentary and trained groups subjected to the I/R protocols are shown in Fig. 3. The amount of lactate released was elevated at the first moments of reperfusion (P < 0.01), but returned quickly to the pre-ischaemic value found at the end of the initial normoxic perfusion, reflecting the restoration of aerobic metabolism during reperfusion. CK release was increased after ischaemia and remained elevated throughout the reperfusion period (P < 0.05). The amount of cTnI released after ischaemia was significantly elevated only at the beginning of reperfusion. No significant differences were found between sedentary and trained hearts for any of the three variables (lactate, CK and cTnI).
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Pre-ischaemic values of myocardial GR, total SOD (tSOD) and mtSOD activities were not different in sedentary and trained hearts (Fig. 5).The I/R protocol did not affect these parameters in sedentary rats, but in trained animals reperfusion elicited a reduction in GR, tSOD and mtSOD activities. CAT and GPX were also analysed, but neither exercise training nor I/R protocol caused any significant change of these activities (data not shown).
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HSP72 levels and ecto-5'-NT activity are depicted in Fig. 6. Of particular interest are the modifications of both parameters observed after 30 min normoxic perfusion. Therefore, the results for sedentary and trained unperfused hearts (C) are also included in Fig. 6. HSP72 level was increased 5-fold in the non-perfused hearts of exercise-trained animals compared with the sedentary ones (P < 0.01). The I/R protocols did not significantly affect the myocardial HSP72 content in either sedentary or trained rats with respect to the corresponding P hearts (after 30 min of normoxic perfusion). Nonetheless, an enhancement in the content of this protein over the value of the respective C hearts was observed after 30 min of normoxic perfusion in sedentary animals. Figure 6B shows that exercise training did not induce changes in ecto-5'-NT activity, but I/R protocols employed in the present work elicited significant modifications of this parameter in both sedentary and trained hearts (P < 0.01). Ecto-5'-NT activity was increased in the P hearts in comparison with unperfused C hearts. The activity remained elevated after 20 min of global ischaemia, but following the reperfusion period the enzymatic activity significantly decreased in comparison to 20i group.
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Discussion |
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Significant controversy exists about the possible mechanisms involved in training-induced myocardial protection against I/R injury. Previous investigations have attributed the protective effect of training to an increase in myocardial antioxidant defence (Powers et al. 1998; Demirel et al. 2001; Hamilton et al. 2001, 2004), whereas other studies have reported preserved cardiac function after I/R injury in trained animals despite no improvement in these systems (Locke et al. 1995; Libonati et al. 1997; Harris & Starnes, 2001). In the present work, training-mediated cardioprotection was achieved although enzymatic and non-enzymatic antioxidant defences were reduced in reperfused trained hearts. Therefore, our results do not support the hypothesis that an increase in antioxidant defences is the mechanism underlying the cardioprotection induced by prolonged exercise-training.
The effects of I/R on the myocardial antioxidant enzymes have been previously analysed in sedentary animals, but the overall results have been conflicting (Dhalla et al. 2000). In this study, reperfusion caused a significant decrease in tSOD, mtSOD and GR activities only in trained hearts. Demirel et al. (2001) have also detected a reduction in myocardial mtSOD and cytosolic SOD activities in treadmill-exercised rats after coronary occlusion in vivo and reperfusion for 30 min. These findings are striking because antioxidant enzymes, and in particular mtSOD, should constitute the first line of defence against the enhanced ROS production that occurs when the ischaemic myocardium is re-oxygenated. Because we did not observe a similar effect in sedentary hearts, our results could lead to the conclusion that, paradoxically, exercise-training is not a protective condition against I/R injury but a deleterious one. However, experimental evidence argues against this idea. Firstly, myocardial levels of oxidative stress markers after the I/R protocol were similar in sedentary and trained animals. Therefore, the reperfused trained hearts showed no more oxidative damage than their sedentary counterparts despite the observed reduction in antioxidant enzyme activities. Secondly, the metabolic recovery after I/R was improved in the heart of trained rats. These results point to the involvement of other cytoprotective mechanism induced by the training programme. The decrease in antioxidant enzymatic activities detected in trained myocardium after I/R, could be related to the fact that mitochondrial function recovery and subsequent ATP synthesis were enhanced in the reperfused trained hearts. An active ATP synthesis might increase the rate of mitochondrial ROS generation linked to electron transport through the respiratory chain, which, in turn, may result in a rapid increase in the local concentration of ROS. In these conditions ROS overproduction could exceed scavenging capacity of the antioxidant enzymes leading to their partial inactivation through a direct ROS attack, or some indirect mechanism. In this respect, thioredoxin, a superoxide scavenger like SOD, has been shown to be inactivated in T cells subjected to oxidative stress due to reversible glutathiolation (Finkel, 2003). Another possible mechanism of inactivation could be peroxynitrite-mediated nitration of critical tyrosine residues within the SOD structure (MacMillan-Crow & Cruthirds, 2001).
Several investigations have related increased myocardial HSP72 content with cardioprotection (for a review see Snoeckx et al. 2001). HSP72 has also been shown to play a key role in exercise-mediated cardioprotection against I/R injury (Locke et al. 1995; Powers et al. 1998; Demirel et al. 2001; Harris & Starnes, 2001; Paroo et al. 2002), although there is not general agreement on the subject (Taylor et al. 1999; Hamilton et al. 2001; Lennon et al. 2004).The augmented HSP72 content detected in our trained animals is in agreement with the results of previous research (Demirel et al. 1998; Powers et al. 1998; Harris & Starnes, 2001). The mechanism by which HSP72 protects myocardium against I/R injury continues to be investigated. Previous research using gene transfection approaches suggests that overexpression of HSP72 in rat hearts confers cardioprotection by increasing ecto-5'-NT activity (Sakaguchi et al. 2000). In our trained rats, however, ecto-5'-NT activity was not significantly modified compared to their sedentary counterparts despite a 5-fold increase in HSP72 with training. Perhaps an explanation for this apparent discrepancy is that exercise-induced overexpression of HSP72, on one hand, and increased expression of the protein by transfection with HSP72 gene, on the other, could elicit different myocardial adaptations as they represent very different stimuli. However more research is needed to clarify this issue.
HSP72 has been implicated in preservation of mitochondrial function, which is essential for cardiomyocyte survival (Snoeckx et al. 2001). Several authors employing different approaches (heat shock and gene transfection) to enhance HSP72 expression have demonstrated that increases in HSP72 content contribute to the recovery of myocardial function after I/R by enhancement of mitochondrial respiratory function (Jayakumar et al. 2001; Sammut & Harrison, 2003). In addition, Polla et al. (1996) have reported that HSP72 protects mitochondria against oxidative stress. Our data support the hypothesis that HSP72 protects mitochondrial energetic capacity because the levels of ATP and CrP were increased in reperfused trained hearts compared with their sedentary counterparts.
In our study, ecto-5'-NT activity and its response to I/R protocols was not modified by training. However, this enzymatic activity increased in hearts from both sedentary and trained animals during the 30 min normoxic perfusion period. In sedentary rats the activation of ecto-5'-NT occurred in parallel with an enhancement in HSP72 content over the perfusion period. The reason for this latter finding is unclear, but the following methodological consideration should be kept in mind. Although the heart isolation procedure was extremely quick in our hands (less than 2 min), it unavoidably caused a mild hypoxic episode in the myocardium as indicated by increased lactate levels and decreased ATP and CrP content in the initial phase of normoxic perfusion (Fig. 2B). Periods of ischaemia as short as 3–5 min followed by reperfusion can induce preconditioning (Kloner & Jennings, 2001). Thus, the short period of ischaemia of hypoxia caused by heart isolation followed by the 30 min period of perfusion could have preconditioned the hearts used in the present investigation. The formation and release of adenosine, a potent cardioprotective substance, is enhanced in ischaemic myocardium, and during ischaemia the major source of adenosine is considered to be the ecto-5'-NT (Mubagwa & Flameng, 2001). Interstitial adenosine levels increase during the brief periods of ischaemia used for heart preconditioning, and decrease during sustained ischaemia in the preconditioned myocardium (Mubagwa & Flameng, 2001). This phenomenon correlates well with our findings, as in both trained and sedentary groups ecto-5'-NT activity was increased during perfusion but decreased after sustained I/R in the reperfused hearts, supporting the idea that heart isolation could have induced preconditioning in our rats.
It is also known that heart preconditioning increases the expression of HSP72 (Kabakov et al. 2002). Although 30 min seems to be a period too short for protein synthesis to occur, previous research has demonstrated that the myocardial HSP72 content can augment quickly (i.e. increases have been reported in only 30 min after different stimuli; Valen et al. 2000; Grünenfelder et al. 2001; Chello et al. 2003). Rapid transcriptional induction of HSP72 gene, stabilization, and/or enhanced translation of HSP72 mRNA after heart exposure to hypoxia are mechanisms that could account for the rapid HSP72 accumulation during the stabilization period. The increased HSP72 levels and ecto-5'-NT activity detected after 30 min of perfusion in our sedentary group could render myocardial cardioprotection, and so diminish differences between sedentary and trained groups in their myocardial response to the I/R protocol employed. In trained hearts, the hypoxia-induced increase in HSP72 was lower and did not reach statistical significance. As the isolation procedure employed by us is a common method to obtain hearts for myocardial I/R studies, we consider that it is necessary to pay more attention to the heart modifications caused by this procedure. In this respect, it is interesting to point out that, in agreement with previous results (Morán et al. 2004), we detected a slight elevation of tSOD and mtSOD activities in non-perfused trained (C) hearts as compared to their sedentary counterparts (not shown). However, these differences between hearts from sedentary and trained groups were attenuated after normoxic perfusion and lacked statistical significance (Fig. 5, P groups). It is possible that the ischaemic episode suffered by the myocardium during heart isolation and manipulation, followed by re-oxygenation during the first minute of the perfusion period, could affect specifically SOD activities in trained hearts in C in a similar way to that occurring during the reperfusion that follows the 20-min period of global ischaemia. Hence, heart alterations induced during the isolation protocol could mask differences between treatments, and/or training state, prior to I/R protocols.
Finally, our results suggest that physiological and pathological stimuli provoke different myocardial responses: training, as a physiological situation, leads to an increase in HSP72 content but no alterations in ecto-5'-NT activity, whereas hypoxia or ischaemia which are pathological conditions, would elicit a quick defensive response that could be mediated in parallel by both HSP72 and ecto-5'-NT.
In conclusion, the response of key antioxidant enzymatic activities to I/R is modified by a prolonged endurance exercise training. The training-induced defence against I/R injury may be secondary, at least partly, to the marked increase in myocardial HSP72 content. In contrast, such training effect is independent of an increase of myocardial antioxidant systems or ecto-5'-NT activity.
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) and cardiac troponin I (cTnI; 
), from the myocardium into coronary effluent throughout the 30-min period of normoxic perfusion. Data are expressed as means ±
S.D.
*P < 0.05, different from the value at 0 min of perfusion for lactate levels. B, changes in myocardial tissue levels of lactate (•), ATP (
) and creatine phosphate (CrP; 
) during the 30 min-period of normoxic perfusion. 
P < 0.05, different from the value at 0 min of perfusion for lactate, ATP and CrP levels.
