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Cardiovascular Research 2000 47(1):74-80; doi:10.1016/S0008-6363(00)00070-5
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Copyright © 2000, European Society of Cardiology

Ecto-5'-nucleotidase plays a role in the cardioprotective effects of heat shock protein 72 in ischemia–reperfusion injury in rat hearts

Taichi Sakaguchia, Yoshiki Sawaa,*, Masafumi Kitakazeb, Ken Suzukia, Motonobu Nishimuraa, Yasufumi Kanedac and Hikaru Matsudaa

aDepartment of Surgery, Course of Interventional Medicine (E1), Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan
bDepartment of Internal Medicine and Therapeutics, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan
cDivision of Gene Therapy Science, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan

* Corresponding author. Tel.: +81-6-6879-3154; fax: +81-6-6879-3163 sawa{at}surg1.med.osaka-u.ac.jp

Received 10 November 1999; accepted 29 February 2000


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 5 Study limitations
 6 Conclusion
 References
 
Objective: Heat shock protein 72 (HSP72) is involved in the myocardial self-preservation system under several conditions such as ischemia–reperfusion injury or late preconditioning. However, its mechanism is not fully understood. Ecto-5'-nucleotidase is a key enzyme for synthesizing adenosine and plays an important role in ischemic preconditioning. In this study, we tested the hypothesis that ecto-5'-nucleotidase plays a role in the cardioprotection of HSP72. Methods: Rat hearts (H group, n=6) were transfected with HSP72 gene by an intracoronary infusion of hemagglutinating virus of Japan (HVJ)–liposome complex. Control hearts (C group, n=6) were transfected with the β-galactosidase gene. Following 30 min of normothermic ischemia, grafts were reperfused using Langendorff apparatus. Results: The activity of ecto-5'-nucleotidase was significantly higher in H group than C group both before and after ischemia–reperfusion (H vs. C; 0.51±0.05 vs. 0.29±0.06, and 1.41±0.15 vs. 0.85±0.11 nmol/mg protein/min, P<0.05). H group also showed significant better functional recoveries than C group (P<0.05), as well as less creatine phosphokinase leakage (4.4±2.8 vs. 14.2±3.4 mU/min, P<0.05) and higher adenosine release (247.5±35.1 vs. 54.3±1.7 pmol/min, P<0.05). Administration of {alpha},β-methylene adenosine diphosphate (AMP-CP), an inhibitor of ecto-5'-nucleotidase, significantly diminished the tolerance to ischemia–reperfusion injury in H group (P<0.05). Conclusion: These results demonstrated that ecto-5'-nucleotidase activated by an overexpression of HSP72 attenuated ischemia–reperfusion injury in the rat myocardium. They suggest that ecto-5'-nucleotidase plays a role in the cardioprotective effects of HSP72 in rat hearts.

KEYWORDS Adenosine; Gene therapy; Hypoxia/anoxia; Preconditioning; Ventricular function


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 5 Study limitations
 6 Conclusion
 References
 
Endogenous myocardial protection against ischemia–reperfusion injury can be induced by acute and delayed mechanisms that have been, respectively, referred to as the early and late phases of ischemic preconditioning [1–3]. Several triggers and mediators such as adenosine, PKC, and KATP channels are involved in the signal transduction cascades of both phases of preconditioning. Among these mediators, ecto-5'-nucleotidase (ecto-5'-N), a key enzyme in synthesizing adenosine, has been reported to play an important role in the early preconditioning [4–6]. Recently, Node et al. [7] reported that ecto-5'-N may also contribute to mediate the late preconditioning showing that ecto-5'-N activity increased 12–24 h after ischemic preconditioning, which contributed to the infarct size-limiting effects via enhanced adenosine release.

The late or ‘second window’ of cardioprotection develops 24–48 h later and lasts up to 72 h after the preconditioning stimulus [2]. Although the signal transduction pathways responsible for the late preconditioning are largely unknown, it has been reported that de novo protein synthesis is associated, and may be required to upregulate the production of those mediators and sustain their activity to affect the protective mechanisms. In this regard, stress proteins are involved in the late preconditioning mechanism. Notably, heat shock proteins (HSPs) may play an important role in the protective mechanism.

There are several reports stating that HSP72 is involved in the delayed phase of cardioprotection. The time course of HSP72 induction, which appears after 24 h of whole body hyperthermia [8] or brief ischemia [9–11], is similar to the degree of tolerance to ischemia. Moreover, a direct correlation between the amount of HSP72 induced and the degree of myocardial protection has been observed [12]. As a mechanism of the cytoprotective effects of HSP72, the chaperon function has been proposed. However, the precise mechanism and role of HSP72 in endogenous myocardial protection are still unclear.

In this study, we hypothesized that ecto-5'-N may be activated by the chaperon function of HSP72 and attenuate ischemia–reperfusion injury in the late preconditioning. We investigated the role of ecto-5'-N in the mechanism of HSP72 cardioprotection using a HSP72 transfected rat heart model.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 5 Study limitations
 6 Conclusion
 References
 
2.1 Animals
Adult male Sprague–Dawley rats were used in this study. The 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 1996). Institutional approval was obtained for our use of the animals and biohazardous materials from the IRB of Osaka University Medical School.

2.2 Construction of the expression vector containing human HSP70 cDNA
A full-length of human HSP72 cDNA [13] was cloned into the EcoRI/BamHI sites of the pcDNA3 expression vector that has a cytomegalovirus promoter (Invitrogen Corporation, San Diego, CA, USA). For the control transfections, pcDNA3 with β-galactosidase cDNA was used.

2.3 Preparation of HVJ–liposome
HVJ–liposome was prepared as previously described [14–17]. Briefly, 10 mg of lipid mixture (L-{alpha}-phosphatidyl ethanolamine, phosphatidylserine, phosphatidylcholine, sphingomyeline, and cholesterol) was deposited on the site of a flask by removing chloroform in a rotary evaporator. The dried lipid was hydrated in 500 µl of balanced salt solution (137.0 mM NaCl, 5.4 mM KCl, 10.0 mM Tris–HCl; pH 7.6) containing a DNA (200 µg)–HMG1 (high mobility group 1 nuclear protein, 50 µg) complex. A liposome–DNA–HMG1 complex suspension was prepared by vortexing, sonication, and shaking to form liposome. The liposome suspension was incubated with 30 000 hemagglutinating units of HVJ, which was inactivated by ultraviolet irradiation, first at 4°C and then at 37°C. Finally, 2 ml of the sucrose gradient layer containing the HVJ–liposome–DNA complex was collected for use.

2.4 In vivo gene transfection by intracoronary infusion
Gene transfection was performed into the hearts of Sprague–Dawley rats (250 g) as described previously [18]. Briefly, the hearts were arrested with a cold crystalloid cardioplegic solution, and were then removed under anesthesia with diethyl ether inhalation and sodium pentobarbital (50 mg/kg, intraperitoneally), with anticoagulation provided by heparin (200 USP Units, intravenously). Those hearts from the group transfected with the HSP72 gene (H group) were infused with 0.7 ml of HVJ–liposome containing pcDNA3 with human HSP72 cDNA via the coronary artery with the vena cavas, pulmonary arteries, and veins ligated. The hearts from the control group (C group) were infused with the same volume of HVJ–liposome containing pcDNA3 with β-galactosidase cDNA. After incubation on ice for 10 min, the hearts were then heterotopically transplanted into the abdomens of recipient rats (300 g) of the same strain by a modification of the Ono and Lindsey technique [19]. These rats were killed on the fourth day after gene transfection, thus allowing the introduced gene to express proteins stably and provide enough time to eliminate the rat intrinsic HSP72 induced by surgical stress.

2.5 Western blot analysis
The transfected hearts (n=5, from each group) were excised from the abdomen of the recipient rats and immediately frozen in liquid nitrogen. The samples were homogenized with a Polytron homogenizer (Brinkmann Instruments, Inc., Westbury, NY) and centrifuged. After determination of the protein concentration by the method of Lowry et al. [20], 100 µg of the proteins in each sample was loaded onto a 10% SDS–PAGE system. The blots were transferred onto a PVDF membrane and then incubated in Tris-buffered saline/Tween 20 (20.0 mM Tris–HCl, pH 7.5, 150.0 mM NaCl, 0.1% Tween 20) containing 3% bovine serum albumin to block the nonspecific binding sites. The membrane was immunoreacted with a 1:1000 dilution of anti-HSP72 antibody (SPA-810; Stress Gen Biotechnologies Co., Victoria, British Columbia, USA), and then incubated with a 1:7500 dilution of alkaline phosphatase-conjugated goat anti-mouse IgG antibody (Promega Co., Madison, WI). Nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate were used as the substrates for visualization of the reaction product.

2.6 Evaluation of cardiac function of the rat heart
Rats from the two groups (n=6 from each group) were anesthetized and anticoagulated as described before. Their hearts were quickly excised and immersed in cold (4°C) buffer. The heart was then mounted on the aortic cannula of the perfusion apparatus. The coronary arteries were perfused with a modified Krebs–Henseleit buffer (120.0 mM NaCl, 4.5 mM KCl, 20.0 mM NaHCO3, 1.2 mM KH2PO4, 1.2 mM MgCl2, 2.5 mM CaCl2, and 10.0 mM glucose; gassed with 95% O2+5% CO2 to obtain pH 7.4 at 37°C), according to the Langendorff technique, at a perfusion pressure equal to 100 cm H2O. Each heart was housed in a controlled heart chamber that was maintained at 37°C. A thin wall latex balloon was inserted into the left ventricle through the left atrium to monitor left ventricular pressure. Following 20 min of stabilization, heart rate (HR), left ventricular developed pressure (LVDP), maximal derivatives of left ventricular pressure (max dP/dt), and coronary flow (CF) were measured, while keeping the left ventricular end-diastolic pressure at 10 mmHg. The rate pressure product (RPP) was calculated by multiplying HR by LVDP. The hearts were then subjected to global ischemia at 37°C for 30 min, followed by 30 min of reperfusion. The balloon was deflated during ischemia. The indices of cardiac function were continuously measured after reperfusion and analyzed. To examine the effects of the inhibition of ecto-5'-N activity, {alpha},β-methyleneadenosine 5'-diphosphate (AMP-CP; Sigma Chemical Co. St. Louis, MO; 30 µM), a selective inhibitor of ecto-5'-N, was administered into the other hearts of both groups (n=6, from each group) during the stabilization and reperfusion period. The coronary effluent was then collected in chilled vials during the first 5-min of reperfusion to measure adenosine release, and at 10 min of reperfusion to measure creatine phosphokinase (CPK) leakage. Experimental protocol is depicted in Fig. 1.


Figure 1
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  		Fig. 1 Schematic diagram of experimental of protochol. H, gene transfected hearts with HSP72; C, control transfected hearts; AMP-CP, {alpha},β-methylene adenosine diphosphate; RPP, rate pressure product; max dP/dt, maximal derivatives of left ventricular pressure; CF, coronary flow; CPK, creatine phosphokinase.

 
2.7 Measurement of ecto-5'-N activity
To measure the ecto-5'-N activity in the transfected hearts after ischemia and reperfusion, the hearts (n=5, from each group) were disconnected from the Langendorff apparatus after reperfusion and immediately frozen in liquid nitrogen. Further, to measure the ecto-5'-N activity without ischemic insult, transfected hearts (n=5, from each group) were excised from the abdomen of recipient rats 4 days after transplantation and frozen in the same way, without mounting onto the Langendorff apparatus. They were then separated into membrane and cytosolic fractions, as described previously [5,6,21]. The activity of ecto-5'-N was assessed by an enzymatic assay technique and is reported in units of nmol per mg protein per min. We defined ecto-5'-N activity in the membrane fractions as ecto-5'-N activity. Protein concentration was measured by the method of Lowry et al. [20], using bovine serum albumin as a standard.

2.8 Statistical analysis
All data are expressed as mean±S.E.M. Student's unpaired t-test for a comparison between two groups, or ANOVA with post hoc analysis using the Bonferroni/Dunn test for a comparison among more than three groups were used to determine significant difference. P values of less than 0.05 were considered statistically significant. All analyses were performed using the Statview statistical package, version J4.5 (Abacus Concepts Inc., Berkely, CA).


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 5 Study limitations
 6 Conclusion
 References
 
3.1 The mortality in the gene transfection method
There was no technical failure or operative death in the 34 consecutive trials for gene transfection in the study.

3.2 Western blot analysis
To confirm transfection of the HSP72 gene into the myocardium, a western blot analysis was performed. It indicated a stronger expression of HSP72 in the H group than in the C group (Fig. 2).


Figure 2
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  		Fig. 2 Western blot analysis of heat shock protein (HSP) 72. H group, gene transfected hearts with HSP72; C group, control transfected hearts.

 
3.3 Evaluation of cardiac function after ischemia
In the perfused heart experiment using the Langendorff perfusion system, no significant difference was seen before ischemia among the groups in terms of HR, LVDP, RPP, max dP/dt, or CF (Table 1).


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  		Table 1 Cardiac parameters before ischemia in Langendorff perfusiona

 
The time courses of percent recovery of RPP, max dP/dt, and CF after ischemia (37°C, 30 min) are shown in Figs. 3 and 4Go. The percent recoveries of RPP and max dP/dt in the H group were significantly higher than those in the C group. However, administration of AMP-CP significantly decreased the percent recovery of both parameters in the H group, whereas AMP-CP caused no significant difference in the C group. As for the recoveries of CF, the H group showed significantly higher percent recoveries of CF than the C group at 10 min after the onset of reperfusion. Administration of AMP-CP significantly decreased the recovery of CF in the H group, whereas AMP-CP caused no significant difference in the C group (Fig. 5).


Figure 3
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  		Fig. 3 Recovery of rate pressure product (RPP) after ischemia. Data are expressed as percentage of basal RPP before ischemia. *P<0.05 vs. C group; {dagger}P<0.05 vs. H with AMP-CP group, n=6 in each group. All values are expressed as mean±S.E.M.

 

Figure 4
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  		Fig. 4 Recovery of maximal derivatives of left ventricular pressure (max dP/dt) after ischemia. Data are expressed as percentage of basal max dP/dt before ischemia. *P<0.05 vs. C group; {dagger}P<0.05 vs. H with AMP-CP group, n=6 in each group. All values are expressed as mean±S.E.M.

 

Figure 5
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  		Fig. 5 Recovery of coronary flow (CF) after ischemia. Data are expressed as percentage of basal CF before ischemia. *P<0.05 vs. C group; {dagger}P<0.05 vs. H with AMP-CP group, n=6 in each group. All values are expressed as mean±S.E.M.

 
CPK leakage at 10 min after the onset of reperfusion was significantly lower in the H group than in the C group (H vs. C, 4.4±2.8 vs. 14.2±3.4 mU/min). Administration of AMP-CP significantly increased CPK leakage in the H group, whereas AMP-CP caused no significant difference in the C group (H with AMP-CP vs. C with AMP-CP, 13.0±3.3 vs. 13.4±2.0 mU/min) (Fig. 6).


Figure 6
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  		Fig. 6 Creatine phosphokinase (CPK) leakage during 5 min of reperfusion. *P<0.05 vs. the other groups, n=6 in each group. All values are expressed as mean±S.E.M.

 
3.4 Adenosine release
Adenosine release during 5 min of reperfusion was significantly higher in the H group than in the C group (H vs. C, 247.5±35.1 vs. 54.3±1.7 pmol/min). Administration of AMP-CP significantly decreased adenosine release in the H group, whereas it made no significant difference in the C group (H with AMP-CP vs. C with AMP-CP, 69.0±10.6 vs. 68.7±10.1 pmol/min) (Fig. 7).


Figure 7
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  		Fig. 7 Adenosine release after 10 min of the onset of reperfusion. *P<0.05 vs. the other groups, n=6 in each group. All values are expressed as mean±S.E.M.

 
3.5 Ecto-5'-N activity
Ecto-5'-N activity was measured at two points, before exposure of ischemia and after reperfusion. Even before exposure of ischemia, Ecto-5'-N activity in the H group was significantly higher than that in the C group (0.51±0.05 vs. 0.29±0.06 nmol/mg protein/min, respectively). Both groups showed a significant increase of ecto-5'-N activity during exposure of ischemia–reperfusion. Moreover, after reperfusion, ecto-5'-N activity in the H group was significantly higher than that in the C group (1.41±0.15 vs. 0.85±0.11 nmol/mg protein/min, respectively) (Fig. 8).


Figure 8
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  		Fig. 8 Ecto-5'-nucleotidase (ecto-5'-N) activity before ischemia and after 30 min of reperfusion. The ecto-5'-N activity in the H group was significantly higher than in the C group at both points. *P<0.05 vs. C group, n=5 in each group. All values are expressed as mean±S.E.M.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
5 Study limitations
 6 Conclusion
 References
 
In this study, we report three lines of evidence supporting the hypothesis that activation of ecto-5'-N is involved in the cardioprotective mechanism of HSP72. First, the activity of ecto-5'-N was significantly increased by overexpression of HSP72. Second, adenosine release during reperfusion was significantly increased in hearts overexpressed with HSP72. Third, the specific inhibitor of ecto-5'-N, AMP-CP, blunted the enhanced tolerance to ischemia–reperfusion injury by overexpression of HSP72, whereas it had no significant effect on the control hearts.

Ecto-5'-nucleotidase is a key enzyme in synthesizing adenosine from adenosine 5'-monophosphate (AMP) in ischemic preconditioning. We have previously reported that ischemic preconditioning increases ecto-5'-N activity and enhances adenosine release during the ischemic preconditioning procedure and early reperfusion after sustained ischemia [4–6]. Furthermore, regarding the late preconditioning, Node et al. showed that the ecto-5'-N activity increased 12–24 hrs after ischemic preconditioning, resulting in infarct-size limiting effect accompanied by increased adenosine level in coronary venous blood and that its infarct-size limiting effect was blunted by either an antagonist of adenosine receptor, 8-sulfophenyl theophyline, or AMP-CP. It may suggest that ecto-5'-N contributes to mediate the second window of cardioprotection via enhanced adenosine release [7]. These data coincide with our results that HSP72, which protects the heart from ischemic damage during the second window of cardioprotection, activates ecto-5'-N as one of the myocardial protective mechanism in ischemia–reperfusion injury.

It has been reported that the activation of ecto-5'-N is mediated by several endogenous substrates such as protein kinase C [22–25], ATP-sensitive K+ channel opener [26], magnesium [27] and cytokines [28]. Although we demonstrated that ecto-5'-N is activated by overexpression of HSP72, the precise mechanism of activation of ecto-5'-N by HSP72 remains unclear. Because that ecto-5'-N activity in the hearts overexpressed HSP72 was elevated even before ischemia, it is reasonable to consider that the activation of ecto-5'-N may be, at least in part, due to increasing its transcriptional level by HSP72 overexpression. However, we could not find significant differences in the expression of ecto-5'-N between the heart overexpressed HSP72 and the control, although there was a tendency that expression of ecto-5'-N was higher in the HSP72 overexpressed heart (data not shown). Considering that HSP72 may enhance myocardial tolerance to ischemia–reperfusion injury by chaperon function [29,30], one possibility is that a post-transcriptional modification of ecto-5'-N activity might occur by overexpression of HSP72, though it is unclear whether HSP72 activates ecto-5'-N directly or indirectly.

In this study, we used a gene transfection model in order to overexpress HSP72 in rat hearts in vivo. The other methods to overexpress HSP72, such as ischemia and heat stress, would tend to have contamination with other factors, inducing the increase in some other cytoprotective proteins, for example, catalase [31], SOD [32], or other members of the HSP family [11]. We, therefore, consider that a method to alter genetic information might be suitable to elucidate the mechanism of the cardioprotective effects of HSP72. We have previously demonstrated that intracoronary infusion of HVJ–liposome provided an efficient gene transfer and overexpression of HSP72 mainly in the cytoplasm of 50–70% of cardiomyocytes. The protein expression was apparent at day 3 and continued until at least 14 days after transfection, and was approximately double (12–20 times higher than the control hearts) compared to heat-stressed hearts (42°C, 20 min). In addition, gene transfected hearts with HSP72 showed significantly better improvement of myocardial tolerance to ischemia–reperfusion injury than heat-stressed hearts [18,33,34]. In this context, the present model with gene transfection using HVJ–liposome seems to be suitable for investigating the direct effect of HSP72 in the myocardium in vivo.


   5 Study limitations
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 5 Study limitations
6 Conclusion
 References
 
Although these results show that ecto-5'-N is one of factors involved in the cardioprotective effects of HSP72, the degree of its contribution in the cardioprotection of HSP72 has not been proved. Furthermore, this gene transfection model may not fully represent physiological condition, such as the late preconditioning. More studies may be required to further identify the contributions of ecto-5'-N in the mechanism of the cardioprotection of HSP72.


   6 Conclusion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 5 Study limitations
 6 Conclusion
References
 
Ecto-5'-N was activated by an overexpression of HSP72 and play a role in the HSP72 induced tolerance to ischemia–reperfusion injury. Our results may also implicate the participation of ecto-5'-N in the late preconditioning, in which HSP72 is considered to have an important role.

Time for primary review 21 days.


   Acknowledgements
 
We acknowledge Akiko Nishimura and Akiko Ogai for technical assistance in preparing HVJ–liposome and measuring 5'-nucleotidase activity.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 5 Study limitations
 6 Conclusion
 References
 

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