Abstract
Objective: The purpose of the present study was to determine a role of β2 adrenergic receptor (β2AR) in ischemic preconditioning (IPC) response. Methods: Post-myocardial infarction (MI) hearts were produced by ligating the left anterior descending coronary artery for 2 weeks. Post-MI hearts were transfected with the empty virus (Empty-vivo) or β2AR cDNA (β2AR-vivo) by intracoronary infusion of hemagglutinating virus of Japan-liposome. Empty-vivo or β2AR-vivo hearts were subjected to Langendorff perfusion as Control or β2AR hearts, respectively. IPC was undertaken in Control(IPC) and β2AR(IPC+β2AR). After global ischemia, seven hearts in each group were reperfused and normalized left ventricular peak developed pressures (LVPDP) and creatine phosphokinase (CPK) leakage were measured. β2AR gene transfection was confirmed by measuring responsiveness to isoproterenol, real time RT-PCR and immunohistochemistory. Results: IPC preserved LVPDP and reduced CPK leakage in IPC+β2AR hearts as compared with IPC hearts. LVPDP was decreased in addition to increase in CPK leakage in β2AR hearts as compared with Control. Expression of β2AR and responsiveness to isoproterenol were increased in β2AR-vivo as compared with Empty-vivo hearts. Conclusion: These results indicate that β2AR are required to generate IPC effects, and that β2AR gene transfection enhances IPC effects in post-MI hearts.
1. Introduction
Short episodes of non-lethal ischemia that protects the myocardium against future prolonged lethal ischemia have been termed ischemic preconditioning (IPC) [1]. IPC offers several cardioprotective effects including reduction in infarct size, preservation of ionic homeostasis and reduction in incidence of ventricular arrhythmias in animal experiments, some of which are clinically relevant [2].
Among the several endogenous signalings responsible for IPC, beta adrenergic signaling (BAS) pathway has been suggested [3]. Lochner et al. have demonstrated that increase in cAMP levels during IPC triggers the IPC response [4]. Beta adrenergic receptor (βAR) stimulation also raises cAMP levels, leading to the effects mimicking IPC [5]. These lines of evidence have implied a close relationship between BAS and IPC.
However, in the process of ventricular remodeling after myocardial infarction (MI) elevated levels of circulating endogenous catecholamines lead to down regulation of BAS consisting of desensitization and phospholylation [6]. These attenuated BAS in post-MI states may interfere with cardioprotective response of IPC. Indeed, recent work demonstrated that IPC fails to exert cardioprotective effects of IPC in post-MI hearts. Moreover, we have recently shown that refractoriness to IPC in the post-MI hearts is attributed to the downregulation of β2AR, and that a potent adenylate cyclase agonist, Forskolin, a downstream effecter of BAS, restores cardioprotective effects of IPC [7]. These results propose a hypothesis that upregulated re-expression of β2AR may restore cardioprotective effects of IPC in post-MI hearts.
Therefore, the present study was undertaken (1) to determine whether intracoronary gene delivery of β2AR enhances cardioprotective effects of IPC in post-MI hearts, and (2) to determine a role of β2AR signaling in IPC response.
2. Materials and methods
2.1. Experimental myocardial infarction
Male Sprague-Dawley (SD) rats (SLC, Shizuoka, Japan) weighing 250 to 300 g were used in this study. Rats were anesthetized with diethyl ether, and maintained on positive-pressure ventilation during the operation. After thoracotomy, MI was produced by ligating the left anterior descending coronary artery (LAD). LAD was ligated 5 mm from its origin with 5-0 nylon suture. Chest was closed and the rats awoke extubated and were returned to the cage. The investigation was carried out in adherence 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) and was approved by the local Animal Care Committee.
2.2. Preparation of HVJ-liposome including β2AR cDNA
The PCR primer pairs used were 5′-TCACCTGCCCGGCCATGGAGC-3′, and 5′-AAAGCCTACACTACAGTGGCG-3′ for construction of cDNA encoding full-length β2AR. The PCR products were subcloned to pCRII vector (Invitrogen, San Diego, CA, USA) and sequenced with M13 sequence primers. The inserts were released from the pCRII vectors by EcoRI digestion and were subsequently cloned into the EcoRI site of pcDNA3.1(-) (Invitrogen, San Diego, CA, USA). HVJ-liposome was prepared as previously described [8]. Briefly, the mixed dried lipid was hydrated in 500 μl of balanced salt solution 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 hemaglutinating units of HVJ. Finally, 2 ml of the sucrose gradient layer containing the HVJ-liposome–DNA complex was collected for use.
2.3. Gene transfection (GT) of β2AR
Gene transfection (GT) was performed into the post-MI hearts as previously described [9,10]. Briefly, the hearts were arrested with a cold crystalloid cardioplegic solution, and were then removed. The isolated hearts were infused with HVJ-liposome–DNA complex including β2AR cDNA via the coronary artery with the vena cavas, pulmonary arteries, and veins ligated β2AR-vivo). The same volume of HVJ-liposome without β2AR cDNA was also infused into the isolated hearts as Empty-vivo. After the incubation on ice for 10 min, the hearts were heterotopically transplanted into the abdomens of recipient SD rats [8]. These rats were killed on the 5th day after the GT.
2.4. Ischemia/reperfusion protocol
Heparine 300 IU was infused into the femoral vein and the transplanted heart was rapidly excised. The aorta was cannulated, and the heart was suspended from the cannula. Then, the aorta was perfused with oxygenated (95%O2/5%CO2) Krebs–Henseleit solution at 37 °C, and the right atrium was incised. Then, the left atrium was excised, and a water-filled balloon was placed into the left ventricle. Left ventricular peak developed pressure (LVPDP) was monitored as the difference between end-systolic and end-diastolic pressure. All hemodynamic data were derived from the average of 10 steady-state cardiac cycles.
The perfusion protocol is shown in Fig. 1A. After stabilization, Empty-vivo (Control, n=7) and β2AR-vivo hearts (β2AR, n=7) were subjected to 20 min of global ischemia followed by 60 min of reperfusion. Empty-vivo (IPC, n=7) and β2AR-vivo hearts (IPC+β2AR, n=7) received IPC before the prolonged ischemia. IPC consisted of 2 cycles of 5 min of complete aortic occlusion and reperfusion. The coronary effluent was collected in chilled vials at 10 min of reperfusion to measure creatine phosphokinase (CPK) leakage.
(A) Langendorff perfusion protocol. Global ischemia occurred from 0 to 20 min and reperfusion from 20 to 80 min. IPC (−) includes the groups designated as ‘Control’ and ‘β2AR’. IPC (+) includes the groups designated as ‘IPC’ and ‘IPC+β2AR’. IPC consisted of 2 cycles of global ischemia and reperfusion. (B). Normalized left ventricular peak developed pressure (NLVPDP) during the experiment. Data are shown as mean±standard deviation. Significant differences during the experiment of P<0.05 versus IPC designated by asterisk, P<0.05 versus Control designated by sharp, and P<0.05 versus β2AR designated by cross.
2.5. Responsiveness to isoproterenol
Empty-vivo or β2AR-vivo hearts were subjected to the perfusion system by the same manner. After stabilization, 2.0×10−8 M of isoproterenol was administrated continuously into the aortic cannula. LVPDP was measured before and 3 min after isoproterenol infusion.
2.6. Real time quantitative-reverse polymerase chain reaction (RT-PCR)
Left ventricular myocardium in empty-vivo or β2AR-vivo hearts were frozen immediately after excluding the region of experimental infarction. After total RNA extraction, first-strand cDNA was synthesized from 1 μg of total RNA with Super-Script II reverse transcriptase (Gibco-BRL, Tokyo, Japan). PCR was carried out in a total volume of 50 μl according to the manufacturer's instruction of Perkin-Elmer Applied Biosystems. The following primers were used for the application:
Sense primer: 5′-GCCACGACATCACTCAGGAAC-3′;
Antisense primer: 5′-CGATAACCGACATGAGGATGG-3′; and
Taq-Man probe; 5′-FAM-CGAAGCGTGGGTGGTGGGCAT-TAMRA-3′.
The ABI Prism 7700 Sequence Detection System (Perkin-Elmer Applied Biosystems, Osaka, Japan) detected the signal from the fluorescent probe during PCR. Linearized cDNA of B2AR was diluted and used as a standard.
2.7. β2AR immunohistochemistry
Sections from Empty-vivo or β2AR-vivo hearts were obtained from the matched original paraffin blocks. For quenching endogenous peroxidase activity, the slides were incubated in 5 mmol/l of peracetic acid for 10 min. After incubation with normal blocking serum, sections were incubated overnight at 4 °C with anti-rat B2AR antiserum (1:200 dilutions in serum diluents). The slides were incubated with fluorescein isothiocyanate-conjugated goat anti-rabbit immunoglobulin G secondary antibody for 45 min, and reacted with ABC reagent (Vectastain Elite ABC reagent, Vector Laboratories, Burlingame, CA, USA). After the incubation in peroxidase substrate solution, the slides were counterstained, rinsed in PBS solution and mounted.
2.8. Statistics
All results are expressed as mean±standard deviation. Student's unpaired t-test, or ANOVA with post hoc analysis using the Scheffe test were used to determine significant difference. Significant changes were considered present when p values were less than 0.05.
3. Results
3.1. Normalized LVPDP (NLVPDP)
Time course of change in NLVPDP is shown in Fig. 1B. NLPDP was indicated as a percentage of the initial value of LVPDP. There were no significant differences among the groups for 15 min after reperfusion. From 30 min after reperfusion, NLVPDP was decreased in the group of β2AR as compared with Control until the end of reperfusion (β2AR=48.9±10.9, Control= 73.8±5.4%, P=0.0008). NLVPDP was preserved in the group of β2AR+IPC as compared with IPC alone (β2AR+IPC=85.0±6.0, IPC=68.6±13.4%, P= 0.034). There were no significant differences in NLVPDP between Control and IPC.
3.2. CPK leakage after reperfusion
CPK leakage at 10 min after the onset of reperfusion is shown in Fig. 2. CPK leakage in IPC+β2AR (6.4±2.2 mU/min) was significantly lower than that in IPC alone (11.3±2.5 mU/min) (P=0.041). In contrast, CPK leakage in β2AR (16.4±4.1 mU/min) was significantly higher than that in Control (10.8±2.5 mU/min) (P=0.015). There were no significant differences in CPK leakage between Control and IPC (P=0.9914).
Creatine phosphokinase (CPK) leakage at 10 min of reperfusion. Data were shown as mean±standard deviation. Significant differences of P<0.05 versus Control are designated by asterisk(*). Significant differences of P<0.05 versus IPC are designated by sharp(#).
3.3. Responsiveness to isoproterenol
Changes in LVPDP by isoproterenol infusion are shown in Fig. 3. Administration of isoproterenol did not increase LVPDP in Empty-vivo hearts (P=ns). The LVPDP before and after administration of isoproterenol was 63.1±7.5 and 67.7±8.0 mmHg, respectively. In β2AR-vivo hearts, administration of isoproterenol increased LVPDP from 68.0±6.6 mmHg to 80.0±6.4 mmHg (P=0.0388). LVPDP after isoproterenol administration was increased in β2AR-vivo, but not in Empty-vivo hearts (P=0.0332). These results indicated that GT of β2AR increased responsiveness to isoproterenol in post-MI hearts.
Increased responsiveness to isoproterenol in β2 GT hearts. Data were shown as mean±standard deviation. Significant differences of P<0.05 versus left ventricular peak developed pressure before isoproterenol infusion are designated by asterisk(*). Significant differences of P<0.05 versus Empty-vivo hearts are designated by sharp(#).
3.4. β2AR mRNA expression
Expression of β2AR mRNA was significantly increased in β2AR-vivo (6.25±0.96×107 copy/μg total RNA) as compared with Empty-vivo hearts (3.26±0.61×107 copy).
3.5. β2AR immunohistochemistry
Representative immunohistochemical findings against antibody of rat β2AR are shown in Fig. 4. As shown in Fig. 4B, immunoreactivity was increased in myocardium without MI in β2AR-vivo as compared with that in Empty-vivo hearts. Fig. 4C shows the myocardium located in border zone between MI (the lower area) and non-MI (the upper area). The increased immunoreactivity against anti-β2AR antibody was found in the myocardium located in border area between MI and non-MI.
Immunohistochemical detection of β2 adrenergic receptor in rat hearts. (A) Empty-vivo hearts (B) β2AR-vivo hearts (C) Borderline zone between myocardial infarction and non-myocardial infarction. Arrows indicate anti-β2AR positive cells.
4. Discussion
The major findings in this study were that IPC preserved LVPDP in relation to reduction of CPK leakage in post-MI hearts in which β2AR cDNA has been transfected, but not in post-MI hearts without β2AR GT. Furthermore, hearts overexpressing β2AR increased susceptibility to reperfusion injury in the absence of IPC. These results suggest that β2AR are a requisite factor to regenerate IPC response in post-MI hearts.
We have previously shown that coronary infusion of HVJ-liposome complex during cardioplegic arrest is feasible to perform GT into whole heart [9,10]. We have also estimated the transformation efficiency of the method using β-galactosidase cDNA. The transfected cDNA was located in the nuclei of more than 70% of the myocytes as well as endothelial cells and its translated product was also found in the cytosol of more than 50% of the myocytes. The efficiency of GT by the HVJ-liposome method is superior to that by direct injection of plasmid DNA [8]. In addition, HVJ-liposome is less inflammatory and immunogenic than adenovirus vector. We have shown that this HVJ-liposome method is beneficial for not only intact hearts but also failing hearts including post-MI [11] and hypertrophied hearts [12]. In this study, GT of β2AR in post-MI hearts successfully increased the expression and the responsiveness of agonist.
IPC elicits cardioprotective effects in intact hearts in terms of reduction in infarct size, CPK leakage and arrhythmic events after reperfusion [1,2]. However, we have recently shown that IPC fails to reduce infarct size in post-MI hearts in which mRNA expression of β2AR reduces, and potent adenylate cyclase agonist forskolin restores cardioprotective effects of IPC [7]. In the present study, IPC also failed to reduce CPK leakage in post-MI hearts. We have also demonstrated that repetitive infusion of phosphodiesterase III inhibitor orprinone before ischemia mimics IPC in post-MI hearts [13]. These sets of evidence suggest that preischemic activation of β2AR or its downstream cascades may be required for generating IPC response.
On the other hand, β2AR gene-transfected hearts increased ischemic susceptibility in the absence of IPC. The result is consistent with Cross's work demonstrating that overexpression of β2AR is deleterious against ischemia reperfusion in isolated mice perfusion models [14]. They suggested that β2AR overexpression resulted in greater energy utilization and increased ischemic injury. Similarly, CPK leakage in β2AR-transfected hearts was the highest among the groups in this study leading to reduction in functional recovery after reperfusion. These sets of evidence imply that β2AR overexpressed hearts may be more susceptible to reperfusion injury than intact hearts, and thereby, resulting in attenuated functional recovery after reperfusion.
Although the step from rat experiments to clinical application is a large one, the present results may have clinical implications. Wu et al. have reported that IPC is clinically beneficial for patients receiving CABG to reduce incidence of arrhythmic events such as ventricular tachycardia, and atrial fibrillation [2]. The IPC effects do not clinically reach to functional benefits after release of aortic cross clamp. There is increasing evidence that β2AR is expected as a molecular targets for new therapeutic strategies including gene therapy [15]. Therefore, the present results indicate that β2AR GT make it possible to provide the advantageous effects for the intrinsitic effects of IPC.
In conclusion, β2AR are required to generate IPC effects and that GT of β2AR enhances cardioprotective effects of IPC in post-MI hearts, although underlying subcellular mechanisms should be further investigated.
