Elderly New Zealand white rabbits (n=64), aged 137±1 weeks and weighing 3.5±0.2 kg, and adult rabbits (n=16), aged 28±1 weeks and weighing 2.8±0.1 kg, were obtained from the SLAC Laboratory Animal Center Co., Ltd. (Shanghai, China). All rabbits were housed separately, fed with a standard laboratory diet and provided with water.

All experiments were approved by the Animal Care and Use Committee of Fujian Medical University (Fuzhou, China). The study was performed in accordance with the Principles of Laboratory Animal Care, proposed by the National Society for Medical Research, and the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication no. 5377-3, 1996). Surgeries were performed under anesthesia and analgesic agents were applied immediately at the end of surgery. All surgeries and associated tests were performed in a blinded manner.

Animals were anesthetized with pentobarbital sodium (30 mg/kg) and heparinized (200 U/Kg) via the marginal ear vein. During the isolation, hearts were perfused at 4°C with cardioplegic St. Thomas II (ST) solution (15 ml/kg; Nanjing Jiancheng Bioengineering Institute, Nanjing, China) for 2 min. Following aortic cannulation and cardiac arrest, the hearts were immersed in ST solution at 4°C. The hearts were subjected to Langendorff perfusion at a constant pressure of 60 mmHg (80 cm H₂O) at 37°C. Cardiac function indices, including systemic arterial pressure (SAP), left ventricular systolic pressure (LVSP), changes in pressure over time (+dp/dt max and -dp/dt max), heart rate (HR) and coronary sinus flow (CSF), were monitored using an ALC_B10-MPA myocardial function analyzing system (Alcott Biotech, Shanghai, China), and well-preserved isolated hearts were then assigned to groups according to the IR process and the different protective strategies. Aortic and CSF data were collected for analyses, and left ventricular apical myocardial tissue was extracted and frozen for further analysis.

Sixteen adult and 16 elderly New Zealand white rabbits were divided into four groups: Adult heart control, adult heart + IPC, aged heart control and aged heart + IPC (n=8 per group).

IPC was performed in hearts from rabbits of the IPC groups. Hearts of the anesthetized aged rabbits were rapidly excised, transferred to a Langendorff apparatus and perfused via aortic cannula with an oxygenated Krebs-Henseleit buffer (Nanjing Jiancheng Bioengineering Institute) containing 118.3 mmol/l NaCl, 2.7 mmol/l KCl, 1.0 mmol/l MgSO₄, 1.4 mmol/l KH₂PO₄, 29.0 mmol/l NaHCO₃, 3.4 mmol/l CaCl₂ and 10 mmol/l glucose, with 70 mU/l insulin and 0.4% bovine serum albumin, at 37°C. Flow was adjusted to achieve a retrograde perfusion pressure of 60 mmHg. All hearts were initially perfused with the oxygenated buffer for a 15-min stabilization period. A subset of the hearts (adult heart + IPC and aged heart + IPC) was subjected to a preconditioning protocol consisting of one cycle of global no-flow ischemia (5 min) followed by normal perfusion (5 min), prior to 120 min of global ischemia, as previously described (13). The remaining hearts were not preconditioned but were instead perfused with a normoxic buffer (Nanjing Jiancheng Bioengineering Institute) for 10 min, prior to being subjected to 120 min of global ischemia. Isolated myocardial tissue from the left ventricular apex was immediately collected for analysis.

Myocardial tissue of the left ventricular apex was extracted from all hearts and immediately preserved in liquid nitrogen. ADO production in the myocardium was detected using a high-performance liquid chromatography system (Beckman System Gold; Beckman Coulter, Beijing, China). ADO standard product was purchased from Sigma-Aldrich (St. Louis, MO, USA) (23).

Total RNA was extracted from the myocardium using the RNAiso Plus reagent (Takara Bio Inc., Shiga, Japan), and cDNA was prepared using the Primescript RT Reagent (Takara). An ABI StepOnePlus™ Real-Time-PCR System (Applied Biosystems; Life Technologies, Carlsbad, CA, USA) was used with the SYBR® Green Master Mix (Applied Biosystems), and primers were obtained from the Beijing Genomics Institute (Shenzhen, China) for the measurement of the expression of ARs (A₁AR, A_2AAR and A₃AR), B-cell lymphoma-2 (Bcl-2) and intercellular adhesion molecule-1 (ICAM-1) (Table I). PCR cycling conditions were as follows: 2 min pre-denaturation at 95°C, 40 cycles of 10 sec denaturation at 95°C, 10 sec annealing at 61°C and a 40-sec extension step at 72°C. GAPDH was used as a reference to obtain the relative fold change for targets using the comparative Ct method.

Cardioprotection strategies are summarized in Fig. 1. Forty-eight elderly New Zealand white rabbits were divided into six groups (n=8 per group). Following exposure to different interventions, the isolated hearts were perfused for 15 min to establish stable hemodynamics. The following six different interventions were undertaken prior to cardiac arrest for 120 min at 15°C: i) ST group, hearts were perfused with Langendorff solution for 10 min at 37°C, followed by protection with ST solution (20 ml/kg) at 4°C. The hearts were stored in the ST solution at 4°C for 120 min; ii) ADO + ST group, hearts were subjected to a 10-ml bolus injection of ADO/KOH (1 mmol/l), followed by Langendorff perfusion for 10 min at 37°C and protection with ST solution (20 ml/kg) at 4°C. The hearts were stored in the ST solution at 4°C for 120 min; iii) APC + ST group, hearts were subjected to a 10-ml bolus injection of ADO/KOH (1 mmol/l), followed by 5 min of ischemia and 5 min of reperfusion at 37°C, then protection with ST solution (20 ml/kg) at 4°C. The hearts were stored in the ST solution at 4°C for 120 min; iv) A1AR agonist 2-chloro-N(6)-cyclopentyladenosine (CCPA) + ST group, rabbits received ear vein injections of CCPA (100 µg/kg) at 48 and 24 h prior to surgery. The hearts were subjected to Langendorff perfusion for 10 min at 37°C followed by protection with ST solution (20 ml/kg) at 4°C. The hearts were stored in ST solution at 4°C for 120 min; v) CCPA + ADO + ST group, rabbits received ear vein injections of CCPA (100 µg/kg) at 48 and 24 h prior to surgery. Hearts were subjected to a 10-ml bolus injection of ADO/KOH (1 mmol/l), followed by Langendorff perfusion for 10 min at 37°C and protection with ST solution (20 ml/kg) at 4°C. The hearts were stored in ST solution at 4°C for 120 min; vi) CCPA + APC + ST group, rabbits received ear vein injection of CCPA (100 µg/kg) at 48 and 24 h prior to surgery. The hearts were subjected to a 10-ml bolus injection of ADO/KOH (1 mmol/l), followed by 5 min ischemia and 5 min reperfusion at 37°C, followed by protection with ST solution (20 ml/kg) at 4°C. The hearts were stored in ST solution at 4°C for 120 min.

Cardiac function indices, including SAP, LVSP, +dp/dt, -dp/dt, HR and CSF, were checked before treatment and 30/60 min post-treatment, using the ALC_B10-MPA Cardiac Function Analysis System (Alcott Biotech), according to the manufacturer's instructions. Post-treatment hemodynamic parameters were measured by catheterization (SRP-320/PVAN 3.2, Millar Instruments, Inc., Houston, TX, USA; Chart 5 software, ADInstruments, Dunedin, New Zealand), as previously described (24).

Coronary venous outflow (2 ml) was collected prior to cardiac arrest and 20 min after reperfusion. The levels of myocardial enzyme leakage in the preserved liquid were measured in duplicate using a creatine kinase (CK) and lactate dehydrogenase (LDH) ELISA testing kit (Nanjing Jiancheng Bioengineering Institute), according to the manufacturer's instructions.

Coronary venous outflow (2 ml) was collected prior to cardiac arrest and 30 min after reperfusion. Levels of NO and endothelin (ET) were detected using NO and ET testing kits (Nanjing Jiancheng Bioengineering Institute), according to the manufacturer's instructions.

Coronary venous outflow (2 ml) was collected prior to cardiac arrest and 60 min after reperfusion. MDA and SOD levels were measured using MDA and SOD testing kits (Nanjing Jiancheng Bioengineering Institute), according to the manufacturer's instructions.

Myocardial tissues of the left ventricular apex were collected 60 min after reperfusion. ATP levels were detected using an ATP testing kit (Beyotime Institute of Biotechnology, Shanghai, China).

Myocardial tissues of the left ventricular apex were collected and fixed in 4% formalin, dehydrated and embedded in paraffin. The samples were then sectioned and stained using standard methods. Samples were observed by microscopy (Olympus Corp., Tokyo, Japan) at x100 and x400 magnifications.

Myocardial tissues of the left ventricular apex were collected and prepared for electron microscopy, as previously described (25).

The apoptotic index of the myocardium was assessed by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method using a TUNEL staining kit (Fuzhou Maxim Biotech, Inc., Fuzhou China), according to the manufacturer's instructions. Samples were examined under microscopy and stained cells were counted in six random visual fields.

Statistical analysis was performed using SPSS 17.1 software (SPSS Inc., Chicago, IL, USA). Data are presented as the mean ± standard error. Two-way repeated-measures analysis of variance was performed to analyze the differences between the groups at different time-points. P<0.05 was considered to indicate a statistically significant difference.

Isolated heart models were successfully established with almost no warm ischemic time in New Zealand white rabbits. All animals had a strong heartbeat during surgery and no cardiac arrest occurred. The duration between surgery and Langendorff perfusion ranged from 9 to 10 min, including 4 min of cold ischemia. Cardiac function was preserved in all isolated hearts, as determined by the following indices: SAP, 83.98±2.18 mmHg; LVSP, 90.01±2.34 mmHg; +dp/dt, 1,840.15±52.74 mmHg/sec; -dp/dt, 1,438.75±62.17 mmHg/sec; HR, 193.94±6.97 bpm and CSF, 60.96±3.09 ml/min. These models were suitable to fulfill the demands of the following experiments.

The baseline levels of endogenous ADO were found to be significantly lower in the aged groups than those in the adult groups (P<0.05, Table II). The increase in ADO in the aged + IPC group was significantly lower than that in the adult + IPC group (P<0.05). These findings indicated that endogenous ADO was associated with the protective effects of IPC in aged rabbit hearts.

As shown in Table II, the baseline levels of A₁AR, A_2AAR and A₃AR were decreased in the aged control group compared with those in the adult control group (P<0.05); however, following IPC, the AR expression was regulated in different manners. The mRNA levels of A₁AR and A₃AR increased more significantly in the aged + IPC group than those in the adult + IPC group (P<0.05). Following IPC, a 4.36-fold increase was observed in the A₁AR and a 1.87-fold increase in the A₃AR mRNA levels in the aged heart tissue, compared with the 1.51-fold increase in the A₁AR mRNA levels and the 1.68-fold increase in the A₃AR mRNA levels in the adult heart tissue. The mRNA levels of A_2AAR were significantly lower in the aged + IPC group than those in the adult + IPC group (P<0.05).

The results in Table III show that the baseline values of SAP, LVSP, +dp/dt and -dp/dt were similar in the CCPA + ST, CCPA + ADO + ST and CCPA + APC + ST groups (P>0.05). Sixty minutes after IR, the values of SAP, LVSP, +dp/dt and -dp/dt were improved in the combination groups, with the most marked change observed in the CCPA + APC + ST group (P<0.05, Fig. 2A). It was also found that, 60 min after IR, the HR and CSF were improved in the combination groups, particularly in the CCPA + APC + ST group, where the improvement was more marked than that in the other groups (P<0.05, Fig. 2B and Table III).

Myocardial samples from the CCPA groups showed an indefinable distribution of hematoxylin and eosin (H&E) stain. Myocardial fibers were well preserved, although moderate regional swelling was observed in the intercellular matrix. In the CCPA + APC + ST group, the fibers were arranged in neat rows, the cell size was uniform and the H&E staining was well distributed. There was no obvious cellular swelling (Fig. 3A).

Transmission electron microscopy revealed that, following IR, myocardial fibers from the different groups varied in size and shape and some exhibited mitochondrial hyperplasia. Both prior to and following IR, the myocardial fibers in the ST group were abnormal, with clear swelling observed in the cytoplasm and mitochondria. By contrast, following IR, myocardial fibers from the CCPA + APC + ST group showed a clear, normal structure and contained rows of mitochondria without evidence of swelling (Fig. 3B).

As shown in Fig. 3C, significantly fewer apoptotic cells were found in the treatment groups, with the exception of the ADO + ST group, compared with the ST group (P<0.05). No significant difference in the apoptotic index was observed between the APC + ST and CCPA + ADO + ST groups; however, the apoptotic index was considerably lower in the CCPA + APC + ST group compared with the other groups (P<0.05), suggesting that both CCPA and APC protected myocardial cells from IR-induced injury, and therefore supporting the combined use of these two agents.

The levels of Bcl-2, ICAM-1, CK and LDH, indicators of cardiac injury or inflammation, were subsequently investigated. As shown in Table IV, the expression of Bcl-2, which is a factor that protects cells from apoptosis, was increased in all treatment groups, with the exception of the ADO + ST group, compared with that in the ST group (P<0.05). The levels of ICAM-1, a factor associated with the induction of inflammation, were also decreased in the treatment groups, with the exception of the ADO + ST group, compared with the ST group (P<0.05). The expression of Bcl-2 in the CCPA + APC + ST group was considerably higher than that in any of the other groups (P<0.05) and the ICAM-1 levels were significantly lower than those in any of the other groups (P<0.05).

CK and LDH are important indicators of myocardial injury. In order to assess the extent of IR injury, coronary venous outflow was collected prior to and 20 min after IR. As shown in Table III, no differences were identified among the groups in the levels of CK and LDH prior to IR (P>0.05); however, 20 min after IR, the CK and LDH leakage was significantly increased in each of the studied groups (P<0.05), but the lowest value was observed in the CCPA + APC + ST group (P<0.05, Fig. 4A and Table III).

NO and ET levels are indicators of endothelial function. As shown in Table III, prior to ischemia, NO levels were higher in the CCPA-treated groups than those in the other groups. Following IR, NO production was significantly reduced, but the best recovery was observed in the CCPA + APC + ST group (P<0.05). Changes in ET levels showed an inverse association; CCPA + APC + ST inhibited the abnormal increase in ET production following IR.

MDA, SOD and ATP, as indicators of oxidative status, were measured prior to and following IR. The results shown in Table III revealed that the mitochondrial function of the myocardium was the most effectively protected, and the levels of reactive oxygen species (ROS) were the lowest, in the CCPA + APC + ST group (P<0.05, Fig. 4B and C).