Adenosine A₁ Receptor Mediates Delayed Cardioprotective Effect of Sildenafil in Mouse

Animals

Adult male C57BL mice were supplied by The Jackson Laboratory (Bar Harbor, ME). The body weight for these animals ranged between 27.1 and 32.7 g. A₁AR-KO mice were obtained from a sub-colony of the original lines generated by Sun et al. (16). A₁AR-KO mice were bred on site at the Virginia Commonwealth University School of Medicine. The deletion of A₁AR gene in the knockout mice was confirmed by RT-PCR.

Langendorff Isolated Perfused Heart Preparation

The methodology of the Langendorff isolated perfused heart has been previously described in detail (17). In brief, the mice were anesthetized with an IP injection of sodium pentobarbital (100 mg/kg containing 33 international units of heparin). The hearts were removed and submerged in ice-cold Krebs-Henseleit (K-H) solution containing heparin. The aorta was secured to a 20 gauge stainless steel blunt needle, and the hearts were perfused at a constant temperature and pressure in a retrograde fashion via the aorta with K-H solution. Following a stabilization period of 30 minutes, I/R injury was produced by subjecting the hearts to 30 minutes of normothermic, no-flow global ischemia followed by reperfusion for 60 minutes. To obtain the developed pressure, a left atrial incision was made to expose the mitral annulas through which a water-filled latex balloon was passed into the left ventricle (LV). The balloon was attached via polyethylene tubing to a Gould pressure transducer that was connected to a PowerLab Acquisition System (ADInstruments 8SP, Australia). The balloon was inflated to adjust the LV end-diastolic pressure (LVEDP) to ~ 10 mmHg. Myocardial ischemic damage was measured using multiple, independent end points of tissue injury. These included infarct size, LV developed pressure (LVDP), LVEDP, rate-pressure product (RPP), heart rate, and coronary flow by timed collection of the perfusate. LVDP was calculated by subtracting LVEDP from the peak systolic pressure. RPP, an index of cardiac work, was calculated by multiplying LVDP with heart rate. The hearts were not paced.

Study Protocol

Mice were randomly assigned into one of the following groups as shown in Figure 1:

• Group I: [saline control, C57BL (WT) mice; n=6]: WT mice received 0.2 ml 0.9 % saline, i.p.;

• Group II: (Sildenafil, WT mice; n=6): Pure sildenafil powder (kindly provided by Pfizer, Inc.) was dissolved in 0.2 ml saline. This preparation was given in WT mice as 0.71 mg/kg i.p. bolus, approximating, on a mg/kg basis, the clinical dose of 50 mg administered to a 70 kg patient;

• Group III: (DPCPX+sildenafil, WT mice; n=6): DPCPX (0.1mg/kg) was administered 30 min prior to sildenafil treatment in WT mice as in Group II;

• Group IV: (DPCPX+saline, WT mice; n=6): WT mice were treated with DPCPX (0.1mg/kg) 30 min prior to saline treatment as in Group I;

• Group V: (DMSO+sildenafil, WT mice, n=6): DMSO (solvent for DPCPX) was administered in WT mice 30 min prior to sildenafil treatment as in group II;

• Group VI: (DMSO+saline, WT mice, n=6): WT mice were given DMSO 30 min prior to saline treatment as in Group I;

• Group VII: (Saline control, A₁AR –KO; n=6): A₁AR -KO mice received 0.2 ml 0.9% saline, i.p.;

• Group VIII: (Sildenafil, A₁AR –KO; n=6): A₁AR-KO mice received 0.71 mg/kg sildenafil as in Group II.

Twenty-four hours later, the hearts were isolated and subjected to I/R as described above.

Isolation of Adult Mouse Ventricular Cardiomyocytes

Adult male C57BL-WT or A₁AR-KO mice were used in this study. The ventricular cardiomyocytes were isolated using an enzymatic technique modified from the previously reported method (18,19). In brief, the animal was anesthetized with pentobarbital sodium (100 mg/kg, i.p.) and the heart was quickly removed. Within 3 min, the aortic opening was cannulated onto a Langendorff perfusion system and the heart was retrogradely perfused (37°C) at a constant pressure of 55 mmHg for ~5 min with a Ca²⁺-free bicarbonate-based buffer containing (in mM): 120 NaCl, 5.4 KCl, 1.2 MgSO₄, 1.2 NaH₂PO₄, 5.6 glucose, 20 NaHCO₃, 10 2,3-butanedione monoxime, and 5 taurine, which was continuously bubbled with 95%O₂ + 5%CO₂. The enzymatic digestion was commenced by adding collagenase type II (Worthington, 0.5 mg/mL each) and protease type XIV (0.02 mg/mL) to the perfusion buffer and continued for ~15 min. 50 μM Ca²⁺ was then added in to the enzyme solution for perfusing the heart for another 10-15 min. The digested ventricular tissue was cut into chunks and gently aspirated with a transfer pipette for facilitating the cell dissociation. The cell pellet was resuspended for a 3-step Ca²⁺ restoration procedure (i.e. 125, 250, 500 μM Ca²⁺). The freshly isolated cardiomyocytes were then suspended in minimal essential medium (Sigma catalogue# 6 M1018, pH 7.35-7.45) containing 1.2 mM Ca²⁺, 12 mM NaHCO₃, 2.5% fetal bovine serum and 1% penicillin-streptomycin. The cells were then plated onto the 35 mm cell culture dishes, which were pre-coated with 20 μg/mL mouse laminin in PBS + 1% penicillin-streptomycin for 1 hour. The cardiomyocytes were cultured in the presence of 5% CO₂ for 1 hour in a humidified incubator at 37°C, which allowed cardiomyocytes to attach to the dish surface prior to the experimental protocol.

Experimental Protocol

The cultured cardiomyocytes were incubated under 37°C and 5% CO₂, for 1 hour with or without 1 μM sildenafil powder dissolved in distilled water. The drug solution was filtered (0.45 μm pore size) before adding into the cell medium. Cardiomyocytes were subjected to simulated ischemia (SI) for 40 minutes by replacing the cell medium with an “ischemia buffer” which contained (in mM): 118 NaCl, 24 NaHCO₃, 1.0 NaH₂PO₄, 2.5 CaCl₂-2H₂O, 1.2 MgCl₂, 20 sodium lactate, 16 KCl, 10 2-deoxyglucose (pH adjusted to 6.2). In addition, the cells were incubated under hypoxic conditions at 37°C during the entire SI period by adjusting the tri-gas incubator to 1-2% O₂ and 5% CO₂. Reoxygenation (RO) was accomplished by replacing the ischemic buffer with normal medium under normoxic conditions. Assessment of cell necrosis was performed after 1 hour of RO.

Evaluation of Cell Viability

Cell viability was assessed by trypan blue exclusion assay. At the end of protocol, 20 μL of 0.4% trypan blue (Sigma-Aldrich) was added into the culture dish. After ~5 min of equilibration, the cells were counted under microscope.

Statistics

All measurements are expressed as means ± SE. The data were analyzed by either unpaired t-test or one-way ANOVA. If a significant value of F was obtained in ANOVA, the Student-Newman-Keuls post hoc test was further used for pair wise comparisons. Paired t-test was used to compare any pair of pre- and post-treatment values for the same parameter. P < 0.05 was considered significant.

Left ventricular cardiac function

There was no significant difference in the basal functional parameters (i.e. heart rate, LVDP and LVEDP) between the groups after 20 min stabilization (Table 1). Except in the A₁AR-KO mice treated with saline, no significant changes in the rate pressure product were observed during post-ischemia (Figure 2) between the groups. Post-ischemic coronary flow was also not significantly different among the groups as shown in Table 2.

Myocardial Infarction

Following I/R, myocardial infarct size (Mean±SEM; % of risk area) was reduced in wild-type mice treated with sildenafil (5.6±0.9) versus saline group (27.3±2.1; P<0.05; n=6/each; Figure 3). In contrast, sildenafil failed to reduce infarct size in the A₁AR-KO mice (31.6±1.9 vs. 32.3±1.5 with saline, p>0.05). Moreover, the selective A₁AR antagonist DPCPX blocked the infarct-limiting effect of sildenafil in the wild-type mice (27.3±3.2, p<0.05). There was no significant difference in the infarct size between controls and animals treated with DPCPX or DMSO, the solvent for DPCPX.

Effect of Sildenafil on Cardiomyocyte Necrosis

Our method for cell preparations yielded at least 70% of the cardiomyocytes with rod shape morphology, which was similar to the previously reported study from our laboratory and others (18,19). After 40 min of SI and 1 hour of RO, the percent of trypan blue positive cardiomyocytes were 53±2.8, (control). Pretreatment with sildenafil for 1 hr resulted in the ~54 % decrease in trypan blue positive cardiomyocytes i.e., 24.5±2.6 % p<0.05, vs control n=3; Figure 4). The A₁AR selective antagonist, DPCPX, abolished the protective effect of sildenafil as shown by increase in the trypan blue positive cells (50.8±3.0; P>0.05 vs. control). DPCPX or its solvent DMSO alone had no effect on trypan blue positive cell count. Furthermore, cardiomyocytes derived from A₁AR-KO mice failed to show protection against SI/RO when treated with sildenafil (46.7±2.3 % vs. 51.9±4.4 non-treated control A₁AR-KO cardiomyocytes; P>0.05).