Clinically-relevant consecutive treatment with isoproterenol and adenosine protects the failing heart against ischaemia and reperfusion

All procedures conform to the Directive 2010/63/EU of the European Parliament and 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). Ethical approval was granted by the University of Bristol, UK. Two months old male Wistar rats (250–260 g) were killed by stunning and cervical dislocation. Hearts (~0.75 g) were rapidly removed into ice-cold Krebs-Henseleit buffer (KH) containing (mm): NaCl 118, NaHCO₃ 25, KCl 4.8, KH₂PO₄ 1.2, MgSO₄ 1.2, glucose 11 and CaCl₂ 1.2, gassed with 95% O₂-5% CO₂ at 37 °C (pH 7.4) and perfused in Langendorff mode. The aorta was rapidly cannulated and the heart perfused at a rate of 8 ml · min⁻¹ · g⁻¹ heart weight with in-line filter using KH. Monitoring of left ventricular pressure was performed with a water-filled balloon inserted into the left ventricle, set to give an initial left ventricular end diastolic pressure (LVEDP) of 2.5–5 mmHg. All hearts were allowed an equilibration period of at least 25 min before any additional treatments was given. Data acquisition and analysis used a PowerLab System (ADInstruments, Australia). Left ventricular developed pressure (LVDP) was calculated as the difference between left ventricular systolic pressure (LVSP) and LVEDP, and work index (RPP) as the product of LVDP and heart rate (HR). Time derivatives of pressure were measured during contraction (+dP/dt) and relaxation (−dP/dt). The haemodynamic parameters were computed using the software Chart 5 (ADInstruments, Australia). Preischaemic values of the parameters of haemodynamic function were measured at the end of the equilibration period prior to any preischaemic intervention. Since the hearts in our experiments contracted spontaneously, haemodynamic function is reflected in both LVDP and HR. For this reason, RPP was considered as the main parameter characterising haemodynamic function. If RPP was less than 15,000 mmHg·beat/min at the end of the preischaemic equilibration period, the heart was excluded from the experiment.

Heart failure was surgically induced by ligation of the left coronary artery as described previously [15] and modified in our laboratory. In brief, 2 month old rats were anesthetized with ketamine (75 mg/kg, i.p.) and medetomidine (0.5 mg/kg, i.p.). Analgesia was induced by buprenorphine (0.05 mg/kg, s.c.). The rats were intubated and ventilated with a respirator for small animals (Harvard Apparatus). Body temperature was maintained at 37°C during the surgical procedure. An incision was made in the left side of the chest to expose the fourth intercostal space. A 7–0 surgical suture was placed under the left coronary artery and tied to complete occlusion of the artery. In sham-operated animals (SO), the coronary artery was not ligated. The wound was closed with a 4–0 surgical suture. Atipamezole (2.5 mg/kg, i.p.) was injected in order to facilitate recovery after the anaesthesia. After the surgery, rats were placed into a recovery chamber (Vet Tech Solutions Ltd) ventilated with oxygen. In coronary artery ligated (CAL) animals, approximately 92% survived and were able to meet the inclusion criteria for the experiments on isolated heart. All sham operated animals survived to 16 weeks after surgery. Animals were monitored and cared for daily. Sixteen weeks after surgery, the animals were used for experiments on isolated heart as shown in the Methods.

After a 25 min period of equilibration followed by preischaemic interventions, global normothermic ischaemia (37°C) was induced for 30 min and then normothermic perfusion was reinstated for 2 hr. Three series of experiments were performed as detailed below and summarised in Figures 1 and 2. In Series 1 and 2, experiments were carried out on healthy male Wistar rats (250–275 g) whilst in Series 3 rats with surgically-induced heart failure or sham-operated rats were employed.In Series 1 (Figure 1A), hearts were divided into a Control group and 4 treatment groups (8–10 hearts/group) according to the preischaemic protocol. Hearts of the Control group were not subjected to any intervention prior to ischaemia. Hearts of the three treatment groups, after the equilibration period, were subjected to the consecutive perfusion with Iso and Ade (3 min with 5 nM Iso and 5 min with 30 μM Ade) followed by a washout period of different duration: 10 min (Iso/Ade-10); 5 min (Iso/Ade-5) and zero min (Iso/Ade-0). Hearts of the fourth treatment group ((Iso/Ade Mix)) were perfused with Ade for 1.5 min and then 3 min with the mixture of Iso and Ade followed by another 1.5 min period of perfusion with Ade (Figure 1A). Analysis of haemodynamic function recovery, lactate dehydrogenase (LDH) release during reperfusion and infarct size, in this and the other series of experiments, was performed in all hearts subjected to ischaemia. Additional hearts (n = 9-10/group) of Control, Iso/Ade-10 and Iso/Ade-0 groups were freeze-clamped following 43 min preischaemia, ground under liquid nitrogen and stored at −80°C for later analysis of PKC activity. Another set of Control, Iso/Ade-0 and Iso/Ade Mix hearts (n = 7-8 in each group) were freeze-clamped at the end of the preishaemic protocol and used for the measurement of glycogen content.In Series 2 experiments, four groups (6–9 hearts/group) were employed (Figure 1B). Two of the groups were Control and Iso/Ade with no washout period. In this series of experiments and in the Series 3 experiments, Iso was used at 10 nM. In preliminary experiments (data not shown), we found that this concentration of Iso might give even higher cardioprotective effect than 5 nM, which is important in terms of optimisation of the intervention, yet was still in the range of normal pharmacological dose. Another two groups of hearts were perfused with 0.2 μM CsA for 8 min prior to ischaemia with or without the Iso/Ade treatment (Iso/Ade + CsA and CsA groups respectively).Series 3 experiments were carried out on CAL and SO rats. Hearts were divided into four groups (5–6 hearts/group): Control and Iso/Ade groups of CAL and SO rats. The experimental protocol of Control and Iso/Ade groups (Figure 2) in this series of experiments was similar to those in Series 2 (Figure 1B). Infarct size was not measured in these experiments because the CAL hearts had already developed an infarct as a result of the left descending coronary artery ligation.

Five additional CAL and 6 SO rats were used to confirm the development of heart failure. Body weight and tibia length were measured before the surgery and 16 weeks after the operation. After 16 weeks post-operation, the rats were killed by stunning and cervical dislocation, the hearts and lungs excised and the wet weight of the hearts, left ventricles and lungs were measured.

PKC activity was determined in freeze-clamped heart powders using a kit supplied by Promega according to the manufacturer’s instructions. The assay relies on a change in charge of the fluorescent PepTag® C1 peptide from +1 to −1 following phosphorylation. Bands were visualized under UV light and the ratio of fluorescence intensity of phosphorylated to non-phosphorylated peptide was quantified using AlphaInotech ChemiImager 4400 with AlphaEase v5.5 software.

Glycogen content was assessed by measuring the glucose released from glycogen breakdown by amyloglucosidase based on the method described by Passonneau [16] and expressed as μmol glucose per gram tissue. A sample (70 mg) of the frozen tissue was suspended in 1 ml of 0.3 M perchloric acid (4°C), homogenized with a Polytron and 50 μl of the suspension were incubated with 0.5 ml of 50 mM Na Acetate Buffer (pH 5.5) containing 0.02% BSA and 50 μg/ml amyloglucosidase (Sigma) at room temperature for 2 hr. The samples were then centrifuged at 10000 g for 10 min and glucose content was assessed using a Glucose (HK) Assay Kit (Sigma) according to the manufacturer’s instruction. Briefly, 0.4 ml of the supernatant was mixed with 1 ml of the glucose assay reagent. Glucose content was measured spectrophotometrically (340 nm) after incubation at room temperature for 15 min.

The quantity of glucose released from glycogen breakdown was determined by subtracting glucose in samples incubated without amyloglucosidase from those incubated with the enzyme.

LDH activity was determined in the effluent perfusate collected from the hearts of all groups prior to ischemia and during each 5 min over the first 30 min of reperfusion as described earlier [17] and modified at our lab: 80 μl of the sample was added to 910 μl of the buffer (pH 7.4) containing 100 mM triethanolamine and 100 μM reduced β-nicotinamide adenine dinucleotide (NADH). After addition of 10 μl of 0.1 M sodium pyruvate to the reaction mixture, the change of absorbance at 340 nm (A₃₄₀) was recorded using a spectrophotometer over 10 min at 37°C. LDH activity was calculated according to the rate of A₃₄₀ decrease.

Infarct size was determined as described previously [18]. Hearts were stained with 1% of triphenyltetrazolium chloride after 2 h of reperfusion, frozen at -20°C and sliced into 6 slices. Necrotic and intact areas of each side for each of heart slices were determined using AlphaEase v5.5 software and the total necrotic and intact area of ventricular myocardium of each heart was calculated. Since the entire heart was at risk from global ischemia, the infarct size was expressed dividing the sum of necrotic areas by the sum of total slice areas of the 6 slices to obtain the percentage of necrosis.