The procedures used in this study were in compliance with the European Convention for the protection of vertebrate animals used for experimental and other purpose.
Hearts from 2–4 months old male Wistar rats were used in all experiments. Animals were divided into three main groups: control, ischemia and ischemia-reperfusion groups. The ischemic and ischemic-reperfusion groups were further divided into several subgroups in which hearts were pre-treated with DETA/NO in the presence/absence of various inhibibitors. All hearts were perfused on a Langendorff perfusion system with Krebs-Henseleit solution (11 mM glucose, 118 mM NaCl, 25 mM NaHCO3, 4.8 mM KCl, 1.2 mM KH2PO4, 1.2 mM CaCl2, 1.7 mM MgSO4 and 0.7 mM Na pyruvate, saturated with 95% O2-5% CO2, pH 7.4 at 37°C) at a pressure of 80 cm H2O. After a 15-minute equilibration period 50 μM DETA/NO (Alexis) was added to the perfusion buffer and hearts were perfused for another 3 min with buffer containing DETA/NO. Control hearts were perfused for 20 min (15 min equilibration plus additional 3–5 min) with buffer only. After the equilibration period (and perfusion with DETA/NO where appropriate) hearts were subjected to 30 minutes stop-flow global ischemia. In reperfusion experiments, hearts were subjected to 30 minutes stop-flow global ischemia followed by 30 min reperfusion. In some experiments where inhibitors were used (1 μM KT 5823, the protein kinase G inhibitor from Calbiochem, 25 μM 1H-[1, 2, 4]oxadiazolo [4,3-a]quinoxalin-1-one (ODQ), the guanylyl cyclase inhibitor from Sigma, or 100 μM 5-hydroxy decanoate (5-HD), the mitochondrial K+ATP channel blocker from Sigma, 0.5 μM Ro 32-0432 and 1 μM Ro 31-8220, the protein kinase C inhibitors from Calbiochem and Sigma, respectively) they were added to the perfusion media 5–15 min prior to perfusion with DETA/NO. Data obtained in each group were compared to control group and ischemic or ischemic/reperfusion group, respectively.
Steady-state level of NO released from 50 μM DETA/NO was measured in 1 ml of oxygenated perfusion buffer at 37°C using NO sensitive electrode and was found to be 281 ± 55 nM.
KCl-based buffers were used for mitochondrial isolation in order to get higher yields of mitochondria. Hearts were cut into small pieces and homogenized with a teflon-glass homogenizer in the isolation buffer (10 ml/g of tissue) containing 180 mM KCl, 20 mM Tris HCl, 1 mM EGTA, pH 7.3 at 4°C temperature. Cytosolic and mitochondrial fractions were separated by differential centrifugation (5 min × 750 g, 10 min × 6800 g). The post-mitochondrial supernatant was additionally centrifuged for 30 min at 10000 g and the resulting supernatant (S10) was used for determination of cytochrome c content in cytosol. Total cytosolic and mitochondrial protein was measured by a Biuret method.
Mitochondrial respiration rate and membrane potential were measured with a Clarke-type oxygen electrode and a tetraphenylphosphonium-selective electrode as described in . The composition of mitochondrial incubation buffer A was 110 mM KCl, 2.24 mM MgCl2, 10 mM Tris HCl, 5 mM KH2PO4, 4 IU/ml creatine kinase, 50 mM creatine (pH 7.2 at 37°C). 5 mM succinate (plus 1 μM rotenone) was used as respiratory substrate. Mitochondrial state 3 respiration rate was achieved by adding 1 mM ATP in the presence of creatine kinase and creatine, which convert the ATP into ADP. The kinetics of the respiratory chain was determined as the dependence of the mitochondrial respiration rate on the membrane potential, when the latter was titrated with carboxyatractyloside (0.4–8 nmol/mg mitochondrial protein). The kinetics of the proton leak was measured as the rate of mitochondrial respiration in the presence of excess oligomycin 1 μg/mg protein (to prevent phosphorylation). This was determined over a range of different values of membrane potential by titrating with malonate (0.33–6 mM) – an inhibitor of the respiratory chain.
Isolated heart mitochondria were preincubated 1–2 min at room temperature in hypotonic conditions: 1 volume of mitochondrial suspension (at 20 mg/ml in isolation buffer) was added to 2 volumes of de-ionized water (so the final buffer contained 60 mM KCl, 6.7 mM Tris, and 0.3 mM EGTA, pH 7.3). Then 500 μM pyruvate plus 500 μM malate, 100 μM cGMP, 100 μM ATP and 40 IU/ml PKG Iα (bovine, recombinant; from Calbiochem), phosphatase inhibitor cocktail (1:100 dilution; from Sigma) were added and incubated 15 min at room temperature. Control mitochondria were preincubated in the same way only without PKG. Afterwards aliquots containing 0.2–0.5 mg mitochondrial protein were taken and added to 1 ml of the mitochondrial incubation buffer B (135 mM KCl, 30 mM Tris, 5 mM KH2PO4, 5 mM nitrilo-triacetic acid (NTA), 1.5 mM MgCl2, 1 mM pyruvate plus 1 mM malate, pH 7.2), and mitochondrial respiration, swelling and calcium accumulation were measured.
Mitochondrial respiration was measured in the incubation buffer B containing 135 mM KCl, 30 mM Tris, 5 mM KH2PO4, 5 mM NTA, 1.5 mM MgCl2, 1 mM pyruvate plus 1 mM malate, pH 7.2 as described above. State 3 respiration rate was initiated by adding 1 mM ADP. Mitochondrial swelling was assayed in buffer B by measuring a decrease in absorbance at 540 nm after addition of 250 μM CaCl2. After 15 min incubation mitochondria were centrifuged at 13000 rpm × 3 min in an Eppendorf centrifuge and supernatants were used for spectrophotometric measurements of cytochrome c as described in [19, 20].
Mitochondrial calcium retention capacity was measured fluorimetrically using Calcium Green-5N (Molecular Probes, excitation at 506 nm, emission at 535 nm) which detects extramitochondrial Ca2+. The incubation buffer C contained 200 mM sucrose, 10 mM Tris-HCl, 1 mM KH2PO4, 10 μM EGTA, 0.3 mM pyruvate plus 0.3 mM malate, pH 7.4, 25°C, final volume 3 ml . Calcium Green concentration in the medium was 100 nM. Calibration of the signal was achieved by the addition of known amounts of Ca2+. Experiments were started by the addition of 0.2 mg/ml mitochondria, then 10 μM CaCl2 pulses were added approximately every 3 min (after ~90% of added calcium was taken up by mitochondria) until opening of permeability transition pore occurred which was recorded as a large increase in fluorescence due to release of accumulated Ca2+ from mitochondria.
For measurement of cytochrome c, mitochondria were solubilized with 1% Triton X-100 (w/v). Sodium hydrosulphite-reduced minus hydrogen-peroxide-oxidized absorption spectra difference was recorded with a Hitachi-557 spectrophotometer. Cytochrome c+c1 content was estimated by using the absorption difference at the wavelength pair 550/535 nm and ε = 14.5 mM-1cm-1 as described in . The cytochrome c content in cytosolic fractions was detected using Quantikinine M rat/mouse Immunoassay ELISA kit (R&D Systems). Cytosolic fraction proteins were dissolved in 0.5% Triton X-100 and further procedures were performed according to the manufacturer's protocol.
Lactate dehydrogenase (LDH) activity in the coronary effluent was measured spectrophotometrically  by monitoring the rate of decrease in NADH (at 340 nm) as pyruvate is converted to lactate. The LDH activity was measured by addition of effluent to 0.1 M Tris-HCl buffer (pH 7.5) containing 0.1 mM NADH and 1 mM Na-pyruvate. A unit of LDH was defined as the amount of enzyme necessary to catalyze oxidation of 1 μmol NADH per min (IU).
The activity of caspases was measured as described in . Cytosolic fractions were prepared from heart homogenates in sucrose-based medium (250 mM sucrose, 5 mM HEPES, 2 mM EGTA, 1 mg/ml albumin) by differential centrifugation: 5 min × 750 g, 10 min × 6800 g, and 30 min × 10000 g. Sucrose-based medium rather than KCl-based buffer was used to avoid possible inhibition of caspase activity by high potassium salt concentrations. 1 mg/ml of total cytosolic protein was incubated for 30 min in buffer containing 10% sucrose, 50 mM HEPES, 1 mM MgCl2, 1 mM ATP (pH 7.4, 37°C) and 0.1 mM z-DEVD-p-nitroanilide (Alexis), a caspase-3 substrate. The hydrolysis of caspase substrate was followed spectrophotometrically at 405 nm and was calibrated with p-nitroanilide. DEVD-cleaving activity was completely suppressed by 0.02 mM DEVD-CHO (Alexis), a reversible inhibitor of caspase-3.
Data are expressed as means ± S.E. of at least 3 separate experiments. Statistical comparison between experimental groups was performed by ANOVA followed by Tukey or LSD tests. A value of p < 0.05 was considered statistically significant.