The Institutional Animal Care and Use Committee of the University Animal Research Laboratory approved this study. All animals received standardized care in accordance with the National Institute of Health Guidelines. Adult male Wistar rats (400 to 450 g, Harlan, Udine, Italy) were used for all experiments.
Rats were anesthetized with 5% isoflurane in 50% O2 in a plastic induction box. After orotracheal intubation with a 14G cannula, the animals were mechanically ventilated (Harvard Model 687, Harvard Apparatus, Holliston, MA, USA). The tidal volume was 7 ml/kg and the respiratory rate was 50 to 60 breaths/minute with an air-oxygen mixture (FiO2 = 0.5). Ventilation was adjusted to keep an arterial carbon dioxide tension (PaCO2) of 35 to 45 mm Hg. Anesthesia was maintained with isoflurane 2% and pancuronium-bromide (2 mg/kg iv) was administered for complete muscle relaxation. Adequate anesthesia was monitored by the withdrawal response to a paw pinch and respiration monitoring. Rats were secured supine on a heating board. ECG was monitored using limb leads. A thermocouple microprobe was inserted into the left femoral artery and advanced into the descending aorta for the measurement of blood temperature. The left femoral artery was cannulated with a heparinized 24-G Teflon catheter to monitor systemic arterial pressure and to collect arterial blood for lactate and gas analysis.
Cardiopulmonary Bypass (CPB) model
Central cannulation was performed as previously described . In brief, after complete sternotomy, a venous cannula (a modified four-hole 16 gauge Angiocath catheter, Delta Med, New York City, NY, USA) was advanced into the right atrium using a right trans-superior vena cava approach, allowing excellent drainage. The left common carotid artery was cannulated using an 18-gauge catheter advanced to the aortic arch and connected to the arterial perfusion line for the CPB circuit. Full heparinization (500 IU/kg) was assured after surgical preparation and immediately before CPB initiation.
CPB was set up as previously described . The setup consisted of a venous reservoir, a roller pump, a hollow-fiber oxygenator (Sorin, Mirandola, Italy), and a vacuum regulator with an applied pressure of 30 mmH2O to facilitate venous drainage, all connected by 1.6 mm internal diameter plastic tubing. Total priming volume was 9.5 mL, the gas exchange surface was 450 cm2, and the heat exchange surface was 15.8 cm2.
CPB was instituted at a flow rate of 120 mL/kg/min. A core temperature of 15°C to 13°C was achieved over 30 minutes using CPB-assisted cooling. The roller pump was turned off and DHCA, as confirmed by asystole and lack of measurable mean arterial pressure, was maintained for 20 minutes at 13°C to 15°C.
With the reinstitution of CPB, rewarming started at a flow rate of 100 mL/kg/min. CPB inflow rate was gradually increased, reaching the full rate of 120 mL/kg/min at the end of rewarming at 36°C in a period of 60 minutes. The temperature gradient between the CPB circuit and body core did not exceed 10°C. After full rewarming, the remaining priming volume was reinfused and animals were weaned from CPB (T1). Then animals were randomly assigned to treatment with levosimendan (0.2 μg/kg/min) or epinephrine (0.1 μg/kg/min) or saline as control. Infusion started immediately after hemodynamic recording and blood sampling post-weaning from CPB (T1) and lasted for 60 minutes in normothermia at 36°C to 37°C (Endpoint = T2).
Hemodynamic parameters were collected continuously during the experiments with a 2 F micro-tip pressure-volume conductance catheter (SPR-838; Millar Instruments, Inc., Houston, TX, USA) inserted into the right carotid artery and advanced into the left ventricle. Signals were continuously recorded at a sampling rate of 1,000 samples/s using a P-V conductance system (MPVS-400; Millar Instruments, Inc.), stored, and displayed on a personal computer by the PowerLab Chart5 Software System (AD Instruments, Colorado Springs, CO, USA). With the use of a special pressure-volume analysis program (PVAN; Millar Instruments, Inc.), heart rate (HR), mean arterial pressure (MAP), maximal slope of the systolic pressure increment (+dP/dt) and the diastolic pressure decrement (−dP/dt), and time constant of left ventricular pressure decay (Tau, according to the Weiss method) were computed and calculated. Stroke volume (SV) and cardiac output (CO) were calculated and corrected according to in vitro and in vivo volume calibrations using PVAN software .
In addition to the above parameters, left ventricle pressure-volume relations were measured by transiently occluding the inferior vena cava (reducing preload) under the diaphragm by tying a snare suture around the vein at baseline condition (T0), after CPB weaning (T1) and at the end of 60 minutes of infusion treatment (T2) .
After euthanasia with a potassium bolus freeze-clamped left ventricle myocardial biopsies were snap-frozen. In particular, the tip of the freeze-clamp tong was pre-cooled in liquid nitrogen prior to taking the biopsies and thereafter the samples were stored at −80°C (protein isolation from tissue extracts) or in liquid nitrogen (high energy phosphates) until analysis.
For Western blot analysis of cTnI phosphorylation, cardiac specimens were pulverized in liquid nitrogen and homogenized in ice-cold lyses buffer (20 mM Hepes, 420 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% NP-40, 20% glycerol) containing a protease inhibitor cocktail (Roche Diagnostics, Monza, Italy) and a phosphatase inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA).
The homogenates were centrifuged (12,000 rpm) for 30 minutes at 4°C, supernatants were recovered, snap-frozen in liquid nitrogen and stored at −80°C. Total protein content was determined with the Bradford method. Equal amounts of each sample were fractionated on 10% polyacrylamide gels by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). Membranes were blocked with 5% BSA (Albumin from Bovine Serum, Sigma-Aldrich) in TBS-0.1% Tween20, and incubated overnight with specific primary antibodies for either phospho-cTnI (Ser23/24) (1:1,000, Cell Signaling Technology, Danvers, MA, USA) or total cTnI (1:1,000, Cell Signaling Technology) at 4°C. A horseradish peroxidase conjugated secondary antibody (Amersham Biosciences, GE Healthcare Europe, Munich, Germany) was used to detect the blots by standard chemiluminescence substrate (LiteAblot PLUS reagent, Euroclone, Siziano, Italy) on Kodak films (Carestream Health Inc., Rochester, NY, USA). Bands were quantified by ImageJ software (National Institutes of Health, Bethesda, MD, USA).
For energy status determination, the sample preparation and high-performance liquid chromatography (HPLC) measurement of ATP, ADP, AMP and phosphocreatine, as well as hypoxanthine and xanthine, were performed as previously described . A piece of frozen tissue (50 to 100 mg) was homogenized with a micro-dismembranator and deproteinized with 400 μL of 0.4 mol/L perchloric acid. After centrifugation (12,000 g), 150 μL of the acid extract was neutralized with 15 to 20 μL of 2 mol/L potassium carbonate (4°C). The supernatant (20 μL injection volume), obtained after centrifugation, was used for HPLC analysis. The pellets of the acid extract are dissolved in 1 mL of 0.1 mol/L sodium hydroxide and further diluted 1:10 with physiological saline for protein determination (BCA Protein Assay, Pierce; Pierce Biotechnology, Rockford, IL, USA). High energy phosphates (ATP, ADP, AMP and phosphocreatine), hypoxanthine and xanthine were measured by HPLC as previously described . In brief, separation was performed on a Hypersil ODS column (5 μm, 250 × 4 mm I.D.) using an L-2200 autosampler, two L-2130 HTA pumps and an L-2450 diode array detector (all: VWR Hitachi, VWR, Vienna, Austria). Detector signals (absorbance at 214 and 254 nm) were recorded, and program EZchrom Elite (VWR) was used for data requisition and analysis.
Energy charge was calculated using the following formula: (ATP + 0.5ADP)/(AMP + ADP + ATP).
Arterial blood samples were collected to measure lactate concentration.
The means ± SD are given. For between-group comparisons, analysis of variance with the Bonferroni post hoc test was used. An unpaired two-tailed Student t- test or paired t test was employed to evaluate differences between groups and within groups versus the baseline, respectively. The statistical software GraphPad Prism 5.0 (GraphPad Software Inc., San Diego, CA, USA) was used. A P-value of <0.05 was considered to be significant.