Experiments followed guidelines of the Canadian Council on Animal Care (CCAC; Ottawa, ON: Vol. 1, 2nd edition, 1993; Vol. 2, 1984). Male Fisher rats (≈3 months, ≈300 g, Charles River Canada, St. Constant, Canada) were heparinized (3000 U/kg intraperitoneal; Pharmaceutical Partners of Canada, Richmond, Canada) and anaesthetized (sodium pentobarbital 160 mg/kg intra-peritoneal; CDMV; Saint-Hyacinthe, Canada) prior to harvest of hearts or cardiomyocytes and were approved by the Dalhousie University Animal Care Committee.
Cardioplegic arrest - isolated cardiomyocytes
Ventricular myocytes were obtained by enzymatic dissociation according to previously described methods [18, 19]. Briefly, hearts were perfused in situ for 5 min with buffer containing (mM): 135.5 NaCl, 4 KCl, 10 HEPES, 1.2 MgSO4, 1.2 KH2PO4 12 glucose and 200 μM CaCl2 (pH 7.4, 37°C, 100% O2). Hearts were then perfused with this solution without CaCl2 (5 min), followed by (20 min) buffer containing 50 μM CaCl2, protease dispase II (0.1 mg/mL, Roche Diagnostics, Laval, Canada), collagenase type 2 (0.56 mg/mL, Worthington, Lakewood, NJ) and trypsin (0.02 mg/mL, Sigma-Aldrich, Oakville, Canada). The ventricles were minced in buffer containing (mM): 45 KCl, 3 MgSO4.7H2O, 30 KH2PO4, 50 L-glutamic acid, 20 taurine, 0.5 EGTA, 10 HEPES and 10 glucose (pH 7.4 with KOH). Quiescent, rod shaped, cardiomyocytes with no visible membrane damage were used. A maximum of two cardiomyocytes per heart were used in any experimental group.
Individual cardiomyocytes were placed in a chamber on an inverted microscope and superfused with buffer containing (mM): 126 NaCl, 20 NaHCO3, 0.9 NaH2PO4, 4 KCl, 0.5 MgSO4, 5.5 glucose and 1.8 CaCl2 (pH 7.4, 37°C, 95% O2, 5% CO2). After equilibration (20 min), cells were superfused with buffer formulated to simulate the cardioplegia we use in the clinic containing (mM): 118 Na+, 18 K+, 5 Mg+, 1.0 Ca2+, and SEA0400 (1 μM, Taisho Pharmaceutical Co, Ltd., Tokyo, Japan, n = 25) or its vehicle, DMSO (0.1%, n = 24). The O2 scavenger sodium dithionite (5 mM) was added to the cardioplegia and it was bubbled with 90% N2 and 10% CO2 and this gas was directed over the chamber during the cardioplegia period to reduce the extracellular pO2 to ≈ 12 mmHG and pH was reduced to 6.8 to simulate conditions at the tissue level during cardioplegic arrest [20, 21]. Cardioplegia used in the isolated cell studies did not contain blood. Following cardioplegic arrest (45 min), cardiomyocytes were reperfused with oxygenated buffer. Ischemic control cells were exposed to hypoxia but not the cardioplegia solution. Myocytes were field stimulated (1 Hz) throughout the protocol. Cell death was identified visually when cells lost the typical cardiomyocyte morphology and rounded up into a ball, and was confirmed by trypan blue staining.
Measurement of intracellular Ca2+ and cardiomyocyte contraction amplitude
At the start of the experimental protocol, cells were loaded with the Ca2+ sensitive dye fura-2 AM (5 μM, 20 min, RT, Invitrogen, Burlington, Canada) and intracellular Ca2+ was measured by whole cell photometry (DeltaRam, Photon Technology International, Birmingham, NJ) according to previously described techniques [18, 19]. The emission ratio at 510 nm, during alternate excitation at 340 and 380 nm was used to determine intracellular Ca2+ concentrations. Background fluorescence was determined at each excitation wavelength and subtracted from the recordings. Emission ratios were converted to intracellular Ca2+ concentrations using an in-vitro calibration curve. Unloaded cell shortening was measured with a video edge detector (Crescent Electronics, Sandy, UT). Ten second trains of contractions were averaged and measured with Clampfit 8.2 (Molecular Devices, Sunnyvale, CA). Contraction amplitude is the difference between systolic and diastolic cell length. All values were normalized to the time point immediately prior to ischemia.
Cardioplegic arrest – isolated hearts
Rats were assigned to their experimental group before the experiment started. Hearts were attached by the aorta to a Langendorff apparatus (AD Instruments Inc., Colorado Springs, CO). Shed blood was collected for mixing with the cardioplegia solution. Electrocardiogram and aortic pressure were recorded continually. Coronary flow was measured using a transit time ultrasound probe (Transonic Systems Inc., Ithica, NY) on the inflow cannula, and coronary vascular resistance was calculated by dividing aortic pressure by flow. A custom non-compliant balloon tipped catheter was placed through the mitral valve into the LV cavity. Hearts were perfused with oxygenated Kreb’s solution (37ºC, 10 mL/min, [Ca2+] = 2.5 mM) in a heated chamber (20 min) then baseline LV function data were obtained (see below). Perfusion was interrupted and cardioplegia delivered through the aorta (20 mL/kg body weight). Cardioplegia was prepared by mixing base solution used in clinical practice (76.1 mM KCl, 20.2 mM MgSO4, 86.5 mM NaHCO3 in 1 L 5% Dextrose/0.225% NaCl) with the shed blood in a ratio of 4 parts blood to 1 part base solution resulting in a final cardioplegia solution with an approximate ionic composition of (mM): 136 Na+, 19 K+, 5 Mg2+, and 1 Ca2+. SEA0400 (1 μM, n = 7) or its vehicle (DMSO 0.1%, n = 6) was added to the cardioplegia, which was cooled on an ice bath (≈4°C) and bubbled with oxygen prior to delivery. After cardioplegia delivery, the heart was exposed to ambient room temperature (22 ± 0.2°C) for the duration of the ischemic period (45 min). After reperfusion with 37°C oxygenated Kreb’s solution (20 min), LV function data were again collected.
Load independent indices were derived from LV pressure recordings during incremental inflation of the intra-ventricular balloon in 25 μL steps to a maximum of 200 μL according to the method described by Li et al. . End-systolic and end-diastolic pressures were plotted against LV volume and the pressure-volume relationships estimated by linear regression. LV work, a load-independent index of ventricular function, was estimated by the area between the systolic and diastolic pressure-volume relationships . LV developed pressure, dP/dt+, and dP/dt- were also recorded.
The electrocardiogram was recorded continuously throughout the experiment using epicardial electrodes. The start time, end time, and incidence of ventricular arrhythmias (tachycardia and fibrillation) during reperfusion were quantified.
Myocardial and mitochondrial injury
Myocardial damage was assessed by measuring troponin I release from timed collections of coronary sinus effluent during reperfusion (ELISA assay, Life Diagnostics, West Chester, PA). Mitochondria were isolated from LV muscle and a blinded observer categorized and counted intact, swollen, and disrupted mitochondria from random electron photomicrographs from each heart [23, 24].
Data are presented as mean ± SEM. Tests for statistical significance included Kaplan-Meier log-rank survival analysis, unpaired t-test, and one-way and two-way repeated measures ANOVA with post-hoc comparisons by Newman-Keuls and Holmes-Sidak method respectively.