Animals
All animal protocols were approved by the Wenzhou Medical University Animal Care and Use Committee (wydw 2015–0121, Zhejiang, China). Adult male Sprague-Dawley rats (SYXK 2015–0150), weighing between 320 and 380 g were provided by the Animal Center of Wenzhou Medical University. The care and handling of animals were in accordance with National Institutes of Health guidelines. All animal’s body were incinerated after the study by the Animal Center of Wenzhou Medical University. A completed ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines checklist is included in Checklist.
Drugs
Bupivacaine hydrochloride (0.5%, Sigma-Aldrich Co., St. Louis, MO, P code: 101524503 B5274-5G), Dexmedetomidine (Sigma-Aldrich Co., St. Louis, MO, P code: 12815 SML0956-10MG) and Yohimbine (Sigma-Aldrich Co., St. Louis, MO, P code: 101509939 Y3125-1G) were used.
Preparation of isolated hearts
The isolated perfused, nonrecirculating Langendorff rat heart preparation was used in our study, as described previously [11]. Rats were anesthetized by the intraperitoneal injection of 80 mg/kg ketamine hydrochloride and 12 mg/kg xylazine, then 1000 U/kg heparin to prevent the formation of intracoronary microthrombi. The rats were euthanized by cervical dislocation, then the hearts were rapidly excised and perfused via the coronary arteries by tying the aorta onto a cannula (ML870B2, AD Instruments, Australia). The constant perfusion pressure was 80 mmHg, and a modified Krebs-Henseleit buffer (KHB) was used and described as follows: NaCl 118 mmol/L, KCl 4.7 mmol/L, MgSO4 1.2 mmol/L, KH2PO4 1.2 mmol/L, NaHCO3 25.0 mmol/L, CaCl2 2.5 mmol/L, glucose 10 mmol/L. The solution was continuously bubbled with 95% O2 and 5% CO2, and pH was maintained at 7.40 ± 0.05. The left ventricular pressure was continuously monitored by a latex balloon placed in the left ventricle. Saline was intermittently injected into the balloon to maintain the left ventricular end-diastolic pressure at 4–10 mmHg. Electrocaridiograph (ECG) electrodes were consistently placed in a “leadII” position. All data were collected using a PowerLab biological signal processing and analysis system (ML870, Australia Ad Instruments) and the Chart 5.5.6 biological signal recording software. The experimental protocol was started after the 10 min of KHB as the steady-state baseline conditions.
Experimental protocol
Forty isolated rat hearts were mounted on the Langendorff system and then randomly assigned to 5 groups (Fig. 1): Group Con, Group Dex, Group Bupi, Group Bupi + Dex and Group Bupi + Dex + Yoh (n = 8). The KHB was continuously perfused into the hearts in all groups until the end. Experimental perfusion was started according to the assigned group after steady state was reached. In group Con, only KHB was perfused; In group Dex, dexmedetomidine and KHB were perfused; In group Bupi, KHB was perfused 25 min and then 50 μmol/L bupivacaine was added, whereas in group Bupi + Dex, 10 nmol/L dexmedetomidine was added after 5 min of KHB perfusion, and 50 μmol/L bupivacaine was added 20 min later. Besides, in group Bupi + Dex + Yoh, 1 μmol/L yohimbine was added immediately after the steady state, then 10 nmol/L dexmedetomidine was added 5 min later, and 50 μmol/L bupivacaine was added after 20 min later. In all groups but the group Con and group Dex, the experimental perfusion was sustained until asystole, and group Con and group Dex were perfused for 35 min. The recorded data were evaluated and the following times were documented for each heart in all groups: the time of the first premature ventricular contraction accompanied by abnormal systole on the pressure trace after the bupivacaine perfusion (Tarrhythmia, Time to the first arrhythmia); the time from the initiation of 50 μmol/L bupivacaine infusion to asystole (> 1 min) (Tasystole, Time to asystole); time to 25, 50 and 75% reductions in heart rate (HR) and rate-pressure product (RPP = HR×(systolic pressure - diastolic pressure)) relative to baseline. Baseline values of hemodynamic were obtained after the 10 min with only KHB perfusion.
Cardiac tissue bupivacaine content [12]
After asystole, heart apex was cut immediately and stored in liquid nitrogen. Frozen hearts were grinded in a precooling glass tissue grinder with ice and homogenized with 0.4 mol/L perchloric acid by 10 mL/g. Precipitated proteins were separated by centrifugation at 4000 g for 15 min, and the supernatant was neutralized with 2 mol/L KOH to adjust pH at 6.0–7.0 by pH meter. Samples were then centrifuged at 4000 g for another 15 min and the supernatant was filtered through a 0.22 μm filter to determine the sample. The above operation was completed at 4 °C. The concentration of bupivacaine in cardiac tissue was determined by HPLC-MS/MS (High performance liquid chromatography-mass spectrometry/mass spectrometry).
Statistical analysis
The power analysis was based on our preliminary study which is mentioned above using the Power Sample Size (PASS 11.0) software program. We compared Tarrhythmia among the various treatment groups. In our preliminary study, we totally used 9 rats: 3 rats in each of 3 groups. The means ± standard deviation(SD) of Tarrhythmia in Bupi, Bupi + Dex and Bupi + Dex + Yoh groups were 161.0 ± 4.9, 283.7 ± 15.2, and 283.3 ± 5.9, respectively. Using a two-tailed type one error at 5% and type two error of 10%(α = 0.05, β = 0.1), the sample size of 6 per group was obtained. Balancing this fact with the desire to reduce the use of animals, we enrolled 8 rats per group for the study. At this point in the development of an assay sample sizing should only be viewed as a guide because the estimates of both the effect size and associated variability are approximate and come with much uncertainty attached to them.
SPSS (version 19.0, Chicago, IL) was used to carry out the computations. The measurement data were tested for normality using the Shapiro-Wilk test, normally distributed data were presented as the means ± SD. One-way ANOVA was used to evaluate differences among groups, and the Tukey test was used as a post hoc test when significance was achieved. Statistical significance was considered as P < 0.05.