| Background:Ventricular fibrillation (VF) is one of the leading causes of sudden cardiac death. During VF, the regular ventricular contraction is depressed, instead of disordered and non-synchronized contraction. It has been proved that there is a relationship between the occurrence, development of VF and electrical, structral remodeling of gap junctions (GJs). Connexin 43 (Cx43) is a connexin that formulate the gap junctions, which are important channels for electrical impulse conducting and signal molecular transmitting between two adjacent cells. Rotigaptide (ZP123), as a novel antiarrhythmic peptide, can enhancing cell-to-cell coupling.Objectives:This study is to observe changes of connexin 43 (Cx43) after CPR on ventricular-fibrillation (VF) swines and the effects of rotigaptide (ZP123) on myocardial Connexin 43 (Cx43)Methods:All 30 pigs of either sex (33.5-38kg) were fasted overnight. Anesthesia was induced with ketamine (10mg/kg, IH), and maintained by pentobarbital- Na (30 mg/kg IV). A continuous infusion of Ringer's solution was given at 3ml/kg/h throughout the experiment. Pigs under anesthesia were placed on the experimental table in the supine position and restrained at the four extremities. The trachea was intubated with a 5.0 cuffed tracheal tube which was then connected to a ventilator. FiO2 was controlled between 50% and 80%. The tidal volume, ventilator rate and inspiration:expiration ratio was 10-15ml/kg,16-20 breaths per minute and 1:1.5-2.0 respectively. Three surface electrodes and arterial oxygen saturation (Sa02) sensor were secured and configured to an automated external defibrillator for electrocardiogram signals and Sa02 monitoring. QT intervals, heart rateand R-R intervals were measured at baseline and after resuscitation. The corrected QT interval (QTc) was calculated by Bazett's formula. Bilateral femoral veins and right femoral artery were punctured on the basis of modified Seldinger's technique. One 6-F pigtail catheter was positioned in the intrathoracic ascending aorta and another in the right atrium. Catheter positions were confirmed by the presence of typical pressure waves and radiograms of the chest. Both catheters were connected to pressure transducers attached to a multipurpose polygraph, by which aortic and right atrial pressures were monitored and recorded continuously. Coronary perfusion pressure (CPP) was calculated by subtracting right atrial diastolic pressure from aortic diastolic pressure. The remaining cannulated femoral vein was used for drug infusion.After baseline measurements, pigs (10 of each group) were assigned in three groups in a randomized fashion:Group 1, ZP123 group (ZP123 1μg/kg bolus+10μg/kg/h pumped by a micro pump for 15min); Group 2, control group (the same dose as Group 2 but only saline); Group 3, sham group, (treated with sham operation). Drugs and saline were used 15min before VF started. Researchers were blinded to the drug given. VF was induced in control group and ZP123 group with an 80-v AC transthoracic shock of 5 seconds using subcutaneous needles. Successfully induced VF was verified by the characteristic ECG waveform and the precipitous fall in aortic pressure (<20 mmHg). Pigs were stopped from ventilation immediately after VF and left untreated for 8 minutes. Then external chest compressions and ventilation with 100% oxygen started. The rate of compression was 100/min, at a depth of 4-6 cm.2 ventilations were delivered after every 30 compressions without interruption of compression. After 2 min of CPR, the rhythm and pulse were checked. Animals with successful restoration of spontaneous circulation (ROSC, defined as a recognized ECG with an arterial systolic pressure of≥80mmHg sustained for≥1min) were treated with advanced life support and observed for 1 hour. If ROSC was not achieved, an immediate defibrillation of 70J biphase was delivered, then another 2 min of CPR was immediately restarted and defibrillation energy was increased to 100J biphase, if not achieved ROSC, then the third CPR was immediately started but the defibrillation energy was increased to 150J, The third CPR was continued until ROSC achieved or the sixth CPR. The CPR would be given up if sixth CPR without achieved successful ROSC. Animals without successful 1 hr survival were excluded from immunofluorescence and Western blot analysis. Those survived for 1 hr and pigs in sham group were euthanized with anⅣinjection of KCL. Thoracotomy was performed immediately and the heart was removed quickly. Myocardial pieces about 0.5 cm3 were cut along the long axis of each left ventricular free wall for immunofluorescence and Western blot analysis.Immunofluorescence:Left ventricular preparations were fixed in 4% paraformaldehyde for 24 hours. Tissue samples were embedded with paraffin, cut into 4μm sections, mounted on gelatin-coated slides, then dewaxed, and rehydrated with graded alcohols. The slides were microwaved in boiling 0.01 M sodium citrate buffer for 15 min to enhance specific immunostaining. For blocking, a goat serum/PBS (1:20) solution was added on the slides for 20 min at room temperature. Then primary antibody (1:100 dilution; Millipore) in PBS was added and incubated overnight at 4℃. Finally, the slides were incubated with a fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse secondary antibody (1:500 dilution) for 30min at 37℃. Samples were examined using a laser scanning confocal microscope. Highintensity Cx43 signal was measured and analyzed with Image-Pro Plus 6.0 software.Western blot:Protein samples were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membrane.β-actin was used as a control for equal protein loading. The PVDF membrane was incubated with the following liquids:5% nonfat milk in PBS containing 0.1% Tween 20 (PBS-T) at room temperature for 2 hours, primary antibody (1:1000 dilution; Milipore) overnight at 4℃, horseradish peroxidase conjugated goat anti-mouse secondary antibody (1:10000 dilution) for 2 hours. The density of protein band was quantified with Quantity One. The amount of Cx43 was defined as the band density corresponding to Cx43 protein normalized toβ-actin (Cx43/β-actin)。Electron microscopy:each heart section was fixed in 3% glutaraldehyde, dehydrated in ethanol and embedded in Spurr's low-viscosity epoxy resin. Ultrathin (50100 nm) sections were cut for electron microscopy after the resin had been polymerized. The intracellular membranous ultrastructures of the left ventricular myocardium in each group were showed by electron microscopy, including the mitochondria, intercalated disk, sarcoplasmic reticulum, were observed.Statistical analysis:All data were expressed as mean±SD. Numerical variables were analyzed by one-way ANOVA. Statistical analysis involved use of SPSS 16.0 (SPSS Inc., Chicago, IL). A two-tailed P< 0.05 was considered statistically significant. Results:The three groups did not differ in baseline characteristics. There were no significant difference in hemodynamic characteristics and QTc between control group and ZP123 group after 30 min of CPR (P>0.05), both of which were differed from sham group (P<0.05). Six of ten pigs and five of ten pigs demonstrated ROSC and 1 hour survival for ZP123 group and control group, respectively. Defibrillation energy was the sum of all shocks required. The average defibrillation energy in ZP123 group was much lower than control group (325±130 J vs.485±149 J, P<0.05).Immunofluorescence analysis results:Immunofluorescence signals for Cx43 in sham group were strong and regularly distributed. In control group, Cx43 signals were weak and distributed in heterogeneity, while in ZP123 group, Cx43 signals were enhanced and their distribution were much more ordered. The percentage area and the integral optical density (IOD) of Cx43 in control group were significantly decreased compared with sham group (1.01±0.07 vs.1.46±0.06; 18800±1772 vs.29600±2160, P<0.05, respectively). The same values in ZP123 group were much higher than control group (1.32±0.05vs.1.01±0.07; 27200±1930 vs.18800±1772, P<0.05, respectively).Western Blot analysis results:All three groups were under qualitative and quantitative immunoblot analysis for Cx43. Statistic analysis revealed that the amount of total Cx43 in control group was significantly decreased compared with ZP123 group and sham group (0.75±0.07 vs.0.95±0.06, P<0.05; 0.75±0.07 vs.1.07±0.04, P<0.05).Electron microscopy results:Representative transmission electron micrograph of the left ventricle in ZP123 group (х7,500). Moderate enlargement and disarray of myocardial fibers were seen. Little disintegrated crystals and almost normal morphology of mitochondria were seen. The intercalated disk was relatively intact. Representative transmission electron micrograph of the left ventricle in control group (×7,500). Myocardial fibers were enlarged and disarrayed and mitochondria were displayed in an anarchic pattern with disintegrated crystals. The intercalated disk lost its continuity and dispersively distributed. Representative transmission electron micrograph of the left ventricle in sham group (×7,500). Normal mitochondria were shown and myocardial fibers were regularly arrayed. The intercalated disks were continuous and intact between two adjacent cardiomyocytes.Conclusions:1. As VF progressed, myocardial intercalated disks became discontinuous and the expression of Cx43 decreased, with disordered distribution.2. Pretreatment with ZP123 could reverse the decreasing expression of Cx43.3. Pretreatment with ZP123 could decrease defibrillation energy as to reduce the myocardial injury. |
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