| BackgroundSudden cardiac death is responsible for significant morbidity and mortality.Ventricular fibrillation (VF) is one important cause of sudden cardiac death. Among VF treatment maneuvers, rescue timing is the most critical factor which can indirectly affect the survival rate, because there is approximately a 10% decrease in survival rate with every minute delayed. Recent study demonstrated that not only the earlier performed resuscitation could increase the survival rate, but also the sequence of therapeutic maneuvers could influence the results of resuscitation from VF. Weisfeldt et al. proposed a 3-phase, time-sensitive model for cardiac arrest (CA) featuring VF which included electrical phase (approximately the first 4 min following CA), circulation phase (approximately 4 to 10 min following CA) and metabolic phase (approximately 10 min after CA) The model suggested that the optimal treatment of VF is phase-specific:immediate defibrillation in the electrical phase would rescue more life, while cardiopulmonary resuscitation (CPR) prior to shock in the circulation phase would be an optimal maneuver. The effectiveness of both immediate defibrillation and CPR followed by defibrillation decreases rapidly and the prognosis is poor.Cobb et al. reported that CPR prior to shock therapy could get better results than immediate defibrillation in prolonged VF (5-10 min). Other studies have confirmed the effectiveness of this model. The pathophysiologic mechanism underlying these observations is unknown. It is supposed that the delayed defibrillation maybe in favor of providing some limited circulation of blood with partial oxygen and nutrient substance, or washout of some deleterious metabolic factors accumulated during ischemia.While in the metablic phase,the accumulation of toxic metabolites, depletion of high-energy phosphate stores, the initiation of coagulation cascades and reperfusion injury may all contribute to the pathophysiology and response to therapy observed during this phase.Beyersdorf treated 14 patients who underwent in-hospital cardiac arrest with intractable VF.After failed standard advanced cardiopulmonary life support therapy,they received cardiopulmonary bypass and alteration of blood composition by using metabolic therapies including a solution with low calcium,increased potassium,and high dextrose.After that,11 of 14 patients survived to hospital discharge.That indicated it was important for us to understand the pathophysiologic mechanism of VF and choose the optimal therapic strategy, while our previous study showed that in the 4-minute VF canine model of cardiac arrest, initial defibrillation or initial chest compression does not affect CPR result.However, there is still no direct evidence supporting step-change theory of the pathophysiological process during VF. Therefore, the present study aimed to examine the whole-blood response and hemodynamics during prolonged VF to investigate whether the physiologic and pathological process presents stepwise changes during prolonged VF.Although initial return of spontaneous circulation (ROSC) is achieved in about 30 to 40% of cases, only 10 to 30% of the patients will be discharged with good outcome. Cardiac dysfunction after CPR has been proved one of the most important reasons for the death in the early period of successful CPR. There are many studies about the mechanisms of cardiac dysfunction after CPR, VF could be understood as ischemia and CPR is equal to the process of reperfusion, characterized as the release of oxygen radicals, cell adhesion molecules including selectins, immunoglobulin family, a variety of inflammatory cytokines such as IL-6, the involvement of calcium overload and so on.Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases, and they regulate the extracellular matrix turnover in a balance with tissue inhibitors of metalloproteinase (TIMPs). They are best known for their roles played in the chronic diseases such as ventricular remodeling of acute myocardial infarction, chronic heart failure, hypertension, occurrence or maintenance of atrial fibrillation and dilated cardiomyopathy. Besides the participation in the synthesis and degradation of extracellular matrix in the chronic cardiovascular diseases, recently, a number of studies demonstrated that TIMPs and MMPs were also involved in acute pathological or physiological processes such as platelet aggregation, the regulation of vascular tone, inflammation and ischemia-reperfusion injury.The imbalance between TIMPs and MMPs in acute pathological conditions could be also observed. Lalu et al. first proposed that there was a rapid (within 10 min) increase in MMP-2, MMP-9 and collagenase activities in the myocardium that was accompanied by a decrease in TIMP-1 and the enhanced MMP-2 and MMP-9 activities and the loss of TIMP-1 correlated with the impaired left ventricular function during the process of i schemia-reperfusion.The present study aimed to determine the changes of physiological and biochemical indexes based on a three-phase model for prolonged ventricular fibrillation in canine and the changes in myocardial TIMP-1 and MMP-9 in the setting of different-duration VF episodes and after CPR and whether the imbalance between TIMP-1 and MMP-9 correlated with the post-resuscitation cardiac dysfunction.ObjectiveVentricular fibrillation (VF) is one important cause of sudden cardiac death. This study aimed to (a) investigate whether physiologic and pathological process present stepwise changes during prolonged VF. (b) there was an imbalance between matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) after CPR in a canine model of prolonged ventricular fibrillation (VF); (c) with the duration of VF, the degree of the imbalance would be greater; and (d) whether there was a relationship between the level of MMP-9 or TIMP-1 and the cardiac function.MethodsThirty dogs (13.5-18.5kg) were anesthetized with pentobarbital-Na (30 mg/kg intravenous, repetition when necessary). Then dogs were placed in the supine position and restrained at the four extremities. Three surface electrodes were placed under three limbs separately to correspond to standard lead II ECG. The right femoral artery and left jugular veins were cannulated. Two 6-F shelters were seperatly positioned in the artery and vein.Two 6-F catheters were positioned in the left ventricle and the right atrium so that we can get the blood samples from the cardiac cave. Both catheters positioned into the root of the ascending aorta and the right atrium were connected to pressure transducers attached to a PROEP recording system by three direct links through which the blood sample can be obtained at the same catheter after the positions were confirmed by the presence of typical pressure waves and verified under the DSA.A bipolar fibrillating electrode was placed subcutaneously across the apex of the heart for the delivery of 30V of accurrent at 60 Hz to induce ventricular fibrillation, VF was confirmed by the characteristic ECG waveform and the precipitous fall in aortic pressure (<30 mmHg),and continued for 12 minutes.Hemodynamic variablesAortic pressure (AOP), right atrial pressure (RAP) were continuously recorded but temporarily paused when the blood samples were obtain from the catheter, and analyzed by the commercially available software (Chart for Windows v5.5, AD Instruments, Castle Hill, Australia).coronary perfusion pressure (CPP) was calculated as the difference between the simultaneously measured aortic and right atrial pressure. Biochemical and coagulation variablesThe blood samples were obtained from the 6-F catheters in the artery and vein at the beginning of the VF, and every minites after the VF until 12 mintues of VF. Arterial blood gases were measured using GEM Premier 3000-blood gas analyzer (IL, America).the concentration of potassium、sodium、calcium、chlorine, serum myocardial zymogram, myocardial troponin I,were recorded by vitros250 chemistry system (JNJ America). Samples for detecting coagulation factors were collected into citrated tubes.After immediately centrifuged at 1,500 g for 10 min,the plasma was stored at -80℃,until analysis. Fibrinogen concentration, antithrombin III(AT)and D-dimer concentration were detected by using STA-R coagulation analyzer (Diagnostica Stago, France).Transthoracic echocardiographic (TTE) studyAll animals before VF and the animals attaining ROSC were examined by TTE (PHILIPS 7500 with a 2.5-3.5 MHz transducer) to measure the left atrium dimension (LAD), left ventricular diastolic dimensions (LVDd) and left ventricular ejection fraction (LVEF), respectively in a parasternal long-axis and M-mode view.Western blot and immunohistochemistryAll the cells were collected, extracted total protein, respectively by SDS-poly acrylamide gel electrophoresis separation, membrane protein, mark, color and other steps, to detect the expression of TIMP-1 and MMP-9,then were analyzed.Results:In this study we analyzed 30 dogs, all of which completed the experimental procedures. The results showed that during 12 min prolonged VF, hemodynamic variables, biochemical factors and coagulation variables presented stepwise changes.In the first 5 min of VF, AP continuously decreased while RAP increased in the first minute of VF and then decreased in the last 4 minutes.The pressure of cornary artery (CPP) rapidly fell to zero. The concentration of fibrinogen decreased after 4 minutes of VF (2.56 mg/ml vs.2.32 mg/ml, P=0.031). Meanwhile, PaO2, PaCO2 and lactate showed no significant differences in the first 5 minutes of VF. After 5 minutes of VF, the change of CPP became slow, and PaO2 lactate showed significant differences compared to the values before VF.In the period before 8 minutes, the arterial blood pH kept on decreasing, while AT concentration did not decrease until 8 min of VF (105.08 ug/ml vs.89.69 ug/ml, P=0.027). Lactate showed significant differences compared to the values before VF. However, the D-dimer concentration did not increase until 12 minutes of VF (0.291 ug/ml vs.1.129 ug/ml, P=0.035,). In addition, the concentration of potassium sodium、 calcium% chlorine, serum myocardial zymogram, myocardial troponin I, showed no significant changes during the VF.Compared with 8-min VF group, the LAD was much larger and the LVEF was much lower in 12-min VF group (P<0.05). Compared with sham controls, dogs under VF and CPR showed significantly decreased level of TIMP-1 (P<0.05), and with the duration of VF, the level of TIMP-1 declined (P<0.05). The level of MMP-9 did not achieve statistical significance in the three groups (P>0.05), however, they were higher in VF and longer-duration VF groups. The ratios of TIMP-1/MMP-9 were lower in VF groups (P<0.05). There was a negative correlation between TIMP-1 and the LAD and LVDd (r=-0.83, r=-0.96, respectively, P<0.05), and a positive correlation between TIMP-1 and the LVEF (r=0.85, P<0.05)Conclusion1. The pathological process of long process ventricular fibrillation presentes phased changes, gradually exists hemodynamic disorders, acid-base balance disorders, coagulation and fibrinolytic.2. Expression imbalance of TIMP-1 and MMP-9 exists in recovery after ventricular fibrillation, and this imbalance may be associated with reduced cardiac function.Background:Ventricular fibrillation (VF) is one of the most common arrhythmias leading to sudden cardiac death. Asynchronous defibrillation, as an effective treatment of ventricular fibrillation at present, however, leads to a low success rate in VF arrest patients. With prolonged ventricular fibrillation time, myocardial ischemia, hypoxia and acidosis aggravate gradually, the number and the thresholds of defibrillation are likely to increase, but the results of high energy defibrillation are not effective.Gap junctions, which constituted by different types of connexin, are the most normal communication between cells. Under pathological conditions, the contents of connexin in the myocardial tissue are changed in size, number, density, spatial distribution, so that the potential between myocardial cells loss coupling, the conduction velocity slows down, the anisotropy ratio changes, the impulse conduction between adjacent cells are blocked, those may lead to the cardiac electrical repolarization become inconsistent, which is easy to form turn-back, providing matrix for various types of arrhythmias.ZP123, a kind of connexin modifier, is a novel anti-arrhythmic peptide, which plays an anti-arrhythmic effect by adjusting the expression of connexin. Studies have demonstrated that after pretreated by ZP123, myocardial cells have prevented coupling loss of the connexin induced by acidosis and restored normal electrical conduction. ZP123 does not affect the cell membrane ion channels, which may lead to the side effect of arrhythmia, so in recent years it has became a hot research. However, there are little current studies for ZP123 about the effect on ventricular fibrillation.Our previous studies for 4 minutes ventricular fibrillation in rabbit model confirmed that ZP123 could reduce ventricular fibrillation defibrillation threshold, however, on long-term process of ventricular fibrillation, the effect of ZP123 on different stages of ventricular fibrillation is needed to further studies. According to the previous clinical and biochemical stage of VF, this study assessed the role of ZP123 on different stages of ventricular fibrillation.Objectives:This study is to observe the changes of connexin 43 (Cx43) after CPR on ventricular-fibrillation (VF) dogs, the effects of rotigaptide (ZP123) on myocardial connexin 43 and the threshold of defibrillation.Methods:All 60 dogs of either sex (13.5-18.5kg) were fasted overnight. Anesthesia was induced with ketamine (lOmg/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. Dogs under anesthesia were placed on the experimental table in the supine position and restrained at the four extremities. The trachea was incubated 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 (SaO2) sensor were secured and configured to an automated external defibrillator for electrocardiogram signals and SaO2 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 dogtail 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, dogs are assigned in five groups in a randomized fashion:Group 1. ZP123 sroup (8min)(ZP123 1μg/ke bolus+10μg/kg/h pumped by a micro pump for 30min); Group 2,ZP123 group (12min) (the same dose as Group 1), Group 3,routine CPR group8min (the same dose as Group 2 but only saline); Group 4, routine CPR group12min (the same dose as Group3);sham group, (treated with sham operation). Drugs and saline were used 30min before VF started. Researchers were blinded to the drug given. VF was induced in routine CPR 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 (<30 mmHg). Dogs were stopped from ventilation immediately after VF and left untreated for 8 or 12minutes. 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 was checked. Animals with successful restoration of spontaneous circulation (ROSC, defined as a recognized ECG with an arterial systolic pressure of> 80mmHg sustained for≥1 min) were treated with advanced life support and observed for 1 hour. If ROSC was not achieved, an immediate defibrillation of 70J bi-phase was delivered, then another 2 min of CPR was immediately restarted and defibrillation energy was increased to 100J bi-phase, 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. The CPR would be given up if the whole course is beyond 30minutes without achieved successful ROSC. Animals without successful 1 hr survival were excluded from immunofiuorescence and Western blot analysis. Those survived for 1 hr and dogs in sham group were executed with an IV injection 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 4 μm 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. P-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 to β-actin (Cx43/β-actin)。Statistical analysis:All data were expressed as mean ± SD. Numerical variables were analyzed by one-way ANOVA. Statistical analysis involved use of SPSS 17.0 (SPSS Inc., Chicago, IL). A two-tailed P< 0.05 was considered statistically significant.Results:The five 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). Eight of ten dogs and six of ten dogs demonstrated ROSC and 1 hour survival for ZP123 group8min and routine CPR group, respectively. Defibrillation energy was the sum of all shocks required. The average defibrillation energy in ZP123 group was much lower than control group (427.2±126.4J vs. 560.7±128.9J,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 (1.31±0.07 vs.1.86±0.06; 14800±1772 vs.19600±2160,, P<0.05, respectively). The same values in ZP123 group were much higher than control group (1.72±0.05 vs 1.31±0.07; 17200±1903vs.14800±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).Conclusions:1. As VF prolonged, myocardial intercalated disks became progressively discontinuous and the expression of Cx43 decreased, with disordered distribution.2. Pretreatment with ZP123 for 30minutes could reverse the decreasing expression of Cx43 in dogs with the prolonged VF3. Pretreatment with ZP123 could decrease defibrillation threshold in dogs with the prolonged VF, meanwhile increase the success rate of defibrillation.4. ZP123 can improve the recovery rate of 8min ventricular fibrillation, this result may be associated with the reducing defibrillation energy and the improving the success rate of defibrillation. |
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