Alterations And Roles Of Connexin43, MMP-2and TIMP-2during Ventricular Fibrillation In Canine

BackgroudVentricular fibrillation (VF) is one of the most common reasons for sudden cardiac death. In recent years, along with the development of electrophysiological mapping technique and in-depth research on the exciting features of heart fibrillation, the mechanism of VF has been great progess. Re-entry is still considered to be the most important mechanism for tachyarrhythmias matintain. Conditions for re-entry include:①the objective existence of anatomical re-entry ring;②unidirectional conduction block;③inconsistence of the conduction velocity and refractory period. In these3basic conditions, the refractory period is determined by the electrical activity of single cells. The electrical activity spread from a single cell to its neighbor cells, thereby causing the synchronization of cardiac tissue excitability, is determined by the electrical coupling which completed through gap junctions (GJ) between cells. GJ is important to the passive electrical activity of myocardium tissues.There are two connection types between GJ:end-to-end and side-to-side.The structural basis for myocardial anisotropy is depend on GJ connections. Normal myocardium is fusiform arrangement, and most of GJs are connecting in end-to-end type at the long axis of cardiomyocytes, the lower junction resistance make the ionic current through ease and electrical excitement conduct fast. But at the short axis direction, where GJs connecting in side-to-side type, electrical excitement conduct slow and spread in "之" shape, cause re-entry easy to be formed. In theory, the increase of side-to-side proportion help to produce and maintain of arrhythmia.Connexins (Cx) are subunits of intercellular GJ. Reduce the expression level and distribution of Cx abnormality can slow down the overall cardiac conduction velocity, changes in conduction anisotropy, thereby forming a re-entrant loop of the anatomical basis induced arrhythmia. In adult ventricle, GJ contain only Cx43and mainly distributed in the intercalated disk. Our previous studies have demonstrated that GJ remodeling caused by decreased expression and myocardial derangement distribution of Cx43were involved in the occurrence and maintenance of VF, and gap junction enhancer ZP123can reduce or reverse the degradation of Cx43and make distribution arrangement trend in uniform, thereby reducing energy defibrillation and make easy to cardioversion in VF.Cx43are phosphoprotein, the function of GJ channel is also affected by the phosphorylation of Cx43. In cardiomyocytes, only phosphorylated Cx43(p-Cx43) can constitute a functional GJ and dephosphorylated Cx43does not have this function. So, whether the change of phosphorylated Cx43occurred in the process and cardioversion of VF? And what kind of influence maintained by these changes for VF? Does the effection of ZP123to enhance the role of gap junctions are also raised the level of phosphorylation of Cx43? These problems did not explore in depth in our previous study, and is also no relevant reports.It was proposed that homogeneous conduction of electrical activity not only relies on cardiomyocytes integration but can be also affected by extracellular matrix (ECM) among the cardiomyocytes. Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases, and they regulate the ECM turnover in a balance with tissue inhibitors of metalloproteinase (TIMPs). This suggests that myocardial MMPs/TIMPs imbalance may be involved in the occurrence and maintenance of VF. But how exactly MMPs and TIMPs changes in the process of VF? And what roles they play in the occurrence and prognosis of VF? These issues are not clear, and are very worthy of study and exploration. Angiotensin Ⅱ (Ang Ⅱ) is the main active ingredient in the rennin-angiotensin system(RAS), which confirmed extensively involved in hypertension, left ventricular hypertrophy, heart failure and many other cardiovascular diseases in previous findings.Recent studies have also found that, Ang II participte the malignant ventricular arrhythmias by affecting left ventricular electrophysiological indicators.And traditional antihypertensive drugs angiotensin converting enzyme inhibitors(ACEI) and angiotensin receptor blockers(ARB) can reduce the incidence of malignant ventricular arrhythmias by inhibiting the biological effects of Ang Ⅱ.Currently the specific mechanism of Ang Ⅱ induced ventricular arrhythmias is still not clear,but studies have reported, Ang Ⅱ can reduce the expression of Cx43and make the electrical conduction between myocardial cells slow, suggesting that GJ remodeling induced by Ang Ⅱ lead to myocardial electrophysiological abnormalities. Is this the protein molecular mechanism that Ang Ⅱ involved in VF?Thus, the present study was to explore the above problems by establishing an animal model of VF.Objectives1. To observe the dynamic changes of the expression, distribution and phosphory lation levels of Cx43in myocardial cell membrane in the process of maintaining and cardioversion of VF.2. To observe the expression of MMP-2/TIMP-2in the process of maintaining and cardioversion of VF, and find the relationship between MMP-2/TIMP-2and the prognosis of VF.3. To observe the effect of gap junction enhancer ZP123to the expression, distribution and phosphory lation levels of Cx43.4. To evaluate the effect of ZP123on cardioversion of VF through analyzing its impact on the defibrillation energy, defibrillation success rete, ROSC and survival rates.5. To explore the protein molecular mechanisms of Ang Ⅱ involving in VF.Methods: Part1. Dynamic alterations of Cx43、p-Cx43, MMP-2and TIMP-2in cardiomyocytes during VF1. Animal preparation(1) Thirty-two mongreal dogs of either sex weighing11.5～17kg were anesthetized with pentobarbital-Na (30mg/kg intravenous, repetition when necessary). After anesthesia, dogs were placed in the supine position on the experimental table and restrained at the four extremities. Three surface electrodes were placed under three limbs separately to correspond to standard lead II electrocardiogram (ECG).(2) All animals before VF were examined by TTE (PHILIPS7500with a2.5-3.5MHz 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.(3) Catheter positions were confirmed by x ray fluoroscopy. Both catheters in the intrathoracic ascending aorta and the right atrium were connected to the pressure transducers attached to a PRO EP recording system (PowerLab/16sp, AD Instruments, Australia) by which aortic systolic pressure (AOSP), diastolic aortic pressure (AODP), right atrial diastolic pressure (RADP) could be recorded during the whole study. Coronary perfusion pressure (CPP) was calculated as AODP minus RADP and mean aortic pressure (MAP) was calculated as1/3AOSP plus2/3AODP.2. Creation of VF model(1) All animals were randomly divided to four groups:sham control group (n=8),8-min VF group (n=8),12-min VF group (n=8) and30-min VF group (n=8). Dogs in the sham control group were only anesthetized and intubated tracheally.(2) VF was induced by delivering a5-s alternating current at80V across the thorax through2needles. Successful VF was defined as a decrease in aortic blood pressure below20mm Hg and the presence of VF waveform on the ECG.3.Measure the expression of Cx43、p-Cx43、MMP-2and TIMP-2(1) Tissue collection:After finishing the experimental protocol, the survival animals were killed by infusing KC1. Tissue samples of the left ventricular myocardium were collected within15s following the death and then immediately snap-frozen in liquid nitrogen (stored at-80℃).(2) Total protein and membrane protein were extracted from cardiomyocytes respactly. Expression of Cx43and p-Cx43in cardiomyocytes membrane and MMP-2/TIMP-2in myocardium were detected by western blot.(3) The expression and distribution of Cx43and p-Cx43were detected by immunofluorescence and confocal laser scaning microscopy, and analyzed with Image-Pro Plus6.0software.(4) Expression levels of MMP-2and TIMP-2were analyzed by immunohisto-chemical staining.Part2. Expressions of Cx43、p-Cx43、MMP-2/TIMP-2and Ang II in the VF cordioversion process were measured and effect of ZP123on Cx43was evaluated, while exploring the relationship between MMP-2/TIMP-2、Ang Ⅱ and prognosis of VF1. Animal preparation(1) Forty-eight mongreal dogs of either sex were anesthetized with pentobarbital-Na (30mg/kg intravenous, repetition when necessary). After anesthesia, dogs were placed in the supine position on the experimental table and restrained at the four extremities. Three surface electrodes and arterial oxygen saturation (SaO2) sensor were secured and configured to an automated external defibrillator for electrocardiogram signals and SaO2monitoring.(2) All animals were examined by TTE to measure LAD, LVDd and LVEF, respectively in a parasternal long-axis and M-mode view before VF and after ROSC. Catheter positions were confirmed by x ray fluoroscopy. Both catheters in the intrathoracic ascending aorta and the right atrium were connected to the pressure transducers attached to a PRO EP recording system by which AOSP, AODP and RADPcould be recorded during the whole study. CPP was calculated as AODP minus RADP and MAP was calculated as1/3AOSP plus2/3AODP.2. Creation of VF model(1) All animals were randomly divided to six groups:8-min VF+ZP123group (n=8),8-min VF+NS group (n=8),12-min VF+ZP123group (n=8),12-min VF+NS group (n=8),30-min VF+ZP123group (n=8) and30-min VF+NS group (n=8).(2) Three ZP123groups were given ZP1231μg/kg bolus+10μg/kg/h pumped by a micro pump for30min, and NS groups were given saline instead of ZP123.(3) VF was induced by delivering a5-s alternating current at80V across the thorax through2needles after the completion of drug pumps.3. CPR and defibrillation(1) External chest compressions and ventilation with100%oxygen started when each group achieve the period of VF. The rate of compression was100-120/min.2ventilations were delivered after every30compressions without interruption of compression.(2) After2min 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 for1hour. If ROSC was not achieved, an immediate defibrillation of70J biphase was delivered, then another2min of CPR was immediately restarted and defibrillation energy was increased to100J biphase, if not achieved ROSC, then the third CPR was immediately started but the defibrillation energy was increased to150J, The third CPR was continued until ROSC achieved. The CPR would be given up after30min.(3) Calculate the average defibrillation energy, defibrillation success rate,the success rate of the previous three defibrillation, ROSC and survival (1hour) rate.4. Measure the expression of Cx43、p-Cx43、MMP-2and TIMP-2(1) Tissue collection:After finishing the experimental protocol, the survival animals were killed by infusing KC1. Tissue samples of the left ventricular myocardium were collected..(2) Expression of Cx43and p-Cx43in cardiomyocytes membrane and MMP-2/TIMP-2in myocardium were detected by western blot.(3) The expression and distribution of Cx43and p-Cx43were detected by immunofluorescence and confocal laser scaning microscopy, and analyzed with Image-Pro Plus6.0software.(4) Expression levels of MMP-2and TIMP-2were analyzed by immunohisto-chemical staining.5. Determination of myocardial Ang II content(1) Preparation of myocardial tissue.(2) Determined the content of Ang II by ELISA.ResultsPart1. Dynamic alterations of Cx43、p-Cx43, MMP-2and TIMP-2in cardiomyocytes during VF1. Baseline characteristicsThere were no statistically significant differences among the animals in body weight, heart rate, LAD, LVDd, LVEF and hemodynamic variables at baseline.2. Immunofluorescence and western blot analysis results of Cx43and p-Cx43(1) High-intensity specific immunoreactive signals of Cx43and p-Cx43were clearly identified and regularly distributed. With the duration of VF, Cx43and p-Cx43signals became weaker and weaker and distributed in heterogeneity.(2) Western blot analysis results showed that compared with the sham control group, Cx43in other three VF groups were significantly decreased, and the amount of Cx43declined with the duration of VF (P<0.05). p-Cx43in12-min VF group and30-min VF group were significantly reduced (P<0.05), while there was no statistical distinction between the sham control group and8-min VF group (P>0.05), in addition, the same change tendency was observed in the ratio of p-Cx43/Cx43.3. Changes of MMP-2, TIMP-2and the ratio of MMP-2/TIMP-2(1) Compared with sham controls, dogs under VF showed significantly decreased contents of TIMP-2, and the level of TIMP-2declined by degrees with the duration of VF(P<0.05). No significant difference was observed between the sham control group and the8-min VF group concerning levels of MMP-2(P>0.05), however, they increased in other two longer-duration VF groups (P<0.05).(2)The ratios of MMP-2/TIMP-2were higher in VF groups, and rose up gradually with the duration of VF (P<0.05).4. Correlation between p-Cx43/Cx43and MMP-2/TIMP-2A remarkable correlation was observed between the ratio of p-Cx43/Cx43and MMP-2/TIMP-2(r=-0.93, P<0.01).Part2. Expressions of Cx43、p-Cx43、MMP-2/TIMP-2and Ang Ⅱ in the VF cordioversion process and the effect of ZP1231. Baseline characteristicsThere were no statistically significant differences among the animals in body weight, heart rate, LAD, LVDd, LVEF and hemodynamic variables at baseline.2. Comparison of TTE and hemodynamic index among survivors in each groupsLAD and LVDd of8-min VF groups and12-min VF groups were significantly increased, and LVEF is significantly decreased compared with that before VF (p<0.05). LAD,LVEF of12-min VF ZP123group and NS group were different compared with8-min VF groups (p<0.05). The difference of LVDd among agoups did not reach statistical significant. Except RADP, other indexes as AOSP, AODP, MAP and CPP in four groups were all significantly lower than that before VF. No statistically analyzed between the30min VF groups because there were too little dogs survival.3. Comparison of defibrillation success rate, the average defibrillation energy and survival rate among groupsAll of8dogs in8-min VF+ZP123group got successful defibrillation, including7in the first three defibrillation, and7fully restored ROSC and survival;8-min VF+NS group has7to succeed defibrillation, including4in the first three defibrillation, and total6restored ROSC and survival for more than1hour. In12-min VF+ZP123group,7dogs got defibrillation,5achieved ROSC and4survived longer than1hour; while12min NS group only5got defibrillation and4recoverd ROSC and3survived.30-min ZP123group treatment group had2dogs to be successful defibrillation, but none survived; while30min NS group only1got defibrillation and survived.Statistical analysis showed that the first three defibrillation success rate in8-min and12-min ZP123groups were significantly higher than the NS control group (p <0.05), and the average defibrillation energy were significantly lower than the control group (p<0.05). Although the difference of ROSC rate and survival rate between ZP123group and control group was not reach statistical significance (p>0.05), but the three ZP123treatment groups had a higher ROSC and survival rate than control groups.4. Immunofluorescence and western blot analysis results of Cx43and p-Cx43(1) High-intensity specific immunoreactive signals of Cx43and p-Cx43were clearly identified and regularly distributed in ZP123groups compared to the control groups. With the duration of VF, Cx43and p-Cx43signals became weaker and weaker and distributed in heterogeneity.(2) Western blot analysis results showed that compared with the sham control group, Cx43in VF groups were significantly decreased (P<0.05or P<0.01). Expression of Cx43in12-min and30-min ZP123groups were more than their control groups(P<0.05), while no difference in8-min groups.With the duration of VF, the expression of p-Cx43decreased, and p-Cx43in ZP123groups were significantly higher than control groups (P<0.05).(3) With the duration of VF, the ratio of p-Cx43/Cx43decreased, and ratio of p-Cx43/Cx43in ZP123groups were significantly higher than control groups (P<0.05).5. Myocardial Ang Ⅱ content in ZP123groups and control groupsCompared with the sham control group, myocardial Ang Ⅱ content in VF groups were all significantly increased (P<0.05or P<0.01), and gradually increase d with the duration of VF. But there was no significantly difference between ZP123group and control group (p>0.05).6. Expression levels of MMP-2, TIMP-2and the ratio of MMP-2/TIMP-2in survival groups and death groups(1) Immunohistochemical staining and western blot analysis results:Statistical analysis showed that there was no significant difference in the expression of MMP-2and TIMP-2between ZP123group and the same process NS group (p>0.05). But TIMP-2in survivals of each groups was significantly higher than the death (P<0.05), while no significant difference in MMP-2(P>0.05).(2) The ratios of MMP-2/TIMP-2in survival groups were lower than the death groups (P<0.05).7. Myocardial Ang II content in survival groups and death groupsStatistical analysis showed that the myocardial Ang Ⅱ content in survivors was significantly lower than the death with the same VF time (P<0.05).8. Correlation analysis between various indicatorsCorrelation analysis showed that there was a significant positive correlation between the expression level of Cx43and the defibrillation success rate (r=0.91, P<0.01); there was a negative correlation between the Cx43level and the mean defibrillation energy (r=-0.854, P<0.01); there was a positive correlation between the phosphorylated Cx43expression level and the previous three defibrillation success rate (r=0.926, P<0.01).In NS control groups, there was a significant negative correlation between myocardial membrane Cx43/p-Cx43and the myocardial Ang II content(r=-0.945,-0.909, P<0.01). And there was a negative correlation between the myocardial TIMP-2level and Ang Ⅱ content (r=-0.947, P<0.01).Conclusions:1. In the development and progression of VF, Cx43remodeling occurs in myocardial membrane,not only the amout of Cx43expression reduce, arrangement and distribution to be disorder, but also the phosphorylated Cx43reduce and dephosphoryled Cx43increase.Cx43remodeling is far more dramatic with prolonged VF. ZP123can reduce Cx43remodeling,including increase the expression of Cx43in membrane,reduce dephosphorylation of Cx43, make its arrangement and distribution tends to uniform, to improving the electrical coupling between gap junctions, thereby reducing the defibrillation energy and improve the defibrillation success rate.2. Extracellular matrix remodeling caused by the imbalance of MMPs/TIMPs is involved in the genesis and maintenance of VF. The expression imbalance of MMPs/TIMPs is associated with the mortality of VF, and its mechanism may be triggered by the myocardial ischemia reperfusion injury during CPR.3. The gap junction remodeling is accompanied with the ECM remodeling in the development process of VF.4. AngⅡboth through reducing the expression of Cx43and decreasing phosphorylated Cx43level to cause GJ remodeling, but also by destroying MMPs/TIMPs balance leading to myocardial ischemia-reperfusion injury. The common result is to promote VF easier to maintain and difficult to cardioversion, increase myocardial injury and make prognosis worsen.The mechanism of ARB in reducing the incidence and mortality of ventricular arrhythmia, perhaps because the arrest of the above effects of Ang Ⅱ5. Prophylactic ARB may reduce the incidence and mortality of ventricular fibrillation.