Effects Of Cx43Remodeling On Ventricular Arrhythmia In Rabbit With Left Ventricular Hypertrophy

BackgroundAccompanied with a gradual increasing of the incidence of hypertension, hypertension-induced left ventricular hypertrophy already has become a major public health problems associated with sudden cardiac death (SCD). Although SCD in patients with left ventricular hypertrophy mainly due to malignant ventricular arrhythmias (MVA), its physiological mechanisms are not entirely clear which bring great difficulties for clinical treatment in patients with MVA induced by left ventricular hypertrophy. The study of these physiological mechanisms associated with MVA can provide us theoretical basis for the clinical evidence-based medicine conclusion and clinical selection of first-line antihypertensive drugs and medication time, and discover effective targets in the drug treatment of LVH patients with MVA. Ultimately, these findings have important social significance and great economic value in reducing SCD and improving the prognosis of patients with LVH.Recently, concept of electrophysiological heterogeneity among transmural ventricle has been proposed along with the study of the left ventricular midmyocardial cells. From the anatomical point of view, there are different electrophysiological characters among the epicardium, midmyocardium, and endocardium cells. Compared with those in the subepicardial myocytes and subendocardial myocytes, action potential refractory period in the midmyocardial myocytes were significantly prolonger at slow frequency electrical stimulation, which will increase repolarization heterogeneity among the left ventricular myocardium in the cross section. Prolongation of action potential duration in the midmyocardial myocytes could increase dispersion of transmural repolarization and induce early afterdepolarizations (EAD), delayed afterdepolarizations (DAD) and triggered activity, which lead to ventricular transmural reentrance and malignant ventricular arrhythmia, such as ventricular tachycardia, ventricular flutter and Torsade de points (Tdp)In the anatomy, there are differences of myocardial structure among the interface of subendocardium, midmyocardium and subepicardium which induce different repolarization and conduction velocity of action potential in the three myocardial lays and is to be important anatomical basis for the formation of transmural reentrance in the left ventricular myocardium. More importantly, not only the individual action potentials but also responses to the variety of stimuli such as myocardial ischemia, heart failure, myocardial reperfusion among three myocardial cells were greatly different. These different responses to pathophysiological stimuli among three myocardial cells could increase dispersion of transmural repolarization, and more prone to reentrant ventricular arrhythmia within the left ventricular myocardium. Therefore, increasing of transmural heterogeneity of repolarization may be an important electrophysiological mechanism of malignant ventricular arrhythmias and sudden cardiac death in hypertension with hypertrophy.Protein molecular mechanisms of dispersion of transmural repolarization are not completely clear. It has been confirmed that connexin (Cx) is the major proteins involved in MVA, and left ventricular myocardial cells mainly express Cx43phenotype, which mediated current conduction among left ventricular myocytes.Gap junction channel composed by Cx43can promote myocardial electrical coupling by regulating ion exchange among myocardial cells, and regulate mechanical coupling of synchronous contraction. In some pathophysiological conditions, the occurrence of ventricular arrhythmias associated with GJ especially Cx43refactoring. A previous study showed that Cx43in the left ventricular myocardium after myocardial infarction was deranged, and result in heterogeneity of electrical conduction among the myocardial cells, which may be important anatomical basis of ventricular arrhythmias. Reduction of Cx43in the left ventricular myocardium would cause a decline in the ability of conduction of electrical coupling, thereby change the myocardial electrical conduction properties, and increase the dispersion of action potential duration and conduction heterosexual in the left ventricular myocardial tissue.Results of the left ventricular myocardial biopsy in patients with severe coronary artery disease and showed left ventricular dysfunction showed that the occurrence of ventricular arrhythmias were associated with reduction of Cx43. The study in heart failure patients also confirmed that ventricular arrhythmias associated to the expression of Cx43. Reduction of Cx43would induce uncoupling among myocardial cells which lead to the heterogeneity of the action potential duration. However, understanding about transmural heterogeneity of Cx protein distribution was not enough in previous studies, and we have not taken Cx as the central link in of research. We can speculate that heterogeneity of Cx43expression in the midmyocardium is an anatomical basis of transmural heterogeneity of repolarization. The stimulus of the pathophysiological factors will expand transmural differences of Cx43expression, and increase transmural heterogeneity and dispersion of repolarization, which may be the protein molecular basis of the occurrence and development of MVA in the LVH.In addition, angiotensin II (Ang II) is a key component of the renin-angiotensin-aldosterone system (RAAS). Past studies have demonstrated that it is widely involved in the occurrence and development of cardiovascular diseases such as hypertension and left ventricular hypertrophy. Recent studies have also recognized that Ang II could affect the left ventricular myocardial electrophysiological parameters and involve in the occurrence of MVA. Traditional antihypertensive drugs including angiotensin-converting enzyme inhibitors (ACEI) and angiotensin receptor blocker (ARB) could inhibit Ang Ⅱ in circulating and local myocardial tissue, and improve ion channel function of midmyocardial cells in the pathological states and reduce the transmural dispersion of repolarization, thus reduce the incidence of MVA. Recent studies show that Ang Ⅱ can also reduce the expression of Cx43, and slow the electrical conduction among myocardial cells. The results have prompted that ang Ⅱ could affect remodeling of gap junction among myocardial cells and lead to electric physiological abnormalities. This may be an important protein molecular mechanism why ang II triggers ventricular arrhythmias.In addition, clinical studies have shown that severe hypokalemia could induce or increase the occurrence of ventricular arrhythmias, which was more significant in pathological conditions. Many organic heart diseases such as hypertension and LVH, heart failure were prone to fatal ventricular arrhythmias, and diuretics were often used to strengthen the control of blood pressure and improve heart function, while the most common side effect of diuretics was hypokalemia. Therefore, studying the effects of hypokalemia on transmural dispersion of repolarization and Cx43expression and exploring the electrophysiological and protein mechanisms of MVA are very important for the prevention and treatment of sudden death in the structural heart diseases. The main content of our topics include:Purpose①To observe the effects of perfusion of Ang II and hypokalemia on transmural dispersion of repolarization and heterogeneity of Cx43expression in the three myocardium, and explore the electrophysiology and protein molecular mechanisms why Ang Ⅱ and hypokalemia is prone to trigger malignant ventricular arrhythmias.②To observe the changes of transmural dispersion of repolarization and heterogeneity of Cx43expression in the rabbit model with left ventricular hypertrophy and the effects of feeding irbesartan and amiodarone on these changes, and investigate the treatment and mechanisms of MVA induced by LVH.Method1. Animal groups:Experiment1:The experimental rabbits were randomly divided into control group (n=10), Ang II perfusion group (n=10) and hypokalemic perfusion group (n=10). Isolated heart was perfuse with simple improvement Tyrode’ solution in the normal control group, and perfuse with normal Tyrode’ solution containing1μM Ang Ⅱ in the ang Ⅱ perfusion group and with hypokalemic Tyrode’ solution in the hypokalemic perfusion group. Experiment2:40rabbits were randomly divided into sham operation group (n=10), LVH group (n=10), irbesartan group (n=10) and amiodarone group (n=10). LVH model was produced with traditional abdominal aortic coarctation in the LVH group and continue to feed for8weeks after surgery. LVH model was also made with traditional abdominal aortic coarctation and given feeding irbesartan tablets (10mg/kg.d) for eight consecutive weeks after surgery in the irbesartan group. In the amiodarone group, LVH model was also made with traditional abdominal aortic coarctation and given feeding amiodarone tablets (50mg/kg.d) for four consecutive weeks after surgery.2. All experimental rabbit hearts were cut and cardioplegia in a4℃Tyrode’ solution, then were perfuse with Langendorff system after heart pruning and aortic cannulation.3. Monophasic action potential of three myocardium among left ventricle was recorded by connecting simple electrode and Bio-signal acquisition and processing system. Then the sinus node was destroyed, and heart was fixed paced with electrophysiological stimulation from the right ventricular wall. Stable individual action potentials of three myocardial tissues were recorded simultaneously after Tyrode’ solution perfusion for20minutes in the normal control group, and various electrophysiological parameters were calculated. Action potentials of three myocardial tissues were recorded after Tyrode’ solution perfusion containing the concentration of1μM Ang Ⅱ for20minutes in the Ang Ⅱ perfusion group. Action potentials of three myocardial tissues were recorded after hypokalemic Tyrode’ solution perfusion for20minutes in the hypokalemic perfusion group.4. By method of fast frozen and slicing, left ventricular subepimyocardium was cut by continuously slicing10times from the surface of the epicardium to endocardium, and the thickness of each slice was20μm. In the same way, subendomyocardium was cut by continuously slicing10times from the surface of the endocardium to epiocardium, and the thickness of each slice was20μm. Midmyocardial tissue was taken away by continuously slicing10times from3mm under the epicardial surface to endocardium, and the thickness of each slice was also20μm.5. Protein in tissue was extracted and sample according to the sample concentration, then subjected to gel electrophoresis and transferred to PVDF membrane. After dropping monoclonal antibody of Cx43and horseradish peroxidase-labeled secondary antibody, PVDF membrane was incubated for2h at room temperature. PVDF was exposure and development on X-ray film after coloration with enhanced chemiluminescence. Image of protein signal strip was got with gel image analysis system, and optical density values of target protein Cx43and internal reference protein β-actin were measured. Their ratio was calculated and seen as Cx43protein expression level.Result1. Comparing of electrophysiological parameters and Cx43protein expression in the Ang Ⅱ perfusion group:(1). Compared with that in the control group, VFT significantly reduced from13.40V to8.30V (P<0.001) in the Ang Ⅱ perfusion group, which showed that perfusion of Ang Ⅱ was more likely to induce ventricular fibrillation in the left ventricular myocardium. APD90of the three myocardium in the Ang Ⅱ perfusion group were longer than those in the control group (P<0.01).△APD90(ms) in the Ang Ⅱ perfusion group was34.70±9.68ms, which was significantly greater than that in the control group (23.7±5.67ms, P<0.01), TDR was also increased from36.10ms to49.30ms (P<0.05). So transmural dispersion of repolarization in the Ang Ⅱ perfusion group was more significant compared to the control group.(2) Relative gray values of Cx43protein expression in the midmyocardium, the subepimyocardium and subendocardium in the Ang Ⅱ perfusion group were0.31±0.06,0.45±0.04,0.49±0.07respectively, which decreased significantly (P<0.05) compared with those in the control group. Difference of relative gray values of Cx43protein expression between the subendomyocardium and midmyocardium (△Cx43) in the Ang Ⅱ perfusion group was higher than that in the control group (＜0.05), and the result showed that Ang II perfusion increased transmural heterogeneity of Cx43protein expression in left ventricular myocardium of rabbit.2. Comparing of electrophysiological parameters and Cx43protein expression in the hypokalemic perfusion group:(1) VFT in the control group and the hypokalemic perfusion group were13.4V and7.0V (P<0.01) respectively, which indicated that hypokalemic perfusion reduced the VFT in the left ventricular myocardium. APD90of the three myocardium in the hypokalemic perfusion were significantly longer compared to those in the control group (P<0.001).△APD90in the hypokalemic perfusion group rise to38.10±10.29ms (P<0.05), and TDR in the hypokalemic perfusion group (52.90ms) significantly increased compared to that in the control group (36.10ms)(P<0.05), which proved hypokalemia further expanded transmural dispersion of of repolarization in left ventricle.(2) Compared to those in the control group, relative gray values of Cx43protein expression in the midmyocardium, the subepimyocardium and subendocardium in the hypokalemic perfusion group were0.22±0.04,0.36±0.06,0.40±0.06respectively and decreased significantly (P＜0.01).△Cx43in the hypokalemic perfusion group increased (P<0.05) compared with that in the control group, so hypokalemic perfusion increased transmural heterogeneity of Cx43protein expression in left ventricular myocardium of rabbit.3. Comparing of electrophysiological parameters and Cx43protein expression in the left ventricular hypertrophy group:(1). Compared to the sham group, mean arterial pressure, heart weight/body mass index and left ventricular mass, thickness of left ventricular free wall in the LVH group significantly increased. Results of pathology biopsy showed that cardiomyocyte in the left ventricle was hypertrophy, disorganized, and in line with the pathological manifestations of left ventricular hypertrophy. All of above changes proved that rabbit model of left ventricular hypertrophy was successfully established in the LVH group.(2) VFT was12.20±1.75V in the sham group and7.50±1.58V in the LVH group, while it was different (P<0.001) and showed that ventricular fibrillation were more likely to cause in the LVH group. APD90in the midmyocardium was longer than those in the subepicardium and subendocardium in the sham group and LVH group.△APD90and TDR in the LVH group were32.40±9.67ms and48.90±11.65ms respectively, and were24.30±5.68ms and36.40±7.40ms in the sham group, which indicated APD90extension of midmyocardium was significantly greater than those of subepimyocardium and endomyocardium in the LVH group (P<0.05and <0.01, respectively), and transmural dispersion of of repolarization increased.(3). Cx43protein expression of midmyocardium were significantly weaker than that of the subepicardium and subendocardium (P<0.01and <0.001, respectively) in the both sham group and LVH group, and there were transmural heterogeneity of Cx43protein expression in the both sham group and LVH group. However, compared with the sham group, Cx43protein expression of all three myocardium decreased significantly (P<0.01) in the LVH group, and decreasing of Cx43protein expression in the midmyocardium was more significant. Compared to that in the sham group,△Cx43in the LVH group was higher, so transmural heterogeneity of Cx43protein expression in the LVH was more apparent.4. Comparing of electrophysiological parameters and Cx43protein expression in the irbesartan group:(1). Mean arterial pressure, heart weight/body mass index and thickness of left ventricular wall in the irbesartan group were significant improved compared with the LVH group (P＜0.001), which showed that irbesartan could reduce mean arterial pressure, heart weight/body mass index and thickness of left ventricular wall, and improve ventricular remodeling in the LVH.(2) VFT in the irbesartan group was significantly higher than that in the LVH group (P<0.05and <0.01, respectively), which proved irbesartan could raise VFT of LVH and decrease occurrence of ventricular fibrillation. Three myocardial APD90, TDR and AAPD90in the irbesartan group significantly reduced compared with the LVH group (P<0.01). These electrophysiological results demonstrated that shorting of APD90in the midmyocardium was more obvious compared to subepimyocardium and midmyocardium after irbesartan treatment, and transmural heterogeneity of repolarization lower.(3). Compare with the LVH group, relative gray values of three myocardial Cx43protein expression in the irbesartan group were significantly increased (P<0.05and <0.001, respectively), especially increasing in the midmyocardium was more significant and△Cx43decline (P<0.01). It showed that irbesartan have the effects of decreasing transmural heterogeneity of Cx43protein expression in the LVH.5. Comparing of electrophysiological parameters and Cx43protein expression in the amiodarone group:(1). Mean arterial pressure, heart weight/body mass index, left ventricular mass and thickness of left ventricular wall were no different (P>0.05) between the LVH group and the amiodarone group, and it indicated that oral amiodarone has no significant effect on blood pressure and heart structure in the LVH.(2). Compared to that in the LVH group, VFT in the amiodarone group was significantly higher (P<0.01), which showed amiodarone could increase VFT of LVH and reduce the incidence of ventricular fibrillation. Although three myocardial APD90in the amiodarone group were significantly longer than those in the LVH group (P＜0.001), TDR and△APD90were significantly lower in the amiodarone group (P<0.05and <0.01, respectively). The results illustrated that effect of amiodarone on extending midmyocardial APD90was less than those on the subendomyocardial and subepimyocardial APD90, which induce decreasing of transmural heterogeneity of repolarization among the three myocardial layers.(3). Relative gray values of three myocardial Cx43protein expression in the amiodarone group were significantly increased (P<0.05and <0.001, respectively) compared to the LVH group, moreover increasing of midmyocardial relative gray values was more significant. So△Cx43decreased (P<0.01). These results instructed that amiodarone could reduce transmural heterogeneity of Cx43protein expression in the LVH.Conclusion1. Ang II perfusion made the left ventricular myocardium more easily to induce ventricular fibrillation, which is related to prolongation of left ventricular three myocardial action potential duration, increasing of transmural dispersion of repolarization.2. Ang II increased transmural heterogeneity of left ventricular myocardial Cx43protein expression in the normal rabbit, which was the basis of the protein which leads to the increasing of transmural dispersion of repolarization.3. Hypokalemic perfusion significantly reduced left ventricular myocardial VFT, and prolonged the three myocardial monophasic action potential and increased transmural heterogeneity of repolarization.4. Hypokalemic perfusion significantly decreased Cx43protein expression of the three myocardial lays, and decreasing of Cx43protein expression in the midmyocardium was more significant. The results instructed that hypokalemia could reduce transmural heterogeneity of Cx43protein expression in the left ventricle.5. Monophasic action potential of three myocardium in the LVH were extended, and the extension in the midmyocardium was more obvious, so transmural dispersion of repolarization was significant, and left ventricle were likely to induce ventricular fibrillation.6. Transmural heterogeneity of left ventricular myocardial Cx43protein expression in the LVH was higher than that in the control group, and this was the reason for the increasing of transmural dispersion of repolarization.7. Irbesartan could reduce mean arterial pressure, heart weight/body mass index and thickness of left ventricular wall, and improve ventricular remodeling in the LVH.8. Irbesartan could shorten three myocardial APD, and shorting of APD90in the midmyocardium was more obvious compared to subepimyocardium and midmyocardium, which reduced transmural heterogeneity of repolarization and increased VFT.9. Irbesartan has the effects of increasing amount and decreasing transmural heterogeneity of Cx43protein expression in the LVH, and improved transmural dispersion of repolarization.10. Effect of amiodarone on extending midmyocardial APD90was less than those on the subendomyocardial and subepimyocardial APD90, which induce decreasing of transmural heterogeneity of repolarization among the three myocardial layers and elevation of VFT.11. Amiodarone could raise three myocardial Cx43protein expression in the left ventricle, and reduce transmural heterogeneity of Cx43protein expression in the LVH, which were reasons of improvement of transmural dispersion of repolarization.