| Electrophysiological remodeling in cardiac hypertropy and failure predisposes the heart to lethal arrhythmias, which account for half of the mortality in patients with heart failure. The renin-angiotensin system (RAS) plays a pivotal role in maintaining cardiovascular homeostasis but may contribute to cardiac arrhythmias in various cardiovascular diseases, including cardiac hypertrophy and heart failure. Large scale clinical trials have provided evidence that inhibition of angiotensin II (Ang II) synthesis by angiotensin-converting enzyme inhibitors (ACEI) or direct blockade of angiotensin receptor type 1 (AT1) with the antagonist losartan results in a significant decrease in sudden cardiac death in patients with heart failure which may be linked to fewer episodes of complex arrhythmias. In addition, experimental studies have demonstrated the efficacy of RAS inhibitors in the treatment of reperfusion-induced arrhythmias. Different mechanisms, including the abolishment of electrophysiological remodeling caused by Ang II, have been postulated to explain the beneficial influence of RAS inhibition.The most prominent performance of cardiac hypertrophy, heart failure is that the repolarization process of cyocardial cells are slower, which led to action potential prolongation, lengthening of the QT interval on the surface ECG, and an increased risk for"after-depolarization"and"torsade de pointes". It has been demonstrated that rats harboring the human renin and angiotensinogen genes always die suddenly because of ventricular tachycardia (VT). Specific overexpress of Ang II gene in mouse heart tissue can prolong action potential and increase the incidence of ventricular arrhythmias, without increasing circulating levels of Ang II. Recently the similar result has been reported by Rivard et al that mice with cardiac-specific overexpression of human angiotensin II type 1 receptor (AT1R) show a significant decrease in the amplitude of transient outward K+ current (Ito), ultra-activated delayed rectifier current (IKur) and inward rectifier current (IK1) with the prolongation of action potential duration (APD) and spontaneous ventricular arrhythmias. Particularly, the young transgenic animals exist delayed repolarization and a high incidence of arrhythmias without cardiac hypertrophic remodelling. The results indicate that electrophysiological changes are not secondary to cardiac remodelling, but a direct response to the stimulation on AT1 receptor. Studies have proved that Ang II down-regulates Ito current density in isolated rat and canine ventricular myocytes or mammalian cells expressed with Kv4.3 channel gene. These results suggest that Ang II directly modulates ion channels and thus contributes to the pathological electrical remodeling. However, K+ currents in ventricular repolarization vary with the animal species. The transient outward K+ current Ito in rats and mice, whereas delayed rectifier K+ current in large animals, is the primary current responsible for repolarization. The delayed rectifier K+ current includes two components IKr and IKs. It is generally considered that, human ether-a-go-go-related gene (hERG or KCNH2) encodes the pore forming subunit of the channel. Up to now, little is known about the modulation of Ang II on IKr, a pivotal repolarizing current in ventricular cardiomycytes. In the previous study we observed the regulatory effect of Ang II on IKr/hERG currents in guinea pig ventricular cells and the heterologous expression system for the first time. We found that Ang II inhibited IKr/HERG current through AT1 receptor, and prolonged the process of repolarization. The effects of Ang II were mediated mainly through the intracellular protein kinase C (PKC) signaling pathway. However, the molecular mechanism underlying the regulation of Ang II on Ikr is still unclear.Therefore , the aim of this study is to observe the distributions of AT1 receptor and HERG channels on cell membrane and their possible co-localization in guinea pig ventricular myocytes, and the effect of Ang II on the expression of HERG channel in guinea pig ventricular myocytes and heterologous expression system by using molecular biological methods. Part1 The expression and co-localization of HERG channels and AT1 receptor in transmural ventricular myocardium in healthy guinea pigObjective: To observe the levels of HERG and AT1 receptor proteins in transmural ventricular myocardium and their possible interaction in healthy adult guinea-pig heart.Methods: Immunoblots were performed using membrane fraction from guinea-pig subepicardial, midcardial and subendocardial ventricular myocardium to determine the protein levels of HERG and AT1 receptor proteins. Immunofluorescence and immunoprecipitation method were used to show the subcellular location and the correlation between HERG and AT1. Results: (1) HERG channel protein in the guinea pig left ventricular is heterogeneously expressed in transmural ventricular myocardium. The level of HERG expression is the least in the endocardial myocardium. There was no significant difference between the epi- and midcardial myocardium. (2) AT1 receptor protein in the guinea pig left ventricular is heterogeneously expressed. The level of AT1 receptor in midcardial myocardium is the least. There was no significant difference between the epi- and endocardial myocardium. (3) Immunoprecipitation result indicated that AT1 receptor formed a complex with HERG channel. (4) Immunofluorescence showed that AT1 receptor had identical distribution to HERG protein, suggesting a co-localization form for two proteins.Conclusions: The expression of HERG channel and AT1 receptor proteins exist transmural heterogeneity in guinea pig left ventricular. However, HERG channel and the AT1 receptor have identical distribution and they may form functional complex.Part2 Effects of Angiotensin II on the expression of mature HERG channel proteinObjective:To observe the effection of Ang II on the expression of mature HERG channel protein in left ventricular cardiomycytes of guinea pig and HEK293 cells co-expressed with HERG and AT1 receptor genes.Methods: With Lipofactamine 2000 transfection kit AT1 cDNA was transfected to steady-state HERG-HEK293 cell line. Western blot test was performed to confirm the successful transfection. The expression of the mature HERG channel protein was assessed after the incubation of different concentrations of Ang II either with transfected HEK293 cells or left ventricular cardiomycytes of guinea pig for different time.Results: (1) AT1 receptor gene transfected to steady-state HERG-HEK293 cells successfully and expressed. (2) Incubated with Ang II 10 nM, 100 nM, 1μM for 24 hours can inhibit the mature HERG channel protein expression, but there was no obvious effect for 10 min. (3) Incubated with 100 nM Ang II for 2 h, 4 h, 8 h, 12 h, 24 h, the mature HERG channel protein expression began to decline after 12 hours. (4) It has no effect on HERG channel protein expression when incubated with 100 nM Ang II for 6 h in guinea pig left ventricular myocardial cells.Conclusion: Ang II produces an inhibitory effect on the mature HERG protein expression in the heterologous expression system. |
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