Modulation And Underlying Signal Transduction Mechanisms Of Angiotensin Ⅱ On I_Kr In Ventricular Myocytes

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 delayed rectifier K+ currents (IK) are the major repolarizing outward currents of ventricular action potentials in mammalian species, including humans and consist of rapidly and slowly activating components (IKr and IKs, respectively). Human ether-a-go-go-related gene (hERG or KCNH2) encodes the pore forming subunit of the channel underlying IKr, which is crucial for the repolarization of cardiac action potentials. A reduction in hERG currents due to either genetic defects, or adverse drug effects can lead to hereditary or acquired long-QT syndrome in humans characterized by action potential prolongation, lengthening of the QT interval on the surface ECG, and an increased risk for"torsade de pointes"ventricular arrhythmias and sudden cardiac death. IKr also represents a target for modulation by autonomic neurotransmitters and hormones. Recent studies have revealed that hERG channels are modulated by G protein-coupled receptors including alpha- and beta-adrenergic receptors through the intracellular second messengers, such as cAMP, PKA and PKC. However, available information is limited regarding the effect of Ang II on repolarizing K+ currents and resultant changes in action potential duration (APD) in cardiac ventricular myocytes.The present study was designed to examine the possible regulation of IKr /hERG currents by Ang II and underlying signal transduction mechanisms in isolated guinea pig ventricular myocytes and heterologous expression system using the whole-cell patch-clamp technique.Part1 Stable transfection of HERG gene into HEK293 cells by liposome method and its identification.Aim: To establish stably transfected HEK 293 cell lines expressing functional HERG channels.Methods: With LIPOFACTAMINE 2000, HERG gene was transfected into HEK293 cells, then cell clones were picked up in the presence of G418. HERG proteins were determined by Western-blot and the function of expressed channel were investigatedb by whole-cell patch-clamp recordings.Results: The colony-like cell clones were formed 4 weeks later after G418 screening. Anti-HERG antibody binding protein was detected by Western-blot and E4031, a specific HERG channel blocker, sensitive currents were recorded in transfected HEK 293 cells.Conclusion: A stable expression of HERG gene was achieved by liposome method in HEK-293 cell lines, which showed a functional channel property.Part2 Inhibition of rapid component of delayed rectifier potassium current in ventricular myocytes by angiotensin II via AT1 receptorAim: To assess the effect of Ang II on IKr in guinea pig ventricular myocytes. Methods: Whole-cell patch-clamp technique was used to record IKr in native cardiocytes or in human embryonic kidney (HEK) 293 cells co-transfected with human ether-a-go-go related gene (hERG) encodingα-subunit of IKr and human Ang II type 1 (AT1) receptor gene.Results: Ang II decreased the amplitude of IKr in a concentration-dependent manner with an IC50 of 8.9 nM. Action potential durations at 50% (APD50) and 90% (APD90) repolarization were prolonged 20% and 16%, respectively by Ang II (100 nM). The inhibition of Ang II on IKr was abolished by AT1 receptor blocker, losartan (1μM). Ang II decreased hERG current in HEK293 cells and significantly delayed channel activation, deactivation and recovery from inactivation. Moreover, PKC inhibitors, stausporine or Bis-1, significantly attenuated the inhibition of Ang II on IKr.Conclusion: Ang II produces an inhibitory effect on IKr /hERG currents via AT1 receptor linked to PKC pathway in ventricular myocytes. The result suggests a potential mechanism by which elevated levels of Ang II may be involved in the occurrence of arrhythmias in cardiac hypertrophy and failure.Part 3 Ang II inhibites Ikr/HERG via PKCεisofornAim: To examine the PKC isoform mediating the inhibitory action of Ang II on Ikr/HERGMethods: Whole-cell patch-clamp technique was used to record IKr in native cardiocytes or in human embryonic kidney (HEK) 293 cells co-transfected with human ether-a-go-go related gene (hERG) encodingα-subunit of IKr and human Ang II type 1 (AT1) receptor gene.Results: Ang II inhibited Ikr/HERG current. In HEK293 cells, the response to Ang II was attenuated by acute inhibition of PKC with stau and bis-1. Chronic down-regulatin of PKC by 24h pretreatment of cells with PMA also attenuated the effect of Ang II. But the response to Ang II was insensitive to intracellular Ca2+. The inhibition on HERG by Ang II was little affected by G?-6976 or G?-6983, furthermore, the inhibitory effect was significantly antagonized by an internal dialysis with PKCε-selective inhibitory peptideεV1-2. In guinea-pig ventricular myocytes, the inhibition of Ang II on Ikr was also significantly attenuated by an internal dialysis withεV1-2. The Ang II-induced effect was attenuated when the PKC-dependent phosphorylation sites in HERG were deleted by mutagenesis.Conclusion: (1) The present study provides experimental evidence to suggest that, in HEK293 cells and native guinea-pig ventricular myocytes, activation of PKCεisoform contributes to Ang II-mediated inhibition on Ikr/HERG. (2) HERG channels are modulated by PKCεthrough a mechanism that involve the 17 of 18 putative phosphorylation sites.SUMMARY1. A stable expression of HERG gene was achieved by liposome method in a HEK-293 cell line, which showed a functional channel property.2. Ang II produces an inhibitory effect on IKr /hERG currents via AT1 receptor linked to PKC pathway in ventricular myocytes. The result suggests a potential mechanism by which elevated levels of Ang II may be involved in the occurrence of arrhythmias in cardiac hypertrophy and failure.3. The present study provides experimental evidence to suggest that, in HEK293 cells and native guinea-pig ventricular myocytes, activation of PKCεisoform contributes to Ang II-mediated inhibition on Ikr/HERG.4. HERG channels are modulated by PKCεthrough a mechanism that involve the 17 of 18 putative phosphorylation sites.