The HERG gene encodes for the delayed rectifier K+ channel in human cardiac tissue and contributes to the repolarization phase of the ventricular action potential. Defects in its activity underlies a cardiac disorders linked to a prolongation in the QT interval known as acquired long QT syndrome. The channel has structural properties that lead to its unintentional inhibition by various classes of drugs and is a source of drug induced cardiac toxicity. To date, no assay has been set as a standard due to variability across laboratories and the use of animals providing variable results due to differences in the ion channels involved in repolarisation. This thesis focuses on the development of testing assays for HERG using animal-free methodology. In Chapter 2, a human embryonic kidney (HEK293) cell line was cultured and transfected with the human HERG gene using animal-free methodologies. The success of the transfection was confirmed using PCR, patch clamp electrophysiology and a non-radioactive rubidium assay. Using a non-radioactive rubidium assay, drug inhibition on the transfected cell line was measured. The IC50 values obtained for a range of drugs were compared to those obtained using electrophysiological studies in the literature and there was a high correlation (r2 = 0.76). In Chapter 3, a human neuroblastoma cell line (SH-SY5Y) was tested for its validity for testing the effect of drugs on the endogenously expressed HERG K+ channel. The drug IC50 values obtained using the Rb+ assay were well correlated (r2= 0.82) with patch clamp studies in HERG transfected HEK293 cells in the literature. Clomipramine a clinically used antidepressant causes prolongation in the QT interval, however its mechanism of action on cardiac cells leading to this cardiotoxic effect is unclear. In this study, clomipramine was tested using HERG transfected HEK293 cells and the neuroblastoma cell line (SH-SY5Y) using a rubidium assay and whole cell patch clamp. Clomipramine inhibited HERG with an IC50 value of 8.35 µM and 2.18 µM in HERG transfected HEK293 cells and the neuroblastoma cell line (SH-SY5Y) using the rubidium assay respectively. Clomipramine inhibited HERG currents with an IC50 value of 0.50 µM using the patch clamp technique in HEK293 cells. The results indicate that the prolongation in the QT interval caused by clomipramine may involve HERG inhibition. The HERG K+ channel is regulated by several protein kinases including protein kinase A and protein kinase B. In Chapter 5, the specific PKC activator and phorbol ester PDA was used to study HERG regulation by PKC in HERG transfected HEK293 cells. PDA caused a reduction in HERG currents in HEK293 cells. The PKC pseudo substrate inhibitor PKC [19-36] did not inhibit the effect of PDA on HERG currents. The results of the study suggest that (1) PDA could be acting directly on the channel and inhibiting its function or (2) PDA is activating other proteins which are affecting HERG currents in the HERG transfected HEK293 cells.
| Identifer | oai:union.ndltd.org:ADTP/210430 |
| Date | January 2007 |
| Creators | Raghib, Hala, halaraghib@yahoo.com |
| Publisher | RMIT University. Medical Sciences |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | http://www.rmit.edu.au/help/disclaimer, Copyright Hala Raghib |
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