Abstract
| Cardiovascular diseases often trigger cardiac arrhythmias, which frequently underlie patient mortality, leading to over 20 million deaths globally. Treatment of cardiac arrhythmias typically involves electrical and/or pharmacological cardioversion; however, neither of these procedures have had any significant developments since the last anti-arrhythmic drug was approved in 2009. This often forces physicians to use in-effective or toxic pharmaceuticals to attempt to treat cardiac arrhythmias. For example, the commonly prescribed drugs flecainide and encainide lack efficacy in clinical trials, while highly effective drugs like amiodarone and phenytoin are often ignored, due to significant side effects. HEK293-RyR2 were grown on glass coverslips, stained with Fluo4-AM, a calcium sensitive dye, and imaged while undergoing perfusion with increasing calcium concentrations. This protocol allowed us to measure calcium leak from the endoplasmic reticulum through RyR2, which is a key cellular mechanism that underpins cardiac arrhythmias. This study confirmed the anti-arrhythmic effects of phenytoin were identifiable by a reduction in calcium leak in HEK293-RyR2 cells. Additionally, this protocol identified two compounds that significantly reduced the number of calcium leak events, while the remaining two compounds exhibit no difference in the calcium leak behaviour. Unblinding confirmed we had correctly identified the anti-arrhythmic characteristics of the four novel compounds, as they mimicked what was seen in human cardiac tissue. This confirmed that HEK293-RyR2 cells can identify RyR2 mediated anti-arrhythmic characteristics of the drug phenytoin, as well as phenytoin-derived novel compounds. In the future, further validation to examine sensitivity and specificity is required, allowing development into a higher throughput method, such as a 96 well plate assay. Overall, these results show a potential method for high throughput analysis of anti-arrhythmic potential for drug discovery in both this library, as well as future RyR2 targeting compounds, potentially leading to accelerated development of much needed anti-arrhythmic drugs. |