'Miniaturising' Calcium Imaging for High-Throughput Screening of RyR2 Genetic Variants and Anti-Arrhythmics

Calcium (Ca²⁺) release through the cardiac isoform of the ryanodine receptor (RyR2) is highly regulated to maintain Ca²⁺ homeostasis in the heart. Genetic variation in the RyR2 gene can result in numerous cardiovascular pathologies, particularly abnormal heart rhythm, termed arrhythmia. This is due to dysregulated intracellular Ca²⁺ signalling known as spontaneous store overload-induced Ca²⁺ release (SOICR). The involvement of RyR2 and RyR2 genetic variants in Ca²⁺ signalling is typically assessed through single cell fluorescent imaging utilising a Ca²⁺-sensitive dye; known as ‘Ca²⁺ imaging’. This technique identifies cells undergoing abnormal RyR2-mediated Ca²⁺-release events such as SOICR, which cause arrhythmia. However, this method is time-consuming, reducing the rate at which new arrhythmia-linked RyR2 variants can be characterised. Here, we aim to develop a ‘miniaturised’ higher-throughput Ca²⁺ imaging assay that can screen several RyR2 genetic variants in parallel. The existing method required cells to be treated with a range of Ca²⁺ concentrations ([Ca²⁺]) to determine the propensity for SOICR to occur, requiring prolonged preparation and experimentation of each sample (>1 hour/sample). Here, we examined if using the frequency of SOICR at a single [Ca²⁺] could replace this. HEK293 cells, expressing either wild-type (WT) RyR2 or an arrhythmia-linked RyR2 variant (R4496C), were loaded with a Ca²⁺-sensitive dye; Fluo-4. SOICR frequency was recorded at 0.3, 0.5 or 1 mM Ca²⁺ for 20 minutes before caffeine-induced intracellular Ca²⁺ depletion. Results showed that compared to WT, R4496C-expressing HEK293 cells displayed a higher SOICR frequency at all three [Ca²⁺] (p <0.0001). The largest difference in SOICR frequency was at 0.5 mM, therefore this was the single [Ca²⁺] used for the novel method. The assay was then optimised to work using a 96-well plate, allowing up to 96 samples to be prepared and imaged in parallel, as opposed to one-at-a-time. WT- and R4496C-expressing cells were imaged in a 96-well plate at 0.5 mM [Ca²⁺] using a high-throughput screening tool; the Opera Phenix. These results showed that R4496C cells had significantly higher SOICR frequency than WT cells (p <0.0001), akin to the results of the existing method. Faster data collection necessitated faster data analysis, therefore ‘automated analysis’ workflows were developed in ‘NIS Elements’, further increasing the time-efficiency of the new Ca²⁺ imaging protocol. These data suggest that switching from SOICR propensity to frequency offers a novel method to characterise RyR2 variants using a multi-well plate reader. This novel method provides a faster way of characterising newly identified RyR2 genetic variants, which is useful for faster mitigation of the existing disparity in Māori and Pasifika genetic research.