Combine use of Intracellular Calcium and Sarcolemma Voltage Measurements to Distinguish Mixed Channel Effects in Human Induced Plutipotent Stem Cells Derived Cardiomyocytes (HIPSC-CMS)

Blockade of the human ether-a-go-go-related gene (hERG) ion channel is a marker for pro-arrhythmic risk. However, the extent of hERG channel inhibition is poorly correlated with action potential duration (APD) prolongation in vitro, due to multi-channel block. Subsequently, compounds which block multiple ion channels may be overlooked by pre-clinical safety screening when measuring voltage alone, with the potential risk of late stage drug attrition and withdrawal. In this work we propose optical measurements of voltage and intracellular calcium ([Ca2+]i) in hiPSC-CMs as an alternative to the current drug screening methods to distinguish mixed action drugs in early stages. We used hiPSC-CMs loaded with 3μM di-4-ANEPPS to record membrane potential and 1μM Fura 4-AM to measure [Ca2+]i from areas of iPSCs using CellOPTIQ platform (Clyde Biosciences, Glasgow UK). Fluorescence signals were digitized at 10kHz and analyzed off-line using proprietary software. Mixed ion channel blockade was mimicked by the co-administration of E-4031 (hERG blocker) and Nifedipine (Ca2+ channel blocker). The results showed that at a critical combined concentrations (e.g. 30nM E4031 + 30nM Nifedipine) which individually prolonged and shortened APD at 75% of repolarisation (APD75) respectively, in combination did not significantly change APD75 from baseline (APD75 6.5 ± 7% of control), whereas [Ca2+]i was markedly reduced (by 18 ± 1.2% change, p<0.05). Low concentrations of Verapamil (30nM), an antiarrhythmic drug with known mixed hERG and L-type Ca2+ channel activity, had no significant effect on APD (APD75 1.5 ± 0.7%) whereas the amplitude of Ca2+ transient was significantly reduced (by 45 ± 8%, p<0.05). In conclusion, simultaneous measurements of [Ca2+]i and voltage provides a convenient indicator of mixed ion channel effects when screening for drug-induced cardiotoxicity.