Development of iPSC-derived human cardiac myocytes for the targeting of SK channels

Abstract

Three isoforms of small conductance, calcium-activated potassium (SK) channel have been identified (SK1-3). SK channel expression is higher in the atria than in the ventricles, therefore inhibition of SK-mediated current (ISK) is of interest as a potential therapeutic strategy for atrial fibrillation. Understanding of human SK channel formation and function is hampered by differences between animal models and the limited availability of healthy human tissue. Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) provide a potential way to study human cardiac tissue, but SK channel activity in iPSC-CMs has not been well characterised. The work undertaken for this thesis therefore aimed to determine whether iPSC-CMs represent a useful model of SK channel function. As SK2 and SK3 are the predominant isoforms within the human atria, additional work using heterologous expression investigated interactions between these.
Retinoic acid (RA) was used to differentiate iPSC-CMs with an atrial-like phenotype (RA-iPSC-CMs), characterised by increased acetylcholine activated K+ current (IK,ACh), the absence of ventricular sarcomeric protein (MLC2v), abbreviated action potential (AP) repolarisation and a less positive AP plateau than observed in iPSC-CMs differentiated without RA (DMSO-iPSC-CMs). However, <40% of isolated RA-iPSC-CMs responded to SK channel inhibition. An iPSC-CM monolayer (iPSC-ML) preparation was developed that allowed whole-cell recording of triggered APs at physiological temperature. RA-iPSC-MLs exhibited a greater response to modulators of the atrial-specific currents ISK, IK,ACh and the ultrarapid outward potassium current than did DMSO-iPSC-MLs. Moreover, all RA-iPSC-MLs responded to SK channel inhibition. Investigation of the interactions between human SK2 and SK3 subunits in HEK293 cells demonstrated (i) preferential formation of heteromeric SK2:SK3 channels and (ii) that co-expression with SK2 limited functional SK3 expression.
Overall, these results demonstrate the feasibility of using RA-iPSC-MLs to study cardiac ISK and its composition. The findings on heteromeric SK2:SK3 channels suggest a limited role for homomeric SK channels in vivo.

Date of Award	9 May 2023
Original language	English
Awarding Institution	University of Bristol
Supervisor	Jules C Hancox (Supervisor), Neil V Marrion (Supervisor) & Raimondo Ascione (Supervisor)