Abstract
"The heart is a made up of different cell types, including cardiomyocytes and non-cardiomyocytes. Cardiac fibroblasts are the pre-dominant non-cardiomyocyte cell type, and differentiate in to myofibroblasts post-myocardial infarction, with these cells responsible for formation of the fibrotic scar. Transplant of human-induced pluripotent stem cell-cardiomyocytes (hiPSC-CMs) is a potential therapeutic strategy post-infarction, but has an associated increased arrhythmogenic risk, with how myofibroblasts affect transplanted hiPSC-CM function relatively unknown. This thesis investigated the hypothesis that myofibroblasts promote pro-arrhythmogenic changes in hiPSC-CMs through paracrine and contact-mediated mechanisms.
HiPSC-CMs were cultured with myofibroblasts for 48 hours in three conditions: with myofibroblast-conditioned medium, cocultured using transwells (noncontact), and cocultured in direct contact. Action potentials and calcium transients were studied using optical mapping, gene expression changes were determined using real-time quantitative PCR, and cytokine analysis of media samples was done using ELISA. Lastly, gap junctional signalling during contact cocultures was investigated through knockdown of myofibroblast connexin-43 with small-interfering-RNA.
Paracrine cocultures were shown to prolong hiPSC-CM APD50/80/90 and time to calcium decay, whilst in contact cocultures hiPSC-CM APD80/90 and time to calcium decay were prolonged, and time to calcium transient peak shortened. IL-6 was shown to be partly responsible for paracrine-mediated changes to hiPSC-CM electrophysiology, with IL-6 blockade able to reduce/prevent pro-arrhythmogenic changes in paracrine cocultures. Knockdown of myofibroblast connexin-43 in contact cocultures promoted changes in hiPSC-CM electrophysiology similar to those observed in paracrine cocultures, with combined connexin-43 knockdown and IL-6 blockade able to reduce/prevent pro-arrhythmogenic changes.
This is the first study to provide an in-depth investigation in to the effects of human myofibroblasts on hiPSC-CMs electrophysiology, and identified distinct myofibroblast paracrine- and contact-mediated mechanisms that promote pro-arrhythmogenic changes in hiPSC-CMs. IL-6 was shown to be partly responsible for pro-arrhythmogenic changes, whilst a potential interaction between gap junctional and paracrine signalling was also identified. "