Deciphering maturation and β-adrenergic dynamics in HiPSC-derived cardiomyocytes via integrated transcriptomic and functional profiling

Thesis

Human iPSC-derived cardiomyocytes (hiPSC-CMs) are a convenient cell source of human cardiomyocytes (CMs), valuable for modelling cardiac disease, drug testing, and regenerative research. However, they remain immature, limiting use in adult onset diseases. Despite many maturation efforts since the 2010s, full maturity hasn’t been achieved. Recent work points to the β-adrenergic pathway’s role, but cAMP nanodomain formation and regulation are still unclear. Therefore this thesis aimed to explore the dynamics between maturation and organisation of the cAMP signalling pathway in hiPSC-CMs.

Specifically, this thesis explored how hiPSC-CM metabolic maturation and left ventricular lineage specification affect cAMP signalling. Single nucleus transcriptomics was used to assess RNA level maturity and cell types produced by differentiations. Functional measurements of action potentials, calcium transients and contractility were correlated with transcriptomic changes. Additionally, β-adrenergic-induced alteration of cellular function was investigated, in relation to changes in activity of PDE3 and PDE4 observed using FRET-based cAMP sensors.

This work found that the proportion of proliferative CMs was reduced with maturation. Transcriptomics showed a high proximity between hiPSC-CM groups, while functional analysis revealed that both protocols produced more mature hiPSC-CMs. Additionally, the left ventricular lineage specification robustly reduced hiPSC-CM functional heterogeneity. An increased reliance on human adult isoform PDE3 was observed in hiPSC-CM from both maturation protocols, and was associated with robust functional responses to β-adrenergic stimulation.

These findings advance our understanding of β-adrenergic signalling in hiPSC-CM maturation. Mapping cAMP nanodomains, and the corresponding enhancement of chronotropic and inotropic responses in hiPSC-CMs, will help establish clear molecular and functional benchmarks of β-adrenergic maturation, that will serve both as a sensitive readout and a driver of hiPSC-CM maturity.

Authors: Charrière, C

• DOI: 10.5287/ora-qqx88p6ny
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: transcriptomics; metabolic maturation; β-adrenergic signalling pathway; left-ventricle cardiomyocyte specification; hiPSC-CM maturation; cAMP nanodomain