Physiologically, cardiomyocytes develop features that enable them to meet the contractile demands of the healthy, adult heart. Among the structural and functional changes during development, there is an engagement of the sarcoplasmic reticulum as the main regulator of cytoplasmic Ca2+ cycling. In human cardiac disease and in ageing, there is progressive disengagement of the sarcoplasmic reticulum, reducing the efficiency of excitation-contraction coupling. In this project, we used human induced pluripotent stem cell- derived cardiomyocytes (hiPSC-CMs) to investigate the role of human cardiac fibroblasts in regulating cardiomyocyte Ca2+ cycling.
In chapter 3, we use hiPSC-CMs with a genetically encoded Ca2+ indicator to perform optical recording of changes in hiPSC-CM intracellular Ca2+ in various co-culture setups with human cardiac fibroblasts. Co-culture setups that only allowed paracrine interactions between the two cell types led to prolongation of the hiPSC-CM Ca2+ transients. There was an abbreviation in Ca2+ transient duration when the two cell types were in direct physical contact, indicating an increase in Ca2+ cycling efficiency.
In chapter 4, we investigated the role of the extracellular matrix in regulating hiPSC- CM Ca2+ cycling. As matrix proteins are known to form interactions with cardiomyocytes via integrin ligand-receptor interactions, we utilised synthetic peptides with the integrin-binding tripeptide motif, Arginine-Glycine-Aspartic Acid, to show that fibril-forming integrin ligands abbreviated hiPSC-CM Ca2+ transients by recruiting the sarcoplasmic reticulum to Ca2+ cycling.
In chapter 5, we focus on the role of extracellular vesicles, which have emerged over the last decade as a major secretory vehicle for non-soluble paracrine interactions. A major limitation in the investigation of extracellular vesicles is that isolation techniques, and thus sample purity, varies considerably between studies. In chapter 5, we validated an ultrafiltration- and chromatography-based technique for the isolation of extracellular vesicles from cardiac fibroblast-conditioned culture media and showed that cardiac fibroblast extracellular vesicles significantly abbreviate the hiPSC-CM Ca2+ transient time to peak, indicating an increase in the efficiency of Ca2+-induced Ca2+-release.
The findings of this project indicate that cardiac fibroblasts have differential effects on hiPSC-CM Ca2+ cycling depending on the modality of interaction. The findings also indicate that fibroblast-mediated modulation of hiPSC-CM Ca2+ cycling can be mediated by fibroblast-regulated turnover of the extracellular matrix. This project demonstrates the importance of the extracellular interactions in utilising hiPSC-CMs and understanding the modulators of cardiomyocyte structure and function.