Heterocellular regulation of cardiomyocyte excitation-contraction coupling

Our understanding of cardiomyocyte electrophysiology and function has been built from experimentation on the single, isolated cardiomyocyte. As a result, a significant body of work has been accumulated describing the mechanisms underlying cardiomyocyte function in health, and how they are altered in disease. The heart however is a sophisticated syncytium of which cardiomyocytes comprise only one third of the cellular content, supported by a number of other cell types. It is the summation of these parts which underlies the effectiveness and adaptability of the heart as an organ, however our understanding of how the non-myocyte niche supports and regulates cardiomyocyte function is limited. Cardiac fibroblasts represent a significant portion of the non-myocyte component of the myocardium, intrinsically linked to extracellular matrix synthesis and turnover as well as secreting significant amounts of bioactive molecules. Multiple aspects of cardiac fibroblast function have been demonstrated to have significant effects on cardiomyocyte electrophysiology, but how these multiple threads combine into a coherent syncytium, as in vivo, is unclear. In this thesis, we investigated the hypothesis that cardiac fibroblasts regulate excitation-contraction coupling in cardiomyocytes, and the effect of this regulation is dependent on the modality of interaction. To do this we utilised cardiac fibroblasts from failing human hearts, and induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) as a surrogate of human cardiomyocytes. Initial work investigating the optimal conditions for iPSC-CM culture conditions identified a significant effect of environment, specifically iPSC-CM seeding density, on their structure and electrophysiological properties. Confluent monolayers of iPSC-CMs were more consistent, stable and functional than isolated cells. As such these formed the basis of further experimentation. iPSC-CM - fibroblast co-cultures were designed in ways to limit the pathways of interaction available between the two cell types. Three were used: fibroblast conditioned medium alone, co-culture in the same well but kept physically separate to allow two-way paracrine interaction, and finally in direct contact with fibroblasts seeded directly on iPSC-CM monolayers. We found that the first two culture conditions broadly slowed iPSC-CM Ca2+ transient properties, while in contrast, physical contact between the two resulted in the opposite, with significantly faster Ca2+ transient kinetics. Assessing these changes in iv more detail with caffeine experiments, we demonstrated that this change in properties was a result of a four-fold increase in sarcoplasmic reticulum Ca2+ uptake when the iPSC-CMs and fibroblasts were in contact, as well as an enhanced ratio of sarcolemmal Ca2+ flux and sarcoplasmic reticulum Ca2+ release. Investigating the potential mechanisms bringing about these changes, we identified a number of cytokines present in the supernatant of the co-cultures that had potential cardio-active effects, however none of these were responsible for the observed contact-induced effects. As fibroblasts actively secrete extracellular matrix, we proceeded to investigate the possible contribution of fibroblast-derived extracellular matrix in this interaction using a small peptide-based model of integrin stimulation. Treatment of the iPSC-CMs with the fibronectin binding sequence peptide GRGDS recapitulated the contact-induced changes in iPSC-CM Ca2+ handling properties as well as significantly shortening the action potential. We further identified that the mechanism through which GRGDS acts was partially a result of stimulation of actin polymerisation and not integrin-associated kinase signalling. This work supports the growing understanding of the fundamental role non-myocytes play in supporting and regulating cardiomyocyte function. In particular we have demonstrated that cardiac fibroblasts stimulate the engagement of the sarcoplasmic reticulum in the process of excitation-contraction coupling in iPSC-CMs. This is not only relevant for the applications of iPSC-CMs in translational medicine and cardiovascular research, but also to understand and target the excitation-contraction coupling machinery in development, health and disease.Open Acces