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Title: The role of the mechanosensitive cation channel Piezo1 in the regulation of cardiac fibroblast function
Author: Blythe, Nicola Marusia
ISNI:       0000 0004 8498 9118
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2019
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Background: Cardiac fibroblasts are fundamental regulators of multiple aspects of cardiac function under both physiological and pathophysiological conditions. These cells contribute to cardiac inflammation, angiogenesis, fibrosis and cardiomyocyte hypertrophy and are therefore a potential therapeutic target for ameliorating adverse cardiac remodelling. Cardiac fibroblasts become activated upon mechanical stimulation; mechanically-activated ion channels have been implicated as sensors of mechanical stress in these cells. The mechanosensitive non-selective cation channel, Piezo1, has widespread physiological importance and although Piezo1 mRNA expression has been detected in the murine heart, its role in the myocardium is poorly understood. The hypothesis is that Piezo1 is important for detecting mechanical stress in cardiac fibroblasts in order to affect cell function and phenotype. Methods and Results: RT-PCR analysis indicated that Piezo1 mRNA is expressed in cardiac fibroblasts at similar levels to endothelial cells and Fura-2 intracellular Ca2+ measurements using the Piezo1 agonist, Yoda1, validated Piezo1 as a functional ion channel in these cells. Yoda1-induced Ca2+ entry can be inhibited by non-specific Piezo1 blockers (gadolinium, ruthenium red) and affected proportionally by alterations in Piezo1 activity or mRNA expression. In addition to using chemical stimulation, Piezo1 could be activated using mechanical force generated by cell-attached patch clamp electrophysiology. Investigation into the effect of Yoda1 on selected cardiac remodelling genes indicated that Piezo1 activation induces the expression of tenascin C (TNC), a mechanosensitive glycoprotein, and interleukin-6 (IL-6), a pro-hypertrophic and pro-fibrotic cytokine, which are both known to be upregulated following cardiac injury. Piezo1 was demonstrated to have a role in regulating basal IL-6 levels; this appeared to be dependent on substrate stiffness and/or composition. Multiplex kinase activity profiling, combined with kinase inhibitor studies and phospho-specific western blotting, established that Ca2+ entry instigated by Piezo1 activation by Yoda1 stimulates concentration-dependent phosphorylation of both p38a MAPK and ERK. It was revealed that it was p38a MAPK which mediated Yoda1-induced IL-6 expression and secretion. Src-family kinases were subsequently discovered to be the link between Piezo1 activation and p38 MAPK phosphorylation; three members of the kinase family were found to be expressed in cardiac fibroblasts. Preliminary data gathered using a myofibroblast-specific Piezo1 KO murine model implied that Piezo1 may promote cardiac remodelling following pressure overload and that its deletion may be advantageous in cardiac dysfunction. Conclusion: In summary, this study reveals that cardiac fibroblasts express functional Piezo1 channels and that utilisation of Yoda1 to stimulate the opening of these channels is coupled to increased secretion of IL-6. This occurs via activation of Src-family kinases and p38 MAP kinase. Improved understanding of Piezo1 channels in cardiac fibroblasts may be necessary for achieving a greater appreciation of how cardiac fibroblasts sense mechanical stimuli in pathological conditions such as fibrosis and hypertrophy.
Supervisor: Turner, Neil A. ; Beech, D. J. ; Drinkhill, M. J. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available