Exploring the regulation of myofilament function in the heart using experimental and computational approaches

Thesis

Precise spatio-temporal control of 3’,5’-cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) is essential for normal cardiac function. Dysregulation of cAMP-dependent, PKA-mediated phosphorylation of myofilament proteins, such as troponin I (TNI) and myosin binding protein C (MYBPC), contributes to cardiac pathologies like heart failure. Current therapies, such as beta-blockers and phosphodiesterase (PDE) inhibitors, often lack specificity and have adverse effects. There is a gap in understanding the spatial and temporal dynamics of cAMP signalling within cardiomyocytes. Specifically, the regulation of the myofilament by multiple distinct nanodomains has not been investigated. Similarly, the impact of PKA phase separation on myofilament function is unknown. The goal of this thesis is to address this knowledge gap.

Computational modelling and experimental techniques were used to investigate the regulation of myofilament function. A new human-induced pluripotent stem cellderived cardiomyocyte (hiPSC-CM) in silico electromechanical model was developed to simulate contraction and electrical activity. cAMP and PKA signalling at the myofilament were investigated using expansion microscopy and FRET cAMP sensors targeted to the myofilament proteins, including a newly developed Myomesin-CUTie sensor. The role of PKA condensation was studied using specific disruption methods.

It was found that myofilament proteins are regulated by distinct cAMP signalling domains where cAMP kinetics in response to β adrenergic activation and PDE involvement are different between the MYBPC and TNI sites. PDE4 was found to regulate cAMP at TNI but not the MYBPC site. PKA RIα and RIIβ were found to undergo condensation in cardiomyocytes. Disruption of these condensates did not affect cAMP compartmentalisation at the myofilament but reduced cytosolic cAMP increase following adrenergic stimulation. An increase in PKA RIα condensates was observed in hypertrophic cells, suggesting a potential role in cardiac disease.

Altogether, the study advances understanding of cAMP-PKA signalling at the myofilament, identifying distinct cAMP nanodomains and elucidating the role of PKA phase separation in modulating the adrenergic response. These findings provide insights into cardiac cell physiology and have implications for the development of more targeted therapies for cardiac dysfunction. The computational models offer a platform for investigating drug effects on cardiac function aiding cardiovascular drug development.

Authors: Folkmanaite, M

• DOI: 10.5287/ora-obagr0dpr
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: modelling and simulation; protein kinase A; liquid-liquid phase separation; myofilament; cAMP
• Subjects: Cardiovascular system