Abstract
The human heart beats more than two billion times over the average human life span. To maintain reliable functioning of this organ throughout a lifetime, the molecular architecture underlying contraction and relaxation of the heart’s muscle cells have evolved to withstand the continuous mechanical strain and to dynamically adapt the workload to the oxygen demand of the body. Such adaption is regulated by biochemical networks which process the information contained in various humoral and mechanical signals.Among the largest molecules in heart muscle cells is the protein obscurin. It provides a direct physical link between the sarcomere, the contractile machinery of the cell, and the sarcoplasmic reticulum from which the calcium transients originate that control contraction. Obscurin also features multiple, yet barely characterised signalling domains including two kinase domains, an SH3 domain and a DH-PH Rho GEF domain tandem, suggesting obscurin may be involved in complex signalling functions. Although increasing numbers of missense mutations in the obscurin protein have been associated with cardiomyopathies and skeletal muscle diseases, the mechanism of action of these potentially pathogenic variants remains unknown. One of the few genetic variants characterised in more detail is the variant Arg4344Gln in obscurin domain Ig58. Although found in about 1 in 7 African Americans, some scholars argue that this variant is pathogenic because it has been reported to cause dilated cardiomyopathy and cardiac arrhythmias in mice due to a strengthened interaction between obscurin and phospholamban. Phospholamban is among the smallest proteins in heart muscle cells and an important mediator of the “fight-or-flight” response. Under resting conditions, phospholamban inhibits the calcium pump SERCA2a. The binding of catecholamines such as adrenaline to the b-adrenergic receptors of cardiomyocytes leads to activation of protein kinase A which phosphorylates phospholamban and thereby releases SERCA2a inhibition, resulting in faster calcium reuptake into the sarcoplasmic reticulum, faster relaxation and stronger contraction of the heart. Phospholamban, too, can harbour genetic mutations, some of which lead to severe and lethal cardiomyopathies. Phospholamban also forms homo-pentamers whose physiological functions are still unknown.
The present work investigates selected biochemical signal processing aspects of obscurin and phospholamban in the context of cardiomyocyte physiology and genetic mutations.
The first project established the recombinant production of obscurin DH and DH-PH domains and provided a first in vitro biochemical characterisation of it's catalytic activity, post-translational modification and binding partners. It is found that recombinant DH and DH-PH domains of obscurin purified from E. coli do not possess in vitro GEF activity towards any classical Rho GTPase but can be phosphorylated and dephosphorylated by several kinases and phosphatases of relevance in cardiac physiology.
A second project systematically investigated the effect of several potentially pathogenic missense mutations in obscurin domains Ig58 and Ig59 on previously reported protein-protein interactions between obscurin and titin, the titin isoform novex-3 and phospholamban. An important finding was that the previously reported interaction with phospholamban is an in vitro artefact and that the phenotype observed in Arg4344Gln mice does likely not translate to humans.
A third project studied the potential signal processing abilities of homo-oligomers with a particular emphasis on the physiological role of phospholamban pentamers. Starting with general mathematical models of homo-oligomerisation, it was found that homo-oligomerisation could provide an unanticipated wealth of non-linear dynamics and signal processing functions including dynamic signal encoding and homeostatic monomer buffering. Furthermore, homo-oligomers are found to constitute pseudo-multisite phosphorylation systems that allow ultrasensitive or bistable steady-state phosphorylation responses in simulations. Applying these concepts to phospholamban demonstrated that phospholamban pentamers indeed cause ultrasensitive and bistable phosphorylation both in numerical simulations and in experiments. Pentamers are moreover found to constitute a substrate competition based low-pass filter for phosphorylation of phospholamban monomers. These mechanisms could contribute to the prevention of cardiac arrhythmias in the context of b-adrenergic stimulation and may be impaired by the pathogenic phospholamban mutation R14del.
Taken together, the presented work contributes to the understanding of several related signal processing functions in heart muscle cells relevant to cardiac physiology and disease.
| Date of Award | 1 Jan 2022 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Mathias Gautel (Supervisor) & Mark Pfuhl (Supervisor) |