Nicole York

Topic

Investigating the role of ankyrin-B p.S646F on cardiomyocyte protein interactions

School of Medical Sciences

Date & location

• Wednesday, July 22, 2026
• 11:00 A.M.
• Hickman Building, Room 110

Examining Committee

Supervisory Committee

• Dr. Leigh Anne Swayne, School of Medical Sciences, University of Victoria (Co-Supervisor)
• Dr. Laura Arbour, School of Medical Sciences, UVic (Co-Supervisor)
• Dr. Dzifa Dordunnoo, Department of Anthropology, UVic (Outside Member)
• Dr. Zachary Laksman, Department of Medicine, University of British Columbia (Outside Member)

External Examiner

• Dr. Michelle Collins, Department of Anatomy, Physiology, and Pharmacology, University of Saskatchewan

Chair of Oral Examination

• Dr. Timothy Iles, Department of Pacific and Asian Studies, UVic

Abstract

Cardiomyocyte structure and function depend on the coordinated organization of ion channels and transporters, signaling proteins, and the cytoskeleton. Ankyrin-B (ANKB) is a scaffolding protein for structurally diverse membrane-associated proteins, including a handful of key ion handling and structural proteins within cardiomyocytes. Moreover, ANKB variants had previously been linked to arrhythmias. A collaborative study from the Swayne and Arbour groups, along with the Gitxsan First Nation, discovered an association between a novel ANKB variant p.S646F and arrythmia, as well as cardiomyopathy, congenital heart malformation, and sudden cardiac death. Furthermore, ANKB p.S646F exhibited decreased expression and altered localization in the H9c2 cardiomyoblast cell line, and also reduced cell viability. While previous work on other ANKB variants focused primarily on electrical disturbances, the association between ANKB p.S646F and cardiomyopathy highlighted ANKB’s potential role in the regulation of cardiomyocyte morphological development, which has been relatively unexplored. Additionally, a comprehensive unbiased proteomics study of ANKB protein interactions in cardiomyocytes had never been performed, hindering fulsome understanding of the potential role for ANKB in cardiomyocyte structure.

I therefore investigated the hypothesis that ANKB participates in protein-protein interaction networks that regulate cardiomyocyte structure, and that these interactions are disrupted by the ANKB p. S646F variant. To properly address this hypothesis, I first needed to determine the appropriate cell culture model. I therefore evaluated two possibilities: differentiation of the rat H9c2 cardiomyoblast cell line and mouse single heart primary cardiomyocyte cultures. As follows, in Chapter 2, I detail my thorough characterization of H9c2 cardiomyoblast cell line differentiation with retinoic acid. In Chapter 3, I outline a novel protocol I developed for neonatal mouse single heart primary cardiomyocytes. Finally, in Chapter 4, I outline the results of my investigation of ANKB protein-protein interactions and the impact of the ANKB p.S646F variant on these interactions. Relatedly, in Chapter 4, I also explore the role of ANKB in cardiomyocyte structure.

In Chapter 2, I determined that differentiated H9c2 cell phenotypes do not fully recapitulate those of mature cardiomyocytes. Notably, key cardiomyocyte structural elements are lacking in differentiated H9c2 cells and therefore this is not an optimal model for the investigation of
the impact of ANKB p. S646F on cardiomyocyte structure. Despite their drawbacks for structural studies, undifferentiated H9c2 cells provided the protein yield essential for the discovery of ANKB protein-protein interactions, performed in Chapter 4. They also allowed for insight into the localization of ANKB and its protein interactors in an immature cardiomyoblast cell context, which is useful for inferring roles in cardiomyocyte structural development. To next test a model more likely to allow for structural analysis, in Chapter 3, I established the first single-heart primary cardiomyocyte culture protocol, which also enables genotype- and sex-specific analysis. In Chapter 3, I characterized the cell type proportions within these cultures, containing fibroblasts and endothelial cells as well as cardiomyocytes and analyzed the baseline protein expression of markers for cardiomyocytes, fibroblasts, and endothelial cells. I used this model in Chapter 4 to explore the role of ANKB in cardiomyocyte structure. I demonstrated that partial and near-complete loss of ANKB disrupts sarcomere organization. Additionally, in Chapter 4, I uncovered novel ANKB protein-protein interactions using an unbiased proteomic approach and investigated how ANKB p.S646F affects these protein-protein interaction networks. My bioinformatics analysis found that the ANKB interactome contains proteins associated with cytoskeletal organization and sarcomere structure. ANKB p.S646F altered these interaction networks; ANKB p.S646F increased a newly-discovered interaction with ⍺-actinin.

Overall, my thesis work established improved and novel protocols for the evaluation of mechanisms underlying cardioymyocyte structure and, importantly, discovered a new role for ANKB in the regulation of cardiomyocyte structure, in part through a novel interaction with ⍺-actinin that is enhanced by ANKB p.S646F. Together this body of work has broad impact for the study of genetic and environmental influences on cardiomyocyte structure, as well as deepening understanding of the role of ANKB in cardiomyocytes, identifying a novel structural role. Finally, this work provides new insight into how ANKB p.S646F affects cardiomyocyte biology, suggesting that it disrupts cardiomyocyte morphological development, which is important for understanding the role of ANKB p.S646F in cardiomyopathy.