Ankyrin-B regulates cardiomyocyte sarcomere organization and its protein-protein interactions are significantly altered by p.S646F expression

Ankyrin-B (ANKB) is a large (220 kDa) multi-domain membrane-localized cytoplasmic protein that acts as a scaffold coordinating cytoskeletal, ion channel, and transporter proteins critical for proper cardiomyocyte structure and function. There is a prevalence of structural cardiac abnormalities within the cohort of the novel ANKB p.S646F variant that we previously identified in two multigenerational Gitxsan First Nation families, suggesting a role for ANKB in cardiac structure. To investigate the impact of ANKB on cardiomyocyte structural organization, we imaged sarcomere structure in primary cardiomyocytes from cardiac-specific ANKB heterozygous (expressing lower levels of ANKB) and full knock-out mice using confocal microscopy. We observed marked disruption of sarcomere structure in primary cardiac cultures associated with lower ANKB expression, suggesting ANKB is required for proper development of sarcomeres. To shed light on the underlying mechanisms, we next employed an unbiased proteomics approach to identify novel ANKB protein-protein interactions. This analysis uncovered a novel ANKB interaction with ⍺-actinin-1, an actin-binding protein that stabilizes focal adhesions essential for development of cardiomyocyte structure. Western blot comparison of ⍺-actinin-1 co-precipitation with wildtype ANKB and ANKB p.S646F, revealed increased ⍺-actinin-1 enrichment with ANKB p.S646F. Bioinformatics analysis of the ANKB interactome revealed enrichment of cytoskeletal organization, sarcomere structure, and chaperone proteins, and marked changes with ANKB p.S646F. Together findings point to a novel role for ANKB in the regulation of cardiomyocyte structural development and support additional investigation into a link between p.S646F and cardiac structural abnormalities.