Ankyrin-B p.S646F undergoes increased proteasome degradation and reduces cell viability in the H9c2 rat ventricular cardiomyoblast cell line

Date

2019-11-21

Authors

Chen, Lena
Choi, Catherine S.W.
Sanchez-Arias, Juan C.
Arbour, Laura T.
Swayne, Leigh Anne

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Abstract

Ankyrin-B (AnkB) is scaffolding protein that anchors integral membrane proteins to the cardiomyocyte cytoskeleton. We recently identified an AnkB variant, AnkB p.S646F (ANK2 c.1937 C>T) associated with a phenotype ranging from predisposition for cardiac arrhythmia to cardiomyopathy. AnkB p.S646F exhibited reduced expression levels in the H9c2 rat ventricular- derived cardiomyoblast cell line relative to wildtype AnkB. Here we demonstrate that AnkB is regulated by proteasomal degradation and proteasome inhibition rescues AnkB p.S646F expression levels in H9c2 cells, although this effect is not conserved with differentiation. We also compared the impact of wildtype AnkB and AnkB p.S646F on cell viability and proliferation. AnkB p.S646F expression resulted in decreased cell viability at 30 hours post-transfection, whereas we observed a greater proportion of cycling, Ki67-positive cells at 48 h post-transfection. Notably, the number of GFP-positive cells was low, and was consistent between wildtype AnkB and AnkB p.S646F expressing cells, suggesting that AnkB and AnkB p.S646F affected paracrine communication between H9c2 cells differentially. In summary, this work reveals AnkB levels are regulated by the proteasome, and that AnkB p.S646F compromises cell viability. Together these findings provide key new insights into the putative cellular and molecular mechanisms of AnkB-related cardiac disease.

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Keywords

Ankyrin-B, proteasome, cardiomyocytes, cell viability

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