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

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 h after transfection, whereas we observed a greater proportion of cycling, Ki67-positive cells at 48 h after 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. This work reveals that 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.

Mechanisms underlying the role of ankyrin-B in cardiac and neurological health and disease

“…Given the cases of structural malformations it is also important to investigate the potential role of ANKB in cardiac cell development. Of note, the p.S6464F variant is less stable and experiences reduced expression only in undifferentiated H9c2 cells suggesting this variant's impact to cells occurs during their development ( 94 ) and provides some additional rationale behind investigating ANKB's roles during cardiac development. As seen in patients with both the p.S646F ( 7 ) and p.E1458G ( 4 ) variants there appears to be an age-related effect.…”

“…Evidence underlying ANKB's role in cell proliferation and survival has been highlighted by the p.S646F variant. In H9c2 cells, a cell model with similar traits of primary cardiomyocytes ( 93 ), expression of the p.S646F variant resulted in decreased cellular viability and proliferation ( 94 ). Using a cardiac-specific conditional knockout model, Roberts et al .…”

“…Evidence underlying ANKB's role in cell proliferation and survival has been highlighted by the p.S646F variant. In H9c2 cells, a cell model with similar traits of primary cardiomyocytes (93), expression of the p.S646F variant resulted in decreased cellular viability and proliferation (94). Using a cardiac-specific conditional knockout model, Roberts et al found that loss of Ank2 in the heart leads to severe cardiac remodeling resulting in ventricular dilation, fibrosis, bradycardia, QTc prolongation, and increased susceptibility to catecholamineinduced ventricular arrythmias (44).…”