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.
Highlights
Ankyrin-B (AnkB) is a large 220 kDa scaffolding protein that plays a critical role in tethering the contractile machinery in a specialized region of the cardiomyocyte cell membrane (Mohler et al 2007b; Koenig and Mohler 2017; El Refaey and Mohler 2017)
To distinguish between two primary pathways of protein degradation, wildtype AnkB-GFP expressing H9c2 cells were incubated with a proteasomal inhibitor, PS-341, or a lysosomal inhibitor, Bafilomycin A1 (BafA)
We investigated the mechanism of AnkB degradation as well as the impact of a novel variant AnkB p.S646F on this process, and on cell viability in H9c2 rat ventricular cardiomyoblast cells
Summary
Ankyrin-B (AnkB) is a large 220 kDa scaffolding protein that plays a critical role in tethering the contractile machinery (i.e. ion channels and transporters) in a specialized region of the cardiomyocyte cell membrane (Mohler et al 2007b; Koenig and Mohler 2017; El Refaey and Mohler 2017). Variants in the ANK2 gene can lead to “Ankyrin-B Syndrome” mainly characterized by a predisposition to cardiac arrhythmia and an increased risk of sudden cardiac death (Mohler et al 2004, 2007b). The phenotype of AnkB+/- mice, partially recapitulated pathologies observed in human ANK2 gene loss of function variants, such as predisposition to arrhythmia (Mohler et al 2007a). AnkB knockout mice are postnatal lethal, where AnkB knockout cardiomyocytes display abnormal contraction as well as irregular calcium homeostasis (Scotland et al 1998; Mohler et al 2002), which could result from altered development at the cellular and molecular level. C>T), a mutation in the membrane binding domain (MBD), whose carriers displayed variety of clinical features including long QT syndrome, dilated cardiomyopathy with associated sudden death, congenital heart malformation, Wolff–Parkinson–White syndrome, and seizures (Swayne et al 2017)
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