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.

中文翻译：

---

锚蛋白B p.S646F在H9c2大鼠心室成肌细胞系中经历增加的蛋白酶体降解并降低细胞活力。

锚蛋白B（AnkB）是将整合膜蛋白锚定在心肌细胞骨架上的支架蛋白。我们最近发现了AnkB变体AnkB p.S646F（ANK2 c.1937 C> T），其表型从心律失常的易感性到心肌病的范围有关。相对于野生型AnkB，AnkB p.S646F在H9c2大鼠心室心肌细胞系中显示出降低的表达水平。在这里，我们证明了AnkB受蛋白酶体降解的调节，蛋白酶体的抑制作用可以挽救H9c2细胞中的AnkB p.S646F表达水平，尽管这种作用在分化过程中并不保守。我们还比较了野生型AnkB和AnkB p.S646F对细胞活力和增殖的影响。AnkB p.S646F表达导致转染后30 h细胞活力降低，而在转染后48小时，我们观察到更大比例的循环Ki67阳性细胞。值得注意的是，GFP阳性细胞的数量很低，并且在野生型AnkB和AnkB p.S646F表达细胞之间是一致的，这表明AnkB和AnkB p.S646F差异地影响H9c2细胞之间的旁分泌通讯。这项工作揭示了蛋白酶体调节AnkB的水平，而AnkB p.S646F损害了细胞的生存能力。在一起，这些发现提供了关键的新见解，推测与AnkB相关的心脏病的细胞和分子机制。S646F差异影响H9c2细胞之间的旁分泌通讯。这项工作揭示了蛋白酶体调节AnkB的水平，而AnkB p.S646F损害了细胞的生存能力。在一起，这些发现提供了关键的新见解，推测与AnkB相关的心脏病的细胞和分子机制。S646F差异影响H9c2细胞之间的旁分泌通讯。这项工作揭示了蛋白酶体调节AnkB的水平，而AnkB p.S646F损害了细胞的生存能力。在一起，这些发现提供了关键的新见解，推测与AnkB相关的心脏病的细胞和分子机制。