Division of Cardiovascular Medicine, Department of Medicine (M.E.L., D.J.-M., H.Z., K.S., D.A., C.Z., A.R., R.M., S.A., S.S., C.C., J.P., E.A.A., M.T.W.), Stanford University School of Medicine, CA.
Cell Sciences Imaging Facility (C.E.), Stanford University School of Medicine, CA.
Circ Genom Precis Med. 2021 Dec;14(6):e003419. doi: 10.1161/CIRCGEN.121.003419. Epub 2021 Nov 22.
ACTN2 (alpha-actinin 2) anchors actin within cardiac sarcomeres. The mechanisms linking mutations to myocardial disease phenotypes are unknown. Here, we characterize patients with novel mutations to reveal insights into the physiological function of ACTN2.
Patients harboring ACTN2 protein-truncating variants were identified using a custom mutation pipeline. In patient-derived iPSC-cardiomyocytes, we investigated transcriptional profiles using RNA sequencing, contractile properties using video-based edge detection, and cellular hypertrophy using immunohistochemistry. Structural changes were analyzed through electron microscopy. For mechanistic studies, we used co-immunoprecipitation for ACTN2, followed by mass-spectrometry to investigate protein-protein interaction, and protein tagging followed by confocal microscopy to investigate introduction of truncated ACTN2 into the sarcomeres.
Patient-derived iPSC-cardiomyocytes were hypertrophic, displayed sarcomeric structural disarray, impaired contractility, and aberrant Ca-signaling. In heterozygous indel cells, the truncated protein incorporates into cardiac sarcomeres, leading to aberrant Z-disc ultrastructure. In homozygous stop-gain cells, affinity-purification mass-spectrometry reveals an intricate ACTN2 interactome with sarcomere and sarcolemma-associated proteins. Loss of the C-terminus of ACTN2 disrupts interaction with ACTN1 (alpha-actinin 1) and GJA1 (gap junction protein alpha 1), 2 sarcolemma-associated proteins, which may contribute to the clinical arrhythmic and relaxation defects. The causality of the stop-gain mutation was verified using CRISPR-Cas9 gene editing.
Together, these data advance our understanding of the role of ACTN2 in the human heart and establish recessive inheritance of truncation as causative of disease.
ACTN2（α-辅肌动蛋白 2）将肌动蛋白锚定在心肌肌节内。将突变与心肌疾病表型联系起来的机制尚不清楚。在这里，我们对携带新突变的患者进行了特征描述，以深入了解 ACTN2 的生理功能。
使用定制的突变分析管道鉴定携带 ACTN2 蛋白截断变异的患者。在患者来源的 iPSC 心肌细胞中，我们使用 RNA 测序研究转录谱，使用基于视频的边缘检测研究收缩性能，使用免疫组织化学研究细胞肥大。通过电子显微镜分析结构变化。对于机制研究，我们使用 ACTN2 的共免疫沉淀，随后进行质谱分析以研究蛋白-蛋白相互作用，以及蛋白标记和共聚焦显微镜以研究截断的 ACTN2 引入肌节。
患者来源的 iPSC 心肌细胞发生肥大，表现出肌节结构紊乱、收缩功能受损和异常的 Ca 信号转导。在杂合插入缺失细胞中，截断蛋白整合到心肌肌节中，导致 Z 盘超微结构异常。在纯合终止增益细胞中，亲和纯化质谱分析揭示了一个复杂的 ACTN2 相互作用组，包括与肌节和肌膜相关的蛋白。ACTN2 的 C 端缺失破坏了与 ACTN1（α-辅肌动蛋白 1）和 GJA1（间隙连接蛋白α 1）的相互作用，这 2 种肌膜相关蛋白可能导致心律失常和舒张功能缺陷的临床表现。使用 CRISPR-Cas9 基因编辑验证了终止增益突变的因果关系。
综上所述，这些数据增进了我们对 ACTN2 在人类心脏中的作用的理解，并确立了截断的隐性遗传是疾病的原因。
