Intracellular calcium current disorder and disease phenotype in OBSCN mutant iPSC-based cardiomyocytes in arrhythmogenic right ventricular cardiomyopathy.
Intracellular calcium current disorder and disease phenotype in OBSCN mutant iPSC-based cardiomyocytes in arrhythmogenic right ventricular cardiomyopathy.
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DOI：
10.7150/thno.45172
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发表时间：
2020
期刊：
影响因子：
12.4
通讯作者：
Zhang S
中科院分区：
文献类型：
--
作者：
Chen P;Xiao Y;Wang Y;Zheng Z;Chen L;Yang X;Li J;Wu W;Zhang S
暗蛋白参与横纹肌的发育和功能性肌浆网的维持。然而，obscurin在致心律失常性右心室心肌病（ARVC）中的作用尚不清楚。我们的目的是研究新的obscurin突变在ARVC的发病机制和潜在的机制。研究方法：我们通过逆转录病毒重编程外周血单个核细胞产生诱导多能干细胞（iPSC），该细胞分离自一名46岁女性，诊断为ARVC，携带OBSCN突变。通过透射电镜、电生理学描述、免疫荧光染色和油红O染色确定细胞分化为功能性iPSC-CMs的表型。通过生物信息学分析进行分子表征，并通过定量实时聚合酶链反应（qRT-PCR）和蛋白质印迹法进行鉴定。结果如下：OBSCN中的ARVC-iPSC-CMs突变显示脂质显著积聚、多形性增加、不规则Z带和L型钙电流增加。功能富集分析确定了参与粘着斑和结构形成的途径; ARVC组中脂肪细胞因子和PPAR信号通路也被激活。此外，我们的超高分辨率显微镜，qRT-PCR和Western blotting结果证实，突变OBSCN蛋白及其锚蛋白Ank 1. 5表现出结构紊乱和表达减少，但有连接蛋白N-Cadherin的表达增加。进一步分析显示，ARVC-iPSC-CM中其他桥粒蛋白的基因表达也降低，但一些脂肪形成途径相关蛋白（PPARγ、C/EBPα和FABP 4）增加。结论：OBSCN中的一种新的移码突变导致表型改变，伴随着其锚定蛋白Ank1.5的破坏定位和表达降低。此外，脂质积聚，脂肪纤维化面积增加，心肌结构紊乱，可能导致钙通道相关心肌收缩节律障碍。这些观察结果表明通过治疗性调节OBSCN表达来减弱ARVC进展的可能性。
Obscurin participates in the development of striated muscles and maintenance of the functional sarcoplasmic reticulum. However, the role of obscurin in arrhythmogenic right ventricular cardiomyopathy (ARVC) is not well understood. We aimed to study the novel obscurin mutations in the pathogenesis of ARVC and the underlying mechanisms. Methods: We generated induced pluripotent stem cells (iPSC) through retroviral reprogramming of peripheral blood mononuclear cells isolated from a 46-year-old female diagnosed with ARVC, carrying a mutation in OBSCN. The cells differentiated into functional iPSC-based cardiomyocytes (iPSC-CMs), whose phenotype was determined by transmission electron microscopy, electrophysiological description, immunofluorescence staining, and Oil Red O staining. Molecular characterization was performed by bioinformatic analyses, and identification by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. Results: ARVC-iPSC-CMs mutation in OBSCN showed significant accumulation of lipids, increased pleomorphism, irregular Z-bands, and increased L type calcium currents. Functional enrichment analysis identified pathways involved in focal adhesion and structure formation; the adipocytokines and PPAR signaling pathways were also activated in the ARVC group. Moreover, our results from ultra-high-resolution microscopy, qRT-PCR and Western blotting confirmed that the mutant OBSCN protein and its anchor protein, Ank1.5, showed structural disorder and decreased expression, but there was increased expression of junctional protein N-Cadherin. Further analysis revealed the gene expression of other desmosomal proteins in ARVC-iPSC-CMs was also decreased but some adipogenesis pathway-related proteins (PPARγ, C/EBPα, and FABP4) were increased. Conclusion: A novel frameshift mutation in OBSCN caused phenotypic alteration accompanied by disrupted localization and decreased expression of its anchoring protein Ank1.5. Furthermore, there was an accumulation of lipids with an increase in fatty fibrosis area and myocardial structural disorder, possibly leading to dysrhythmia in calcium channel-related myocardial contraction. These observations suggested the possibility of attenuating ARVC progression by therapeutic modulation of OBSCN expression.
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Marston S
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64.8
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Kontrogianni-Konstantopoulos A;Ackermann MA;Bowman AL;Yap SV;Bloch RJ
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