Department of Cellular and Molecular Medicine and Sarver Molecular Cardiovascular Research Program (M.C., S.M.N., C.C., A.A.W., C.C.G.).
Department of Medicine (I.R.C., E.B.J.).
Circulation. 2018 Feb 6;137(6):605-618. doi: 10.1161/CIRCULATIONAHA.117.028976. Epub 2017 Nov 3.
Gap junction remodeling is well established as a consistent feature of human heart disease involving spontaneous ventricular arrhythmia. The mechanisms responsible for gap junction remodeling that include alterations in the distribution of, and protein expression within, gap junctions are still debated. Studies reveal that multiple transcriptional and posttranscriptional regulatory pathways are triggered in response to cardiac disease, such as those involving RNA-binding proteins. The expression levels of FXR1 (fragile X mental retardation autosomal homolog 1), an RNA-binding protein, are critical to maintain proper cardiac muscle function; however, the connection between FXR1 and disease is not clear.
To identify the mechanisms regulating gap junction remodeling in cardiac disease, we sought to identify the functional properties of FXR1 expression, direct targets of FXR1 in human left ventricle dilated cardiomyopathy (DCM) biopsy samples and mouse models of DCM through BioID proximity assay and RNA immunoprecipitation, how FXR1 regulates its targets through RNA stability and luciferase assays, and functional consequences of altering the levels of this important RNA-binding protein through the analysis of cardiac-specific FXR1 knockout mice and mice injected with 3xMyc-FXR1 adeno-associated virus.
FXR1 expression is significantly increased in tissue samples from human and mouse models of DCM via Western blot analysis. FXR1 associates with intercalated discs, and integral gap junction proteins Cx43 (connexin 43), Cx45 (connexin 45), and ZO-1 (zonula occludens-1) were identified as novel mRNA targets of FXR1 by using a BioID proximity assay and RNA immunoprecipitation. Our findings show that FXR1 is a multifunctional protein involved in translational regulation and stabilization of its mRNA targets in heart muscle. In addition, introduction of 3xMyc-FXR1 via adeno-associated virus into mice leads to the redistribution of gap junctions and promotes ventricular tachycardia, showing the functional significance of FXR1 upregulation observed in DCM.
In DCM, increased FXR1 expression appears to play an important role in disease progression by regulating gap junction remodeling. Together this study provides a novel function of FXR1, namely, that it directly regulates major gap junction components, contributing to proper cell-cell communication in the heart.
缝隙连接重塑是涉及自发性室性心律失常的人类心脏病的一个一致特征。导致缝隙连接重塑的机制仍存在争议，包括缝隙连接分布和蛋白表达的改变。研究表明，多种转录和转录后调控途径被触发以应对心脏疾病，例如涉及 RNA 结合蛋白的途径。RNA 结合蛋白 FXR1（脆性 X 智力低下综合征 1 号同源物）的表达水平对维持心肌的正常功能至关重要；然而，FXR1 与疾病之间的联系尚不清楚。
为了确定调节心脏疾病中缝隙连接重塑的机制，我们试图通过生物素邻近测定法和 RNA 免疫沉淀法鉴定 FXR1 在人左心室扩张型心肌病（DCM）活检样本和 DCM 小鼠模型中的功能特性以及 FXR1 的直接靶标，研究 FXR1 如何通过 RNA 稳定性和荧光素酶测定来调节其靶标，以及通过分析心脏特异性 FXR1 敲除小鼠和注射 3xMyc-FXR1 腺相关病毒的小鼠来改变这种重要的 RNA 结合蛋白的水平所产生的功能后果。
通过 Western blot 分析，发现 FXR1 在人源和鼠源 DCM 模型的组织样本中的表达显著增加。FXR1 与闰盘相关，通过生物素邻近测定法和 RNA 免疫沉淀法鉴定到 Cx43（连接蛋白 43）、Cx45（连接蛋白 45）和 ZO-1（紧密连接蛋白-1）等间隙连接蛋白是 FXR1 的新型 mRNA 靶标。我们的研究结果表明，FXR1 是一种多功能蛋白，参与心脏肌肉中其 mRNA 靶标的翻译调控和稳定。此外，通过腺相关病毒将 3xMyc-FXR1 导入小鼠会导致缝隙连接的重新分布并促进室性心动过速，这表明在 DCM 中观察到的 FXR1 上调具有功能意义。
在 DCM 中，FXR1 表达的增加似乎通过调节缝隙连接重塑在疾病进展中发挥重要作用。总之，这项研究提供了 FXR1 的一个新功能，即它直接调节主要的缝隙连接成分，有助于心脏细胞间的正常通讯。
