原理:心肌细胞(CMs)在出生后成熟时经历了巨大的结构和功能变化;然而,监管机制仍然非常不清楚。Cypher/Z带选择性剪接的PDZ基序蛋白(ZASP)是维持Z盘稳定性的必需肌节成分。小鼠Cypher的缺失和人ZASP中的突变导致扩张型心肌病(DCM)。尚未回答Cypher/ZASP是否参与CM成熟并因此影响心脏功能。方法:免疫荧光,透射电子显微镜,实时定量PCR,并利用Westernblot鉴定Cypher在CM成熟中的作用。随后,RNA测序和生物信息学分析预测血清反应因子(SRF)是关键调节因子。使用编码SRF的腺病毒或腺相关病毒进行抢救实验,在体外和体内。通过G-肌动蛋白/F-肌动蛋白分级分离阐明了分子机制,核-细胞质提取,肌动蛋白分解分析,和共沉淀测定。结果:Cypher缺失导致线粒体肌节同工型开关受损和形态异常,横小管,和插层光盘。RNA测序分析揭示了与肌节组装相关的关键基因的显著失调,线粒体代谢,和没有Cypher的电生理学。此外,预测SRF是介导转录差异的关键转录因子。随后的抢救实验表明,在出生后的关键时期,SRF的重新表达有效地纠正了Cypher耗竭小鼠的CM成熟缺陷,并显着改善了心脏功能。机械上,Cypher缺乏导致F-肌动蛋白的不稳定和G-肌动蛋白水平的显着增加,从而阻碍肌钙蛋白相关转录因子A(MRTFA)的核定位,并随后启动SRF转录。结论:Cypher/ZASP通过肌动蛋白介导的MRTFA-SRF信号在CM成熟中起着至关重要的作用。提示了CM成熟异常与DCM迟发之间的联系,提供对DCM发病机制和潜在治疗策略的进一步见解。
Rationale: Cardiomyocytes (CMs) undergo dramatic structural and functional changes in postnatal maturation; however, the regulatory mechanisms remain greatly unclear. Cypher/Z-band alternatively spliced PDZ-motif protein (ZASP) is an essential sarcomere component maintaining Z-disc stability. Deletion of mouse Cypher and mutation in human ZASP result in dilated cardiomyopathy (DCM). Whether Cypher/ZASP participates in CM maturation and thereby affects cardiac function has not been answered. Methods: Immunofluorescence, transmission electron microscopy, real-time quantitative PCR, and Western blot were utilized to identify the role of Cypher in CM maturation. Subsequently, RNA sequencing and bioinformatics analysis predicted serum response factor (SRF) as the key regulator. Rescue experiments were conducted using adenovirus or adeno-associated viruses encoding SRF, both in vitro and in vivo. The molecular mechanisms were elucidated through G-actin/F-actin fractionation, nuclear-cytoplasmic extraction, actin disassembly assays, and co-sedimentation assays. Results: Cypher deletion led to impaired sarcomere isoform switch and morphological abnormalities in mitochondria, transverse-tubules, and intercalated discs. RNA-sequencing analysis revealed significant dysregulation of crucial genes related to sarcomere assembly, mitochondrial metabolism, and electrophysiology in the absence of Cypher. Furthermore, SRF was predicted as key transcription factor mediating the transcriptional differences. Subsequent rescue experiments showed that SRF re-expression during the critical postnatal period effectively rectified CM maturation defects and notably improved cardiac function in Cypher-depleted mice. Mechanistically, Cypher deficiency resulted in the destabilization of F-actin and a notable increase in G-actin levels, thereby impeding the nuclear localisation of myocardin-related transcription factor A (MRTFA) and subsequently initiating SRF transcription. Conclusion: Cypher/ZASP plays a crucial role in CM maturation through actin-mediated MRTFA-SRF signalling. The linkage between CM maturation abnormalities and the late-onset of DCM is suggested, providing further insights into the pathogenesis of DCM and potential treatment strategies.