Fam13c功能及其P181L突变致扩张型心肌病的分子机制研究

Genetic factor is essential for dilated cardiomyopathy (DCM) pathogenesis. Recently, the P181L mutation of fam13c gene has been identified by our group through the Whole Exome Sequencing (WES) analysis in a pedigree of familial DCM. The P181L mutation showed loss of function with dominant-negative effect in H9c2 cells. IP-protein mass-spectrography analysis identified 430 interacting protein including cytoskeleton actin and DCM signaling proteins. Zebrafish embryos with fam13c knockout by CRISPR-Cas9 system show significant dilated heart, less cardiac muscle fibers and reduced actin/TPM3 expression appear in the mutant embryo. These results suggest that P181L leads function loss and DCM phenotype through downregulation of cytoskeleton actin and DCM signaling proteins. We plan to study the gene function by analyzing fam13c gene mutation, transfecting mutant plasmid and establishing knockout or mutant mouse and zebrafish. Echocardiography and MRI analysis will be used to investigate the role of fam13c and its mutation for cardiac structure and function in model organism. We are also going to use assays of Co-IP, IF, electron microscope, confocal microscope and transcriptome to identify the interacting proteins. In addition, we plan to perform recovery experiments to explore the possibility of DCM therapy by fam13c using microinjection wild type fam13c to embryo or injection of adeno associated virus to mouse heart muscle.

遗传因素在扩张型心肌病(DCM)发生发展中起着重要作用。课题组前期发现：fam13c基因新突变P181L伴随DCM表型分离；H9c2细胞中研究发现P181L功能缺失，具“显性负效应”；IP-质谱鉴定到以actin细胞骨架通路及DCM通路为主的互作蛋白430个；CRISPR-Cas9敲除fam13c斑马鱼胚胎心脏扩大、电镜发现心肌纤维减少、qPCR发现actin、TPM3等mRNA下调。前期工作表明P181L致fam13c功能降低/缺失，可能影响actin细胞骨架通路及DCM通路等，导致DCM。本项目拟通过突变分析、质粒转染、基因敲除小鼠/斑马鱼和突变小鼠、超声、MRI、Co-IP、免疫荧光、电镜、转录组测序等研究，明确fam13c功能、关键互作蛋白及P181L致DCM的具体分子机制；野生型fam13c显微注射胚胎/腺相关病毒心肌内注射小鼠恢复试验等研究，探索fam13c治疗DCM的可行性。

遗传因素在扩张型心肌病(DCM)发生发展中起着重要作用。本项目在DCM患者中新发现了Fam13c P181L突变。构建了Fam13c基因敲除(KO)斑马鱼模型，发现其胚胎心脏扩大，类似DCM表型。构建了P201L(对应人类Fam13c P181L)突变敲入及KO小鼠模型，发现突变及KO小鼠左心室功能随年龄增长逐渐下降，左心室扩大，出现DCM表型。电镜检测发现突变及敲除小鼠心肌肌小节肿胀、疏松，结构紊乱。转录组测序(RNA-Seq)分析结果发现，与野生型(WT)小鼠相比，KO小鼠心脏中基因表达差异显著小于突变小鼠，且突变小鼠心脏中差异基因富集到“心肌收缩”信号通路，表明Fam13c基因在心脏发育和功能调控中发挥重要作用，敲除后导致心脏中基因表达异常，而P201L突变通过影响“心肌收缩”信号通路中大量关键基因表达，进一步导致其功能障碍。采用同位素标记相对和绝对定量(iTRAQ)蛋白组学检测小鼠心脏中差异蛋白，发现与WT小鼠相比，突变及KO小鼠心脏组织中心肌收缩纤维成分、肌小节、I带、Z盘、A带等蛋白显著降低。进行了染色质免疫共沉淀-测序(ChIP-Seq)分析，发现了Fam13c可能调控的多个基因，包括已报道的DCM致病基因，如SGCD、CACNA1S、CACNA1C、NRXN1及ADAM17等。构建了Fam13c过表达载体，转染细胞后进行免疫沉淀(IP)，蛋白质谱鉴定到Fam13c相互作用候选蛋白35个，其中多个为DNA和RNA结合蛋白，在基因表达调控中发挥关键作用。采用免疫共沉淀(Co-IP)、免疫荧光(IF)等，验证到HNRNPU等与Fam13c相互作用，且在细胞内有共定位。HNRNPU主要位于胞核内，Fam13c过表达后，HNRNPU与Fam13c在胞浆内大量重叠，表明Fam13c诱导其表达由胞核迁移至胞浆。有文献报道心脏中敲除HNRNPU的小鼠会发展成DCM并致死。本项目研究结果表明Fam13c可能通过结合HNRNPU等蛋白参与基因表达调控，在心脏发育和功能调控中发挥重要作用，其P181L突变影响其功能，导致心肌收缩关键基因表达降低，心脏收缩功能受损，最终导致DCM。研究结果为阐明Fam13c基因功能及其突变导致DCM机制的基础与转化研究奠定了基础。