The Cardiac Role of Filamin C

项目总结 细丝C(Flc)是一种肌肉特异性肌动蛋白结合蛋白，定位于Z盘和间盘 并与β-1整合素和肌聚糖在胞间相互作用。中的多个突变 FlnC与人类心肌病有关，强调了它对心脏功能的重要性。个人 伴有FlnC基因(F106L/R991*(无效等位基因))复合杂合变异的儿童发生扩张症 心肌病(DCM)。有趣的是，只携带一种FlnC(F106L或R991*)变异的个体不会 表现出任何心肌病的迹象；表明F106L是一个功能丧失的突变。很少有人知道TO F106L突变导致心肌细胞缺失的机制 以及携带FlnC F106/空复合杂合等位基因的个体是如何发生DCM的。删除 在小鼠中，FlnC基因的最后8个外显子导致围产期死亡。然而，鉴于多个不同FLNC 突变会导致心肌病，在这种FlnC缺陷小鼠模型中缺乏心脏表型是 令人费解。正如作者注意到的，截短的FlnC蛋白在这个突变的小鼠中仍然表达，这表明 突变的FlnC等位基因是亚型的。因此，一个真正的空的FLNC鼠标模型和一个特定于FLNC CM的 基因敲除(KO)小鼠模型对于全面了解FlnC在心脏中的作用是必不可少的。在……里面 此外，对CMS和活体动物模型的研究对于理解其潜在的分子基础是必不可少的。 F106L/空突变引起的弥漫性心肌病为了解决这些问题，我们生成了一个 构建了FLNC小鼠系，并用其生成了一个FLNC全局KO(GKO)小鼠模型 (CKO)和可诱导(IcKO)FLNC CM特异性KO小鼠。与已报道的Flnc亚型小鼠不同， 我们的FLNC GKO小鼠模型在E10.5是致命的，并表现出严重的胸水和CM减少 扩散。此外，FLNC CKO小鼠在E10.5-E11.5之间死亡，并表现出基本上相同的 在FLNC GKO小鼠中观察到的形态表型，表明致死的主要原因 FLONC GKO小鼠是由于FLNC在CMS中丢失所致。我们还观察到成年Flnc icKO小鼠发生DCM 和进行性心力衰竭。因此，我们的假设是，FLNC在维持 肌节和胞间的完整性、心脏形态发生和正常心功能，以及 FlnC F106L突变是一种功能缺失突变，会破坏FlnC与肌动蛋白之间的特异性相互作用。 我们的具体目标是：1.通过分析FlnC在发育和成人心肌中的作用，确定FlnC在发育中和成年心肌中的作用 CKO和icKO FLNC CM特异性KO小鼠心脏形态发生、结构和功能的研究 心肌病的进展；以及2.阐明F106L Flc突变导致心肌病的机制 功能丧失以及携带F106L/空复合杂合等位基因的个体如何发展为儿童扩张型心肌病 F107L/Null和F107L/F107L突变敲入小鼠和人胚胎干细胞来源CMS的分析 包含FLNC F106L/-、FLNC F106L/F106L或FLNC-/-突变。

PROJECT SUMMARY Filamin C (FLNC) is a muscle-specific actin-binding protein, which localizes to the Z-disc and intercalated disc of cardiac muscle, and interacts with β1 integrin and sarcoglycans at the costamere. Multiple mutations in FLNC are associated with human cardiomyopathies, highlighting its importance for cardiac function. Individuals with compound heterozygous variants in the FLNC gene (F106L/R991*(a null allele)) develop pediatric dilated cardiomyopathy (DCM). Interestingly, individuals carrying only one FLNC (F106L or R991*) variant do not exhibit any signs of cardiomyopathy; suggesting that F106L is a loss-of-function mutation. Little is known as to the specific role of FLNC in cardiomyocytes (CMs), or mechanisms by which the F106L mutation leads to loss of function, and how individuals with FLNC F106/null compound heterozygous alleles develop DCM. Deletion of the last 8 exons of FLNC in mice results in perinatal lethality. However, given that multiple distinct FLNC mutations lead to cardiomyopathy, the absence of a cardiac phenotype in this FLNC-deficient mouse model is puzzling. As the authors noted, a truncated FLNC protein is still expressed in this mutant mice, suggesting that the mutant FLNC allele is hypomorphic. Thus, a true null FLNC mouse model and a FLNC CM-specific knockout (KO) mouse model are essential to comprehensively understand the role of FLNC in the heart. In addition, studies in CMs and in vivo animal models are essential to understand the molecular basis underlying the DCM caused by the F106L/null mutations in FLNC. To address these questions, we have generated a floxed FLNC mouse line and used it to generate a FLNC global KO (gKO) mouse model, as well as constitutive (cKO) and inducible (icKO) FLNC CM-specific KO mice. In contrast to the reported FLNC hypomorphic mouse, our FLNC gKO mouse model is lethal at E10.5 and exhibits severe chest edema and decreased CM proliferation. Moreover, FLNC cKO mice die between E10.5-E11.5 and exhibit an essentially identical morphological phenotype as observed in FLNC gKO mice, suggesting that the primary cause of lethality in FLNC gKO mice is due to loss of FLNC in CMs. We also observed that adult FLNC icKO mice develop DCM and progressive heart failure. Accordingly, our hypothesis is that FLNC plays an essential role in maintaining CM sarcomere and costamere integrity, cardiac morphogenesis, and normal cardiac function, and that the FLNC F106L mutation is a loss-of-function mutation and impairs specific interaction between FLNC and actin. Our Specific Aims are: 1. To determine the role of FLNC in the developing and adult myocardium by analyzing cKO and icKO FLNC CM-specific KO mice for heart morphogenesis, structure and function, and the progression of cardiomyopathy; and 2. To elucidate mechanisms by which the F106L FLNC mutation leads to loss of function and how individuals with F106L/null compound heterozygous alleles develop pediatric DCM by analysis of F107L/null and F107L/F107L mutation knock-in mice and human embryonic stem cell-derived CMs containing FLNC F106L/-, FLNC F106L/F106L, or FLNC -/- mutations.