The Cardiac Role of Filamin C

项目摘要 细丝蛋白C（Filamin C，FLNC）是一种肌肉特异性肌动蛋白结合蛋白，定位于Z盘和闰盘 与β1整合素和肌聚糖在肋节处相互作用。中的多个突变 FLNC与人类心肌病相关，突出了其对心脏功能的重要性。个人 FLNC基因（F106 L/R991*（无效等位基因））中的复合杂合变体发生小儿扩张 心肌病（DCM）。有趣的是，仅携带一种FLNC（F106 L或R991*）变体的个体不 表现出任何心肌病的迹象;这表明F106 L是一种功能缺失突变。很少有人知道， FLNC在心肌细胞（CM）中的特定作用，或F106 L突变导致缺失的机制 以及具有FLNC F106/null复合杂合等位基因的个体如何发展DCM。删除 小鼠FLNC最后8个外显子的缺失导致围产期死亡。然而，考虑到多个不同的FLNC 突变导致心肌病，在这种FLNC缺陷小鼠模型中缺乏心脏表型， 令人费解。正如作者所指出的，截短的FLNC蛋白在这种突变小鼠中仍然表达，这表明， 突变的FLNC等位基因是亚型的。因此，真正的无效FLNC小鼠模型和FLNC CM特异性的小鼠模型可以在小鼠中表达。 基因敲除（KO）小鼠模型对于全面了解FLNC在心脏中的作用至关重要。在 此外，在CM和体内动物模型中的研究对于理解潜在的分子基础是必不可少的。 FLNC中F106 L/无效突变引起的DCM。为了解决这些问题，我们创建了一个 floxed FLNC小鼠系，并将其用于产生FLNC全局KO（gKO）小鼠模型，以及组成型 (cKO)和诱导型（icKO）FLNC CM特异性KO小鼠。与已报道的FLNC亚型小鼠相反， 我们的FLNC gKO小鼠模型在E10.5时是致死的，并表现出严重的胸部水肿和CM降低 增殖此外，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学表型，表明在FLNC gKO小鼠中致死的主要原因， FLNC gKO小鼠是由于CM中FLNC的丢失。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 CM肌节和肋节完整性、心脏形态发生和正常心脏功能， FLNC F106 L突变是一种功能丧失型突变，它损害了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在发育和成年心肌中的作用， cKO和icKO FLNC CM特异性KO小鼠的心脏形态发生、结构和功能，以及 心肌病进展;和2.为了阐明F106 L FLNC突变导致 功能丧失以及F106 L/null复合杂合等位基因个体如何通过以下途径发展为儿科DCM： F107 L/null和F107 L/F107 L突变敲入小鼠和人胚胎干细胞衍生的CM的分析 含有FLNC F106 L/-、FLNC F106 L/F106 L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.