Pathogenic Role of Selected Cardiac Myocyte- and Fibroblast-Specific Epigenetic Changes in Laminopathies

 描述(申请人提供)：目的是确定特定的表观遗传修饰在扩张型心肌病(DCM)椎板病变中的致病作用，该疾病包括由编码层蛋白A/C(LMNA)的LMNA基因突变引起的一系列疾病。心脏受累是影响横纹肌的一组患者的主要死亡原因。主要表现为扩张型心肌梗死、传导缺陷、心律失常和心源性猝死。染色质状态以特定细胞类型的方式调节基因表达。染色质的动态修饰对心脏发育至关重要，对成人心脏重构也很重要。在哺乳动物基因组中，LMNA以一种细胞类型特异性的方式与染色质相互作用，在~1,100-1,400个LMNA相关结构域(LAD)。LMNA参与了SUV39H1和EHMT2(G9a)对H3K9的三甲基化、HDACs对组蛋白的去乙酰化和DNMTs对DNA的甲基化。LADS主要位于异染色质基因座，富含H3K9me3和H3K27me3抑制性标记，少量激活组蛋白标记。初步数据显示，两周大的Lmna-/-小鼠心脏出现明显的表观遗传学改变，出现心功能障碍。LMNA和几个染色质标记在新生儿和成人心肌细胞(CMS)以及CMS和心脏成纤维细胞(CFs)中的表达存在差异。LMNAD300N在新生儿CMS中的表达会导致DCM和过早死亡，而其在成人CM中的表达会导致轻微的表型，这一发现与表观遗传学在新生儿CMS中的显著作用相一致，并与HDAC3缺失的表型效应相平行。最后，AAV9介导的LMNA表达改善了心功能和存活率。因此，我们推测LMNA在新生儿和成人CMS和CFs中具有不同的细胞类型特异性表观遗传学功能，这在LMNA突变引起的DCM中是致病的。为了验证这一假设，将使用特定的Cre缺失器小鼠，在新生(P3)和成年(P60)CMS和CFS这两种常见的心脏细胞类型中有条件地删除Lmna。在分离的CMS和CFS中，将通过特异组蛋白标记、CpG甲基化和整个转录组在心脏功能障碍和纤维化开始前后的全基因组启动子占有率的特征来确定DCM表型依赖和自主效应的表观遗传学基础(目标1)。在目标2中，将识别新生儿和成人CMS中负责人类DCM的LMNAD300A表达的不同表型效应的表观决定因素(与目标1中相同)，以及它们在开启和关闭LMNAD300N表达时的诱导和逆转。在目标3中，预防和逆转表观遗传修饰(如在AM 1中)和随后的DCM将取决于AAV9介导的LMNAWT在CM特异性和系统性LmNA-/-小鼠心脏中的表达。这些发现将深入了解LMNA在心脏结构和功能的细胞类型特异性表观遗传调节中的作用，以及表观遗传修饰在扩张型心肌病中的致病作用，并为靶向操作选定的表观遗传标记以精确控制、预防和逆转由LMNA突变引起的扩张型心肌病奠定基础。

 DESCRIPTION (provided by applicant): The objective is to determine the pathogenic role of specific epigenetic modifications in dilated cardiomyopathy (DCM) in laminopathies, which encompass a wide range of diseases caused by mutations in LMNA gene, encoding lamin A/C (LMNA). Cardiac involvement is the primary cause of death in a subset that affects striated muscles. It manifests as DCM, conduction defects, arrhythmias, and sudden cardiac death. Chromatin state regulates gene expression in a cell type-specific manner. Dynamic chromatin modifications are critical for cardiac development and important for adult heart remodeling. In the mammalian genomes, LMNA interacts with chromatin, in a cell-type specific manner, at ~ 1,100 - 1,400 LMNA-associated domains (LADs). LMNA is implicated in H3K9 trimethylation by SUV39H1 and EHMT2 (G9a), histone deacetylation by HDACs and DNA methylation by DNMTs. LADs, mostly located in the heterochromatin loci, are enriched in H3K9me3 and H3K27me3 repressive and to a lesser extent activation histone marks. Preliminary data show marked epigenetic alterations in the heart of 2-week old Lmna-/- mice, preceding cardiac dysfunction. LMNA and several chromatin marks are differentially expressed in neonatal vs. adult cardiac myocytes (CMs) and in CMs vs. cardiac fibroblasts (CFs). Switching on expression of LMNAD300N in neonatal CMs leads to DCM and premature death, while its expression in adult CM induces a mild phenotype, findings in accord with the prominent role of epigenetics in neonatal CMs and parallel the phenotypic effects of Hdac3 deletion. Finally, AAV9 mediated expression of LMNA improves cardiac function and survival. Thus, we posit LMNA has distinct cell-type specific epigenetic functions in neonatal and adult CMs and CFs, which are pathogenic in DCM caused by LMNA mutations. To test this hypothesis, Lmna will be conditionally deleted in neonatal (P3) and adult (P60) CMs and CFs, two common cell types in the heart, using specific Cre deleter mice. Epigenetic basis of both cell type-dependent and -autonomous effects on DCM phenotype will be determined in isolated CMs and CFs by characterizing genome-wide promoter occupancy by specific histone marks, CpG methylation, and the whole transcriptome, prior to and after the onset of cardiac dysfunction and fibrosis (aim 1). In aim 2, epigenetic determinants of differential phenotypic effects upon expression of LMNAD300A, responsible for human DCM, in neonatal and adult CMs will be identified (as in aim 1), along with their induction and reversal upon switching on and off LMNAD300N expression, respectively. In aim 3, prevention and reversal of the epigenetic modifications (as in am 1) and the ensuing DCM will be determined upon AAV9- mediated expression the LMNAWT in the heart of CM-specific and systemic Lmna-/- mice. The findings will provide insight into the role of LMNA in cell type-specific epigenetic regulation of cardiac structure and function, the pathogenic role of the epigenetic modifications in DCM, and set the stage for targeted manipulation of selected epigenetic marks for the precise control, prevention, and reversion of DCM due to LMNA mutations.