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

 描述（由申请人提供）：目的是确定扩张型心肌病（DCM）中特定表观遗传修饰在核纤层蛋白病中的致病作用，核纤层蛋白病包括由编码核纤层蛋白A/C（LMNA）的LMNA基因突变引起的广泛疾病。心脏受累是影响横纹肌的一个亚组的主要死亡原因。它表现为扩张型心肌病、传导缺陷、心律失常和心源性猝死。染色质状态以细胞类型特异性方式调节基因表达。动态染色质修饰对心脏发育至关重要，对成人心脏重塑也很重要。在哺乳动物基因组中，LMNA以细胞类型特异性方式在约1，100 - 1，400个LMNA相关结构域（LAD）处与染色质相互作用。LMNA参与SUV 39 H1和EHMT 2（G9 a）的H3 K9三甲基化、HDAC的组蛋白脱乙酰化和DNMT的DNA甲基化。LAD主要位于异染色质基因座，富含H3 K9 me 3和H3 K27 me 3抑制性组蛋白标记，并在较小程度上激活组蛋白标记。初步数据显示，2周龄Lmna-/-小鼠的心脏中存在明显的表观遗传学改变，发生在心功能障碍之前。LMNA和几种染色质标记在新生儿与成人心肌细胞（CM）和CM与心脏成纤维细胞（CF）中差异表达。在新生儿CM中开启LMNAD 300 N的表达导致DCM和过早死亡，而其在成人CM中的表达诱导轻度表型，这一发现与表观遗传学在新生儿CM中的突出作用雅阁，并与Hdac 3缺失的表型效应平行。最后，AAV 9介导的LMNA表达改善了心脏功能和存活率。因此，我们发现LMNA在新生儿和成人CM和CF中具有不同的细胞类型特异性表观遗传功能，这些功能在由LMNA突变引起的DCM中是致病的。为了检验这一假设，将使用特定的Cre删除小鼠在新生儿（P3）和成人（P60）CM和CF（心脏中的两种常见细胞类型）中有条件地删除Lmna。在心功能不全和纤维化发作之前和之后，通过特异性组蛋白标记、CpG甲基化和整个转录组表征全基因组启动子占用，将在分离的CM和CF中确定细胞类型依赖性和自主性对DCM表型影响的表观遗传基础（目的1）。在目标2中，将鉴定新生儿和成人CM中负责人DCM的LMNAD 300 A表达时差异表型效应的表观遗传决定子（如目标1中所述），沿着它们分别在开启和关闭LMNAD 300 N表达时的诱导和逆转。在目的3中，表观遗传修饰（如在am 1中）和随后的DCM的预防和逆转将在CM特异性和全身性Lmna-/-小鼠的心脏中的AAV 9介导的LMNAWT表达时确定。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，表观遗传修饰在DCM中的致病作用，并为靶向操纵选定的表观遗传标记以精确控制，预防和逆转由于LMNA突变导致的DCM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.