Mechanical Regulation of Gene Expression in Ventricular Myocytes

项目摘要 心脏压力和应变的慢性变化与驱动基因程序的激活有关 病理性生长和重塑。这些基因的长期上调可导致心肌肥厚和 疾病，然而机械力的变化如何传递到基因表达的变化是很差的 明白了。最近对非肌肉细胞的研究发现，菌株通过连接子传播到细胞核 核和细胞骨架(LINC)复合体的表达可能是力依赖的基因表达的关键。The LINC 复合体形成蛋白质-蛋白质相互作用，将细胞骨架与内核膜连接起来，并 相关的染色质，形成一条潜在的依赖于力的基因调控途径。有趣的是，基因突变 许多细胞骨架、核骨架和LINC复合蛋白与扩张型心肌病有关 (DCM)。本提案中概述的实验将定义LINC综合体的哪些组件和 在成年心肌细胞(CMS)中，细胞骨架将应变传递到细胞核，以及这些成分是否 参与菌株依赖的基因表达。根据我的初步数据，我假设肌肉- 特定的中间丝(IF)结蛋白和心脏微管(MTS)是菌株传播和 调节成年CMS中依赖菌株的基因表达。 这项建议的第一个目标是确定哪些LINC和细胞骨架组件传递应力和应变 到CM核。根据我的初步证据，我预测desmin和MTS将对这一过程至关重要 MT网络的解聚减少了传递到细胞核的应变。要确定哪些其他组件 对向原子核传递应变很重要，我将评估原子核形变的程度 控制心肌细胞的伸展/收缩以及干扰结蛋白、肌动蛋白和肌动蛋白成分的操作 LINC建筑群。 这项建议的第二个目标是确定LINC复合体和细胞骨架是否导致压力- 介导的基因表达。我假设结蛋白和MTS通过调节依赖菌株的基因表达 LINC建筑群。我将通过周期性地扩展孤立的CMS种群并测量 在对照和细胞骨架和LINC条件下使用RNAseq的基因表达的变化 建筑群被打乱了。为了补充这种细胞方法，我将确定这些组件是否也 在完整的心脏中调节依赖于应变的基因表达。 这些实验将首次确定CM LINC复合体和细胞骨架的哪些成分 调节菌株向细胞核的传播和菌株诱导的基因表达。如果成功，此操作的结果 这项工作将提供对心脏压力和紧张的慢性变化机制的关键洞察 诱导基因表达的变化。

Project Abstract Chronic changes in stress and strain on the heart are associated with the activation of gene programs that drive pathological growth and remodeling. Long-term upregulation of these genes can lead to cardiac hypertrophy and disease, yet how a change in mechanical force is transduced to a change in gene expression is poorly understood. Recent studies in non-muscle cells have found that strain transmission to the nucleus via the Linkers of the Nucleo- and Cytoskeleton (LINC) complex may be critical for force-dependent gene expression. The LINC complex forms protein-protein interactions that connect the cytoskeleton to the inner nuclear membrane and associated chromatin, forming a potential route for force-dependent gene regulation. Interestingly, mutations in many cytoskeletal, nucleoskeletal and LINC complex proteins have been linked to dilated cardiomyopathy (DCM). The experiments outlined in this proposal will define which components of the LINC complex and cytoskeleton transmit strain to the nucleus in adult cardiomyocytes (CMs), and whether those components are involved in strain-dependent gene expression. Based on my preliminary data, I hypothesize that the muscle- specific intermediate filament (IF) desmin and cardiac microtubules (MTs) are critical for strain transmission and regulate strain-dependent gene expression in adult CMs. The first goal of this proposal is to determine which LINC and cytoskeletal components transmit stress and strain to CM nuclei. I predict desmin and MTs will be critical for this process based on my preliminary evidence that depolymerization of the MT network reduces strain transmitted to nuclei. To determine which other components are important for strain transmission to the nucleus, I will assess the degree of nuclear deformation upon stretch/contraction in control myocytes and upon manipulations that disrupt desmin, actin, and components of the LINC complex. The second goal of this proposal is to determine whether the LINC complex and cytoskeleton contribute to strain- mediated gene expression. I hypothesize that desmin and MTs regulate strain-dependent gene expression via the LINC complex. I will test this hypothesis by cyclically stretching populations of isolated CMs and measuring alternations in gene expression using RNAseq both in control and in conditions where the cytoskeleton and LINC complex are disrupted. To complement this cellular approach, I will determine whether these components also regulate strain-dependent gene expression in an intact heart. These experiments will be the first to identify which components of the CM LINC complex and cytoskeleton regulate strain transmission to the nucleus and strain-induced gene expression. If successful, the results of this work will provide key insight into the mechanism by which chronic changes in stress and strain on the heart induce changes in gene expression.