Nuclear mechanics and mechanotransduction in muscular laminopathies

项目摘要 该项目的长期目标是确定人类LMNA基因编码的核转录因子的突变是如何发生的。 包膜（NE）蛋白核纤层蛋白A和C引起严重的心脏和骨骼肌疾病。本研究是 重要的是，因为LMNA基因的突变导致了大约10%的遗传性疾病， 扩张型心肌病和LMNA相关心肌病的病例预后特别差。 患有LMNA相关肌营养不良症的个体患有使人衰弱的进行性肌肉萎缩， 死于扩张型心肌病到目前为止，对于心脏和骨骼， LMNA突变引起的肌肉缺陷，目前尚不清楚LMNA突变如何导致肌肉缺陷。 具体缺陷。对疾病发病机制的不完全理解是治疗的主要障碍。 开发有效的治疗方法。根据最近公布的大量初步数据，该项目 提出了一个新的假说，发病机制层粘连蛋白病影响心脏和骨骼肌：LMNA 与横纹肌疾病相关的突变降低了核的机械稳定性。在骨骼和心脏 组织中，脆弱的细胞核则易于对NE产生机械诱导的损伤（“NE破裂”）， 在肌细胞收缩和成熟过程中作用于肌核的细胞骨架力。NE断裂导致 DNA损伤和DNA损伤反应途径的激活，导致细胞死亡，衰老， 导致进行性心脏和骨骼肌缺陷的代谢损伤。额外 机制，如转录调节紊乱和活性氧产生增加， 可能进一步导致DNA损伤的增加和DNA损伤反应途径的激活 LMNA突变肌肉细胞。拟议的工作利用了几种体外和体内模型， 在PI实验室定制开发的测定，以测量核力学和机械 诱导活细胞和固定组织的损伤。该项目的目标是：（1）确定分子和 导致NE断裂、DNA损伤和DNA损伤反应增加的生物物理机制 核纤层蛋白病中骨骼肌和心肌细胞的激活;（2）确定导致核纤层蛋白病的分子途径 NE断裂、DNA损伤和DNA损伤反应激活横纹肌细胞死亡和功能障碍; 和（3）评估是否抑制过度活跃的DNA损伤反应途径或减少对细胞的物理应激， 核改善LMNA相关疾病小鼠模型的心脏和骨骼肌健康。的 提出的研究将建立核损伤在核纤层蛋白病中观察到的功能作用，并提供新的 深入了解影响心脏和骨骼肌的层粘连蛋白病的发病机制， 发现新的治疗靶点和策略，可以显着提高可用的治疗方案 对于受影响的个人。

Project Summary The long-term goal of this project is to determine how mutations in the human LMNA gene encoding the nuclear envelope (NE) proteins lamin A and C cause severe cardiac and skeletal muscle diseases. This research is important because mutations in the LMNA gene are responsible for approximately 10% of all genetically inherited cases of dilated cardiomyopathy, and LMNA-associated cardiomyopathies have a particularly poor prognosis. Individuals with LMNA-associated muscular dystrophy suffer from debilitating progressive muscle wasting and die from dilated cardiomyopathy. To date, no effective treatments are available for the cardiac and skeletal muscle defects caused by LMNA mutations, and it remains unclear how LMNA mutations result in muscle- specific defects. The incomplete understanding of the disease pathogenesis presents a major hurdle in the development of effective treatments. Building on recently published and extensive preliminary data, this project proposes a novel hypothesis for the pathogenesis of laminopathies affecting cardiac and skeletal muscle: LMNA mutations associated with striated muscle disease reduce nuclear mechanical stability. In skeletal and cardiac tissues, the fragile nuclei are then prone to mechanically induced damage to the NE (‘NE rupture’) due to cytoskeletal forces acting on myonuclei during muscle cell contraction and maturation. The NE rupture results in DNA damage and activation of DNA damage response pathways, which lead to cell death, senescence, and metabolic impairment responsible for the progressive cardiac and skeletal muscle defects. Additional mechanisms, such as disturbed transcriptional regulation and increased production of reactive oxygen species, may further contribute to the increased DNA damage and activation of DNA damage response pathways in the LMNA mutant muscle cells. The proposed work takes advantage of several in vitro and in vivo models and assays that were custom-developed in the PI’s laboratory to measure nuclear mechanics and mechanically induced damage in living cells and fixed tissues. The proposed project aims to: (1) determine the molecular and biophysical mechanisms that cause the increased NE rupture, DNA damage, and DNA damage response activation in skeletal and cardiac muscle cells in laminopathies; (2) identify the molecular pathways leading from NE rupture, DNA damage, and DNA damage response activation to striated muscle cell death and dysfunction; and (3) evaluate if inhibiting hyperactive DNA damage response pathways or reducing physical stress on the nucleus improves cardiac and skeletal muscle health in mouse models of LMNA-associated disease. The proposed studies will establish the functional role of nuclear damage observed in laminopathies and provide new insights into the pathogenesis of laminopathies affecting cardiac and skeletal muscle, with the potential to discover novel therapeutic targets and strategies that could significantly enhance the treatment options available for affected individuals.