Regulation of skeletal muscle extracellular matrix remodeling by satellite cells

描述（由申请人提供）：骨骼肌质量的维持以及响应负荷而增加质量的能力对于全身功能和生活质量至关重要。卫星细胞或肌肉干细胞在肌肉质量调节中的作用是模棱两可的。有效的肌肉肥大可以独立于卫星细胞而发生;然而，在没有卫星细胞的情况下的长期肌肉适应尚未被探索。初步数据表明，在卫星细胞的情况下，肌肉变得纤维化和肥大高原。这提出了激活的卫星细胞在调节肌纤维生态位中发挥旁分泌作用的可能性。在肌营养不良症和衰老中观察到的过量细胞外基质（ECM）产生和肌肉纤维化对肌肉可塑性和功能产生负面影响。活化的卫星细胞可能通过调节转化生长因子（TGF-β）信号传导介导骨骼肌ECM的产生。TGF-β促进肌肉成纤维细胞中ECM的产生增加，卫星细胞可能阻止TGF-β下游信号传导，从而影响成纤维细胞活性和ECM的产生。据推测，在没有激活的卫星细胞的情况下，通过肌肉成纤维细胞的ECM的产生通过TGF-β信号传导的激活而增加，导致肌肉纤维化和肌肉生长和功能减弱。此外，我们建议测试新的假设，肌肉纤维化，而不是肌纤维结构域上限，限制肌肉肥大的卫星细胞耗尽的肌肉。在两种新的小鼠品系中，跖肌功能超负荷将决定激活的卫星细胞在 调节细胞外环境以实现肥大。Pax 7-DTA株能够特异性地去除卫星细胞，Pax 7-N-WASp株能够活化卫星细胞 和增殖，而不是融合。目的1将评估TGF-β信号传导活性、成纤维细胞增殖、ECM组分的表达、纤维化的功能后果以及Pax 7-DTA小鼠中卫星细胞耗竭后骨骼肌中响应于过载的肥大反应。将进行免疫组织化学、荧光激活细胞分选、qRT-PCR、蛋白质印迹、生化测定和全肌肉功能评估。增加的成纤维细胞增殖，ECM成分的表达和与卫星细胞耗尽的肌肉中生长减少相关的抑制力产生将证明激活的卫星细胞在控制纤维细胞外环境中的调节作用。目的2将利用Pax 7-N-WASp小鼠来确定激活的卫星细胞在肥大反应中的作用，而不管它们贡献新的核生长纤维的能力如何。如目的1所述，在肌肉超负荷期间评估成纤维细胞增殖和ECM产生的活化卫星细胞调节。Pax 7-N-WASp小鼠肥大的恢复将证明激活的卫星细胞在成人骨骼肌适应性中的新作用，这可能与它们在肌纤维再生中的良好特征作用一样重要。

DESCRIPTION (provided by applicant): The maintenance of skeletal muscle mass, and the ability to increase mass in response to load, are crucial for whole body function and quality of life. The role of satellite cells, or muscle stem cells, in muscle mass regulation is equivocal. Effective muscle hypertrophy can occur independently of satellite cells; however, long- term muscle adaptation in the absence of satellite cells has not been explored. Preliminary data suggest that in the absence of satellite cells, muscle becomes fibrotic and hypertrophy plateaus. This raises the possibility that activated satellite cells play a paracrine role in regulating the muscle fiber niche. Excess extracellular matrix (ECM) production and muscle fibrosis, observed in muscular dystrophies and in aging, negatively impact muscle plasticity and function. Activated satellite cells may mediate skeletal muscle ECM production through modulation of transforming growth factor ¿ (TGF-¿) signaling. TGF-¿ promotes increased production of ECM in muscle fibroblasts, and satellite cells may prevent TGF-¿ downstream signaling, thereby influencing fibroblast activity and ECM production. It is hypothesized that in the absence of activated satellite cells, production of ECM by muscle fibroblasts increases via activation of TGF-¿ signaling, resulting in muscle fibrosis and attenuated muscle growth and function. Further, we propose to test the novel hypothesis that muscle fibrosis, rather than a myonuclear domain ceiling, limits muscle hypertrophy in satellite cell-depleted muscle. Functional overload of plantaris muscle in two novel mouse strains will determine the role of activated satellite cells in modulating the extracellular environment to enable hypertrophy. The Pax7-DTA strain enables specific depletion of satellite cells, and the Pax7-N-WASp strain enables satellite cell activation and proliferation, but not fusion. Aim 1 will assess TGF-¿ signaling activity, fibroblast proliferation, expression of ECM components, functional consequences of fibrosis, and the hypertrophic response in skeletal muscle following satellite cell depletion in the Pax7-DTA mouse in response to overload. Immunohistochemistry, fluorescent activated cell sorting, qRT-PCR, western blotting, biochemical assays and whole muscle function assessment will be performed. Increased fibroblast proliferation, expression of ECM components and depressed force production associated with reduced growth in satellite cell-depleted muscle will demonstrate a regulatory role for activated satellite cells in controlling the fiber extracellular environment. Aim 2 will utilize the Pax7-N-WASp mouse to determine the role of activated satellite cells in the hypertrophic response irrespective of their ability to contribute new nucleito growing fibers. Activated satellite cell regulation of fibroblast proliferation and ECM production will be assessed during muscle overload as in Aim 1. Restoration of hypertrophy in Pax7-N-WASp mice will demonstrate a novel role for activated satellite cells in adult skeletal muscle adaptability that may prove equally as important as their well-characterized role in muscle fiber regeneration.