The role of satellite cells in adult skeletal muscle growth and maintenance

描述（由申请人提供）：本次修订的目的是扩大母基金的范围，以确定肌肉干细胞（卫星细胞）在严重肢体缺血期间肌肉组织血运重建中的作用。通过跨学科研究小组成员之间的初步讨论，我们最初假设卫星细胞是肌肉从缺血诱导的肌肉损伤中恢复所必需的。McClung和Kontos博士最近的一项研究支持了这一假设，该研究表明，不同近交系小鼠中生肌基因表达的差异激活是肌肉对缺血反应的遗传调节的关键组成部分。为了验证这一新的假设，我们利用我们在骨骼肌和血管生物学方面的跨学科专业知识进行了初步研究，以直接评估卫星细胞在骨骼肌对股动脉结扎和切除的标准模型后肢缺血（HLI）反应中的作用。使用Pax 7-DTA小鼠，其能够响应于他莫昔芬（在父母资助中开发的）诱导卫星细胞消融，我们比较了含有卫星细胞的肌肉（载体组）与卫星细胞耗尽（>90%）的肌肉（他莫昔芬组）的缺血反应。HLI后一周，由McClung博士进行，在媒介物组（n=4）的肌肉中观察到的损伤水平与Kontos和McClung先前在野生型C57 BL/6小鼠中报道的一致。相比之下，在他莫昔芬组中，卫星细胞已经被基因消融，一组小鼠似乎几乎完全没有坏死，这与我们最初的假设完全相反。尽管一些他莫昔芬处理的小鼠显示出与溶剂处理组相似的肌肉损伤，但Pax 7免疫组织化学显示，这些小鼠在缺血期间显著恢复了PAX 7+细胞数量，并且似乎具有Pax 7+细胞的不完全缺失或Pax 7表达细胞的恢复。在与这些手术同时进行的已知“不良反应者”BALB/c亲本品系（阳性对照）队列中，手术后流量测量、完全动脉切除的收获后解剖验证和肢体坏死证实了手术缺血，排除了手术变异作为这一惊人观察结果的解释。最后，与溶剂处理的小鼠相比，保护免受缺血诱导的损伤的小鼠显示出更大数量的CD 31免疫染色毛细血管的趋势。这些结果表明，有趣的可能性，卫星细胞有一个意想不到的和新的作用，在骨骼肌对缺血的反应，可能包括肌肉血管反应的调节。因此，本次修订的具体目的是在更大的小鼠队列中对这种反应进行详细表征，以便就卫星细胞在严重肢体缺血的肌肉血管和再生反应中的作用得出明确结论。

DESCRIPTION (provided by applicant): The purpose of this revision is to expand the scope of the parent grant to determine the role of muscle stem cells (satellite cells) in revascularization f muscle tissue during critical limb ischemia. Through initial discussions among members of the interdisciplinary research team, we originally hypothesized that satellite cells are required for muscle to recover from ischemia-induced muscle injury. Support for this hypothesis came from a recent study by Drs. McClung and Kontos demonstrating that differential activation of myogenic gene expression in different inbred strains of mice is a key component of the genetic regulation of the muscle response to ischemia. To test this novel hypothesis, we utilized our interdisciplinary expertise in both skeletal muscle and vascular biology to perform a preliminary study to directly assess the role of satellite cells in the response of skeletal muscle to hind lim ischemia (HLI) using the standard model of femoral artery ligation and excision. Using the Pax7-DTA mouse, which enables inducible satellite cell ablation in response to tamoxifen (developed in the parental grant), we compared the ischemic response of muscle that contained satellite cells (vehicle group) to muscle depleted (>90%) of satellite cells (tamoxifen group). One week after HLI, performed by Dr. McClung, the level of damage observed in muscle of the vehicle group (n=4) was consistent with that previously reported by Kontos and McClung in wild-type C57BL/6 mice. In contrast, in the tamoxifen group, in which satellite cells had been genetically ablated, a cohort of mice appeared to be almost completely protected from necrosis, completely contrary to our original hypothesis. Although some of the tamoxifen-treated mice showed muscle injury similar to that of the vehicle-treated group, Pax7 immunohistochemistry revealed that the these mice significantly recovered PAX7+ cell number during ischemia, and appear to have had either incomplete deletion of Pax7+ cells or recovery of Pax7-expressing cells. Post-surgical flow measurements, post harvest anatomic verification of complete arterial resection, and limb necrosis in a known "poor responder" BALB/c parental strain (positive control) cohort performed in concert with these surgeries confirmed surgical ischemia, ruling out surgical variation as an explanation for this stunning observation. Finally, the mice that were protected against ischemia-induced damage showed a trend towards a greater number of CD31 immunostained capillaries compared to vehicle-treated mice. These results suggest the intriguing possibility that satellite cells have an unanticipated and novel role in the skeletal muscle response to ischemia that may include the regulation of the muscle vascular response. Thus, the specific aim of this revision is to perform a detailed characterization of this response n a larger cohort of mice so that a definitive conclusion can be reached regarding the role of satellite cells in the muscle vascular and regenerative response to critical limb ischemia.