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.
Peripheral artery disease affects 8-12 million Americans and results in limb ischemia that produces a skeletal muscle myopathy. The purpose of this revision is to determine if skeletal muscle stem cells (satellite cells) have a role in muscle and vascular remodeling in response to ischemia.
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