Suppressing Hippo signaling in the stem cell niche promotes skeletal muscle regeneration

Qi Liu, Su Pan, Shijie Liu, Sui Zhang, James T. Willerson, James F. Martin, Richard A. F. Dixon

Lack of blood flow to the lower extremities in peripheral arterial disease causes oxygen and nutrient deprivation in ischemic skeletal muscles, leading to functional impairment. Treatment options for muscle regeneration in this scenario are lacking. Here, we selectively targeted the Hippo pathway in myofibers, which provide architectural support for muscle stem cell niches, to facilitate functional muscle recovery in ischemic extremities by promoting angiogenesis, neovascularization, and myogenesis. We knocked down the core Hippo pathway component, Salvador (SAV1), by using an adeno-associated virus 9 (AAV9) vector expressing a miR30-based triple short-hairpin RNA (shRNA), controlled by a muscle-specific promoter. In a mouse hindlimb-ischemia model, AAV9 SAV1 shRNA administration in ischemic muscles induced nuclear localization of the Hippo effector YAP, accelerated perfusion restoration, and increased exercise endurance. Intravascular lectin labeling of the vasculature revealed enhanced angiogenesis. Using 5-ethynyl-2'-deoxyuridine to label replicating cellular DNA in vivo, we found SAV1 knockdown concurrently increased paired box transcription factor Pax7+ muscle satellite cell and CD31+ endothelial cell proliferation in ischemic muscles. To further study Hippo suppression in skeletal muscle regeneration, we used a cardiotoxin-induced muscle damage model in adult (12-15 weeks old) and aged mice (26-month old). Two weeks after delivery of AAV9 SAV1 shRNA into injured muscles, the distribution of regenerative myofibers shifted toward a larger cross-sectional area and increased capillary density compared with mice receiving AAV9 control. Together, these findings suggest our approach may have clinical promise in regenerative therapy for leg ischemia and muscle injury. © AlphaMed Press 2021 SIGNIFICANCE STATEMENT: The age-associated progressive decline in skeletal muscle regeneration limits our ability to treat skeletal muscle injury. The regeneration of skeletal muscles depends on the activity of resident stem cells that are protected by myofibers in a unique anatomical niche and that reside adjacent to the endothelial cells. This study developed a novel treatment regimen that targets the Hippo signaling pathway in the muscle stem cell niche to promote myogenesis simultaneously with angiogenesis and neovascularization. This strategy may overcome the current limitation of functional skeletal muscle regeneration in aged populations.