Targeting p21 to stimulate irradiated muscle stem cell function and muscle regeneration

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Abstract Over the last 30 years, the survival rate of juvenile cancer patients has improved dramatically. Today, 80% of children and adolescents will survive five years beyond initial diagnosis. However, as juvenile cancer survivors live longer, they experience the after-effects of cytotoxic cancer therapies into adulthood. To this end, juvenile cancer survivors suffer from early-onset sarcopenia. Sarcopenia, normally afflicting the elderly population, is the severe and accelerated loss of skeletal muscle. Because radiation therapy can target active progenitors, it is possible that irradiation of juvenile muscle can have long-term consequences. Skeletal muscle is endowed with a population of muscle stem cells (satellite cells, SCs). In adult skeletal muscle, SCs reside in a quiescent state. However during postnatal growth, SCs contribute myonuclei to the development and maturation of juvenile skeletal muscle. We have recently demonstrated a role for SC-derived myonuclear contribution to the development of prepubertal murine skeletal muscle (Bachman et al, Development 2018). Loss of juvenile SCs results in immediate and significant deficits in muscle fiber size and force generation capacity. It is has not been established if irradiation to juvenile murine skeletal muscle can disrupt the cycling SC population. Additionally, neither the consequences nor mechanism of irradiated SC dysfunction have been elucidated. Our preliminary data indicates that that a fractionated radiation treatment (8.2 Gy MWF) can result in an immediate reduction in the juvenile SC pool. However, a population of radio-resistant SCs does persist. These radio- resistant SCs have a reduced capacity to proliferate and undergo myogenic commitment in vitro. Consistent with these deficits, irradiated juvenile skeletal muscle has impaired regenerative potential. Intrinsic irradiated SC dysfunction coincides with upregulated expression of the cell cycle inhibitor and sensor of DNA stress p21 (Cdkn1a). Thus, the goal of my proposal is to determine the impact of irradiation on the juvenile SC pool and identify if elevated p21 expression is responsible for irradiated SC dysfunction. We will address these questions in the following aims: Aim 1) To determine if the radio-resistant SC pool primarily consists of infrequently dividing label-retaining cells (LRCs) Aim 2) To examine whether fractionated irradiation of juvenile skeletal muscle leads to severely delayed regeneration due to intrinsic SC dysfunction Aim 3) To determine if knockdown of p21 stimulates irradiated SC function and muscle regeneration. Together, these aims will shed light on the cellular and molecular mechanisms of juvenile radiotherapy-mediated skeletal muscle decline and potential therapies.