Fibro/Adipogenic Progenitor-Derived Follistatin-Like 1 Expression is Reduced by Irradiation to Regulate Fibro/Adipogenic Progenitor Fate Decisions

Abstract

Introduction: Five-year cancer survivorship is increasing; however, the long-term effects of treatments are detrimental to skeletal muscle. We have found that fibro-adipogenic progenitors (FAPs) contribute to irradiation-induced muscle atrophy and fibrosis, in part, through alterations to their secretome that impairs muscle stem (satellite) cell (MuSC) fate. It is unknown; however, which specific FAP secreted factors are responsible for the irradiation-induced defect in MuSC differentiation and fusion. Preliminary data from our lab and others suggests that Follistatin-like 1 (FSTL1) may be a key, irradiation-sensitive secreted factor. We aimed to understand how irradiation impacts FSTL1 expression in FAPs and elucidate how FSTL1 expression impacts the MuSC niche.

Methods: Fstl1 in vitro gene and protein expression was analyzed on irradiated C3H10T1/2 (C3H) fibroblasts (a FAP cell line) and primary FAPs. In vitro FAP differentiation was analyzed on FSTL1 knockdown C3H cells. Fstl1 in vivo gene expression was analyzed on mice that had one hindlimb irradiated. The effect of FSTL1 repletion was analyzed in vivo on mice that had both hindlimbs irradiated and rFSTL1 injected intravenously.

Results: In C3H cells, Fstl1 was significantly lower at 24-hours (p<0.05) and FSTL1 was significantly higher at 48-hours post-irradiation (p<0.01). In primary FAPs, FSTL1 was significantly lower at 12- and 48-hours post-irradiation (p<0.01). FSTL1 knockdown significantly lowered the area (p<0.01) and mean fluorescence intensity per cell (p<0.0001) of fibrogenically differentiated C3H cells. FSTL1 knockdown (p=0.0701) and irradiation (p<0.01) lowered the percentage of total C3H cells that were adipogenically differentiated. In vivo fractionated irradiation did not induce alterations in muscle-resident mononuclear cell populations, muscle fibrosis, and muscle cross-sectional area (CSA) at 3-days. Intravenous repletion of rFSTL1 raised FSTL1 in circulation (p<0.0910) but not in skeletal muscle.

Significance: The present study demonstrates that Fstl1 expression is decreased by in vitro irradiation, that it upregulates FAP fibrogenic differentiation, and downregulates FAP adipogenic differentiation. We identified FSTL1 as a compelling candidate for mitigating cancer treatment-induced skeletal muscle atrophy and fibrosis. Future work using a novel intramuscular FSTL1 repletion protocol has the potential to unveil benefits in in vivo myogenesis following irradiation.