FGF-2-Dependent Signaling Activated in Aged Human Skeletal Muscle Promotes Intramuscular Adipogenesis

Abstract

Background: A unique feature of muscle during aging, obesity and type 2 diabetes is the appearance of adipose tissue between skeletal muscle fibers, the intramuscular adipose tissue (IMAT). IMAT is generally associated with systemic insulin resistance, decreased muscle strength and, in older adults, impaired mobility. A large body of work over the last years have addressed the origin of IMAT. Skeletal muscle contains preadipocyte progenitors termed fibro/adipogenic progenitors (FAPs) that normally do not form adipocytes, but proliferate during muscle injury to support the commitment of myogenic progenitor cells during muscle repair. However, under the conditions outlined above, FAPs differentiate to adipocytes and give rise to IMAT. The molecular cues that trigger this pathogenesis are unknown and strategies that can modulate the fate of FAPs and their propensity to differentiate to adipocytes in disease states are urgently needed. Methods: We have used molecular cloning and genome editing techniques together with a variety of biochemical, molecular, genomic, transcriptomic and proteomic analyses in pre-adipocyte and muscle cell lines, primary cells isolated from human and mouse skeletal muscle, human skeletal muscle biopsies, and animal models. Results: Using multiple screening assays upstream and downstream of a master regulator of myogenesis, microRNA (miR)-29a, we located the secreted protein and adipogenic inhibitor SPARC to an FGF-2 signaling pathway that is conserved between skeletal muscle cells from mice and humans and that is activated in skeletal muscle of aged mice and humans. We identified FGF-2 as an upstream regulator that increases miR-29a promoter activity and gene expression via MEK1/2/MAPK signaling and consequently decreases the conserved miR-29a target SPARC. FGF-2 induces the miR-29a/SPARC axis through transcriptional activation of FRA-1, which binds and activates an evolutionary conserved AP-1 site element proximal in the miR-29a promoter. CRISPR/Cas9-mediated genomic deletions of miR-29a recognition elements located in the Sparc 3´UTR of muscle cells and AAV-mediated overexpression of FGF-2 or SPARC in mouse skeletal muscle revealed that this axis regulates differentiation of FAPs in vitro and IMAT formation in vivo. Skeletal muscle from human donors aged > 75 years versus < 55 years showed activation of FGF-2-dependent signaling and increased IMAT. Conclusion: FGF-2 not only stimulates muscle growth, but also promotes intramuscular adipogenesis via targeting the fate of FAPs. Thus, our data highlights a disparate role of FGF-2 in adult skeletal muscle and reveals a novel pathway, which has the potential to provide innovative diagnostic and therapeutic approaches to combat fat accumulation in aged human skeletal muscle.??