Mechanisms of adipogenic and fibrotic degeneration of muscle

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Fibrotic and adipogenic infiltration are hallmarks of injured, diseased, and aged skeletal muscle. This fibrofatty degeneration (“FFD”) results not only in functional decline of skeletal muscle but also to the increased prevalence of metabolic disorders. The origins of the major cellular contributors (fibrogenic and adipogenic progenitors) of this FFD remain to be definitively identified, but recent evidence (including Preliminary Studies included herein) suggest a population of mesenchymal progenitors termed “fibroadipogenic progenitors” (FAPs) are major sources. FAPs reside in the muscle interstitium and display robust fibrogenic and adipogenic potential in vitro and in vivo following transplantation. Studies in vivo to directly test whether endogenous FAPs are responsible for fibrosis and adiposity in the setting of injury and disease have been limited by the lack of specific tools to genetically label and target FAPs. We have recently developed such tools by taking advantage of the highly specific expression of PDGFRα in FAPs among the mononucleated cells of muscle. Our Preliminary Studies using a PDGFRαCreER strain that we developed to either genetically label or specifically deplete FAPs support the hypothesis that FAPs are sources of both fibrogenic and adipogenic cells in the setting of aberrant muscle regeneration associated with FFD. In Preliminary Studies, we also identified a microRNA, miR-206, as a candidate regulator of FAP adipogenic differentiation, and a transcription factor, Runx1, as a likely target of mIR-206 in this process. We have, in addition, identified candidate microRNAs involved in the regulation of FAP fibrogenic differentiation. In the studies of this proposal, we will explore the regulation of FAP adipogenic and fibrogenic differentiation, and the essential role of FAPs in FFD in two clinically relevant models. In the studies of Aim 1, we will examine the role of the miR-206/Runx1 axis in FAP adipogenic differentiation in vitro and in fatty infiltration in glycerol-induced muscle injury in vivo. In the studies of Aim 2, we will work collaboratively with our colleague, Dr. Brian Feeley, at UCSF to explore the role of FAPs in general, and of the miR-206/Runx1 axis in particular, in the fatty infiltration that occurs in the setting of rotator cuff injury (RCI). Dr. Feeley has developed a robust murine model of RCI that exhibits the kind of fatty infiltration and muscle atrophy seen in humans. We will use a novel tamoxifen analog delivery method we have developed to allow for the depletion of FAPs only in the region of the RCI. In the studies of Aim 3, we will examine the regulation of FAP fibrogenic differentiation, again focusing on the key role of miRNAs in such cell fate decisions. We will identify functional targets of candidate miRNAs using a recently developed pull-down technology combine with RNA sequencing (LAMP- seq). Furthermore, we will address the role of FAPs in the extensive fibrosis seen in the common extremity traumatic injury experience by soldiers and treated in Veterans, volumetric muscle loss (VML). We have extensive experience with a murine model of VML, and we will examine both the development of fibrosis and interventions to prevent fibrosis based on our understanding of FAP differentiation. Through our studies of FAPs and the regulatory processes that control their differentiation to adipogenic and fibrogenic cells, we aim to understand the mechanisms that give rise to FFD and the subsequent muscle dysfunction. Our investigation will both capitalize on new experimental tools to study this population and lend insight into therapeutic strategies to prevent FFD. This will have direct relevance to Veterans who have experienced skeletal muscle injuries, injuries that have limited their functional capacity and that, to date, have little hope of functional recovery. Our goal is to develop therapeutic approaches to enhance muscle repair and prevent muscle degeneration based upon a thorough understanding of the basic stem cell biology. These goals are based upon a firm commitment to a mission to improve the health and quality of life of Veterans whose function is limited by the lack of effective therapeutic options.