In the rare genetic disease fibrodysplasia ossificans progressiva (FOP), progenitor cells are mis-regulated to differentiate to heterotopic extra-skeletal bone in connective tissues. Mutations in the BMP type I receptor ACVR1/ALK2 cause FOP, with the R206H mutation as the most prevalent. This increases BMP signaling to promote increased downstream chondro-/osteogenic gene expression and heterotopic ossification (HO) formation in FOP patients. HO formation is often initiated by injury to skeletal muscle. HO in FOP patients is qualitatively normal endochondral bone tissue, however the ACVR1 mutation leads to misdirected cell fate decisions by tissue-resident mesenchymal stem cells. In addition to ligand-receptor signaling, mechanical cues from the physical environment direct cell fates. While roles for BMPs are established, less is known about signals from the surrounding tissue stiffness or interactions with mechanical effectors. Cells perceive physical cues like substrate stiffness through surface mechanoreceptors; these mechanical inputs modulate cell morphology and lineage through cytoskeletal and chromatin organization. Softer substrates support adipo-/myogenesis, while stiffer substrates promote chondro-/osteogenesis. Utilizing an established mesenchymal stem cell model of mouse embryonic fibroblasts (MEFs) from our Acvr1R206H mouse model, we demonstrated that activation of the mechanotransductive effectors RHO/ROCK and YAP/TAZ were increased in Acvr1R206H MEFs. We found that on softer substrates, morphology of Acvr1R206H MEFs is similar to the morphology of control MEFs on stiffer substrates and Acvr1R206H MEFs have a propensity for osteogenic differentiation. Our data support that the combination of increased BMP signaling, misinterpretation of soft substrates, and overall reduced sensitivity to mechanical stimuli lowers the threshold of Acvr1R206H cells for commitment to chondro-/osteogenic lineages.
HO develops within skeletal muscle following cardiotoxin (CTX) injury in the conditional knock-in mouse model for ACVR1R206H. Additionally, injured Acvr1R206H/+ muscle tissue appears more fibrotic and does not repair as efficiently as Acvr1+/+ muscle tissue, indicating that skeletal muscle repair is impeded by the ACVR1R206Hmutation. The regenerative potential of skeletal muscle is dependent on the function of muscle stem cells (MuSCs). Additionally, non-myogenic mesenchymal progenitor cells (or fibro/adipogenic progenitors, FAPs) are in close association with regenerating muscle fibers and support myogenesis; these cells, considered mesenchymal progenitors based on their ability to differentiate to adipocytes and osteoblasts, are a source of pro-myogenic signals that support muscle regeneration. We examined the effect of the ACVR1R206H mutation on MuSCs and FAPs alone by isolating the two populations using fluorescent activated cell sorting (FACS) and analyzed proliferation. Acvr1+/+ and Acvr1R206H/+ MuSCs and FAPs proliferated similarly after CTX injury. We investigated the ability of Acvr1+/+ and Acvr1R206H/+ MuSCs to differentiate in vitro. Acvr1+/+ MuSCs cultured in myogenic media differentiate normally and form branching myofibers (high fusion index) by day 7 of culture, but Acvr1R206H/+ MuSCs form underdeveloped fibers that fail to fuse (low fusion index). Acvr1+/+ FAPs cultured with Acvr1R206H/+ MuSCs leads to proper myofibers formation and fusion, while Acvr1R206H/+ FAPs cultured with Acvr1+/+ MuSCs form undeveloped fibers with a low fusion index. This suggests that the FAP population under the influence of the ACVR1R206H mutation contributes largely to the poor muscle regeneration seen in FOP lesions. Taken together, our data support the impact of the ACVR1R206H FOP mutation on the differentiation capacity of MuSCs to regenerate skeletal muscle and the impact of FAPs on the function of MuSCs. This work was supported by the International Fibrodysplasia Ossificans Progressiva Association (IFOPA), NIH R01 AR071399 and NIH F31 AR069982 (to AS).
Full text of Dr A. Stanley’s thesis is available from HERE