Date of Award

2019

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Cell & Molecular Biology

First Advisor

Eileen Shore

Second Advisor

Foteini Mourkioti

Abstract

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease characterized by formation of extra-skeletal bone, or heterotopic ossification (HO), in soft connective tissues like skeletal muscle. All cases with classic clinical features of FOP carry a mutation in ACVR1 (R206H; c.617G>A), a cell surface receptor that mediates bone morphogenetic protein (BMP) signaling, which is recognized for its chondro/osteogenic-induction potential. HO in FOP patients is qualitatively normal bone tissue; the ACVR1R206H mutation induces misdirected cell fate decisions by tissue-resident mesenchymal stem cells to form this ectopic bone.

In addition to ligand-receptor signaling, mechanical cues from the physical environment direct cell fates. While BMP ligands are established signaling molecules, less is known about signals from the surrounding tissue physical properties or interactions with mechanical effectors by the BMP-pSmad1/5 pathway. 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. Primary mouse embryonic fibroblasts (MEFs) isolated from our Acvr1R206H/+ mice that mimics the human disease are used as an in vitro model system of mesenchymal stem cells (MSCs), including their ability to differentiate into adipogenic, chondrogenic, and osteogenic lineages. We previously showed increased BMP signaling in Acvr1R206H/+ MEFs as measured by phosphorylated Smad1/5/8 (pSmad1/5/8) protein levels in the presence or absence of BMP ligand. We also found increased expression of direct downstream chondro/osteogenic target genes of BMP such as Msx2, Id1, Sox9, and Runx2. Utilizing MEFs, we determined that activation of the mechanotransductive effectors RHO/ROCK and YAP/TAZ were increased in Acvr1R206H/+ MEFs. We found that Acvr1R206H/+ MEFs on softer substrates acquire a morphology, and other responses to the physical environment, similar to control MEFs on stiffer substrates and that Acvr1R206H/+ MEFs have a basal propensity for osteogenic differentiation. Our data support that the combination of increased BMP pathway signaling, misinterpretation of soft substrates, and overall reduced sensitivity to mechanical stimuli lower the threshold of Acvr1R206H/+ cells for commitment to chondro/osteogenic lineages.

HO formation is often initiated by injury to skeletal muscle. In normal tissue, injury leads to recruitment of fibroblasts to provide repair signals and stimulation of muscle progenitor cells (muscle stem cells, MuSCs) to undergo myogenic differentiation to restore muscle tissue. Our lab previously demonstrated that in FOP, repair initiates normally, however appears to diverge from the normal wound healing response at the fibroproliferative stage. We have established that our Acvr1R206H/+ mouse model develops HO after cardiotoxin (CTX) injury, and that injured skeletal muscle tissue from Acvr1R206H/+ is more fibrotic and severely damaged compared to muscle of injured Acvr1+/+ animals, indicating that muscle repair is impeded by the ACVR1R206H mutation. The regenerative potential of skeletal muscle is dependent on the function of MuSCs. The regeneration process following CTX injury in Acvr1+/+ mouse skeletal muscle tissue results in the formation of small, centri-nucleated myofibers 7-10-days post-injury. However, in Acvr1R206H/+ muscle tissue, we find persistence of damaged myofibers at later time points and subsequent formation of HO. Additionally, fibro/adipogenic progenitors (FAPs), another muscle-resident progenitor cell, 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 demonstrated that Acvr1+/+ and Acvr1R206H/+ MuSCs and FAPs proliferate at similar levels after injury. While Acvr1+/+ MuSCs differentiate normally and form branching myofibers, Acvr1R206H/+ MuSCs form underdeveloped fibers that fail to fuse in vitro. Acvr1+/+ FAPs cultured with Acvr1R206H/+ MuSCs leads to proper myofibers formation and fusion, while Acvr1R206H/+ FAPs cultured with Acvr1+/+ MuSCs form undeveloped fibers. This suggests that the FAP population under the influence of the ACVR1R206H mutation contributes largely to the poor muscle regeneration seen in FOP lesions. The ACVR1R206H mutation also creates a hostile environment for repair, hindering engraftment of transplanted Acvr1+/+ MuSCs. Taken together, our data exhibit the impact of the ACVR1R206H FOP mutation on the differentiation capacity of myogenic progenitor cells to regenerate skeletal muscle and the importance of environment during skeletal muscle regeneration.

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