Developing a Fibrotic Phenotype in a 3D Human Skeletal Muscle Microphysiological System

Abstract

Muscle fibrosis is caused by muscle injury, dystrophy, sarcopenia, and rheumatoid arthritis. This condition is characterized by hardening and scarring, which impairs contractile muscle function. To understand how fibrotic disease affects muscular function, we created a model of human skeletal muscle fibrosis using three-dimensional engineered skeletal muscle (myobundles). Furthermore, to investigate the effect of skeletal muscle fibrosis on the vascular system, we integrated the fibrotic skeletal muscle with tissue engineered blood vessels. Treating myobundles with Transforming Growth Factor β1 (TGF-β1) reproduced key characteristics of fibrotic skeletal muscle including reduced contractile force, disrupted contractile protein organization, increased stiffness, and expression of profibrotic genes. Treatment with a selective inhibitor (SB525334) of TGF-β1 receptor (ALK5, TGF-βRI) increased contractile function and decreased ECM deposition, consistent with animal studies in the literature. We also observed endogenous secretion of TGF-β1 in our myobundles which is of novel biological significance. siRNA knockdown of TGF-β1 increased contractile force. Testing anti-fibrotic drug Nintedanib in this model, showed an increase in tetanus force production in 2 out of 3 donors and reduction of pro-fibrotic ECM accumulation of collagen 1 and fibronectin. Western blot analysis of Nintedanib also providence evidence of its inhibition of TGF-β1 signaling by the reduction of phosphorylated Smad2/3. Repositioned anti-fibrotic drug Suramin treatment of fibrotic myobundles resulted in increase of tetanus force production in all three donors and reduction of pro-fibrotic ECM accumulation of collagen 1 and fibronectin. Suramin’s influence on TGF-β1 signaling in our system was found not to be as targeted as Nintedanib as there was only reduction in Smad3 phosphorylation and not Smad2 phosphorylation. Anti-fibrotic drug testing in our model was also able to wean out donor specific sensitivity to the drugs with donor 3. Skeletal myobundles were integrated with Tissue Engineered Blood Vessels (TEBVs) to identify the effect of skeletal muscle fibrosis on blood vessels or the human vasculature. Integrated TEBVs with 5 ng/ml TGF-β1 dosed myobundles showed reduced function, increased mesenchymal markers such as vimentin and alpha smooth muscle actin, and increased endothelial cell inflammation. Our results suggest a detrimental effect of skeletal muscle fibrosis on blood vessels and show an interaction between the skeletal muscle fibrosis and the human vasculature This model provides a platform to study skeletal muscle fibrosis alone or its effect on the vasculature and allows for testing anti-fibrotic drugs and assessing myobundle function along with disease influence on human vasculature.