Browsing by Author "Ananthakumar, Anandita"

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.

Advances in tissue engineering have led to the development of 3D biomimetic models of human skeletal muscle (myobundles), which have a wide range of applications in regenerative medicine, predictive drug testing and toxicology screening, and development of therapeutics for muscular disorders. Statins are the most commonly used medications to lower cholesterol and prevent cardiovascular diseases. Long term treatment using statins may lead to musculoskeletal side effects in the form of myopathy, myalgia or rhabdomyolysis. Currently, there are no reliable diagnostic measures of statin-induced myopathy since only some of the individuals complaining of the symptoms of statin myopathy exhibit elevated creatine kinase levels. This study examined if the engineered human skeletal myobundles using cells derived from patients with a history of statin induced myopathy exhibit statin-dependent defects in muscle physiology when exposed to varying concentrations of statins in vitro. Utilizing a microphysiological system for skeletal muscle, that uses patient-derived tissue to form engineered myobundles, to recapitulate the organization and function of native muscle is a novel way to understand the functional changes within individuals that experience statin induced myopathy. To create myobundles, myoblasts were encapsulated in a matrigel/fibrin matrix. The myobundles were cultured in 3D human growth media for 4 days and shifted to low amino acid differentiation media for another 4 days and then dosed for 5 days with varying concentrations of statins in low amino acid differentiation media. These were then force tested to see if there is a difference in twitch, tetanus and fatigue force response between different statin types, concentration levels, case and donor and dose exposure periods. A repeated measures multiple linear regression of the raw tetanus force versus statin dose, myopathy or control, and type of statin revealed a negative relationship between tetanus force response and statin concentration and myopathy. It was also seen that force production for the myopathy donors dosed with simvastatin when compared to their control pair was much lower which can be due to muscle weakness in these donors. In addition, immunofluorescence of the myobundles showed there is a structural difference in myotubule formation between the myopathy donors and control donors which can be correlated to force production. The donors that exhibited fewer myotubules or those that had a frayed appearance and lacked structural definition had lower force production. Overall, myobundles prepared with myoblasts from the myopathy and control donors exhibited functional changes associated with reported statin myopathy, while absence of myofibers or degradation of myofibers was associated with very low levels of force production.