(L-465) Excessive Stiffness Promotes Fibrosis and Impairs Regeneration in Muscle Stem Cells from Children with Cerebral Palsy

Introduction:: Cerebral Palsy (CP) is the most common movement disorder in children, affecting approximately 2 in every 1,00 live births. CP is a group of neuromuscular disorders that result in muscle weakness, involuntary movements, muscle contractures, and fibrosis. Fibrosis is the pathological accumulation of extracellular matrix components. A defining characteristic of muscle contractures and fibrosis is an excessive increase in stiffness. However, how this excessive stiffness affects cells within skeletal muscle is not well understood. Muscle satellite cells (MuSCs) and fibro-adipogenic progenitors (FAPs), two types of muscle-resident stem cells, are known to be mechanosensitive. Stiff substrates inhibit self-renewal in MuSCs and promote myofibroblast activation, a marker of fibrosis, in FAPs. The mechanosensitivity of muscle-resident stem cells has not been investigated in the context of muscle contractures. Understanding the mechanosensitivity of these cells provides context to the progressive nature of fibrosis and mechanistic targets for treatments of fibrosis and muscle contractures. We hypothesized that MuSCs would have reduced differentiation, and FAPS would have enhanced myofibroblast activation on stiffer substrates.


Materials and Methods:: A small biopsy from the gracilis muscle was collected from typically developing (TD) children and children with cerebral palsy (CP) undergoing surgeries for ACL reconstruction and hamstring lengthening, respectively. The muscle biopsies were digested using mechanical and enzymatic digestion, and the FAPs and MuSCs were isolated using magentic-activated cell sorting. Polyacrylamide gels at stiffnesses of 0.2, 8, and 25 kPa were coated with collagen-I, and cells were cultured on the gels for 5 days in differentiation media. Cells were then fixed in 4% PFA, permeabilized using 0.5 % Triton-X, blocked with 5% BSA, and stained with primary antibodies in 5% BSA overnight. MuSCs were stained with MF20, a myosin heavy chain antibody, and FAPs were stained with αSMA, a marker for myofibroblasts. Secondary antibodies were applied the next day in 0.1% BSA for 90 minutes, cells were washed, stained with Hoescht for 15 minutes, and washed in PBS. Plates were imaged using inverted Leica DMi8. Images were captured with a dry 20X/0.40 objective using a Leica DFC9000 GTC camera and LAS X software. Images were analyzed using FIJI: ImageJ and custom macro scripts to obtain differentiation index in the MuSCs and myofibroblast activation in the FAPs.


Results, Conclusions, and Discussions:: Contrary to our hypothesis, there was no significant difference in MuSCs' ability to differentiate into muscle between the CP and TD cells. However, in both cases MuSCs had altered cell morphology when differentiating on higher stiffnesses. There was a significant increase in nuclear clustering, with myonuclei clustering in large groups rather than spreading evenly across the myotube (Fig. 1). This suggests that in cases of fibrosis and muscle contractures, like CP, MuSCs have altered differentiation due to the increase in stiffness. This nuclear clustering may affect the myonuclear domain of the myotubes, making the muscle weaker and more prone to injury. Inhibiting mechanosensing is a potential avenue to improve regeneration in CP.

In FAPs, myofibroblast activation was significantly higher across all CP samples compared to TD samples (Fig 2.). This suggests that the FAPs in CP are in a more profibrotic state and could be the reason for increase ECM deposition and progressive fibrosis. TD samples appeared sensitive to substrate stiffness, with higher myofibroblast activation on stiffer substrates, suggesting that as a tissue becomes fibrotic, it may induce FAPs to activate into myofibroblasts leading to progressively degenerative fibrosis.

This research has implications across various fibrotic muscle diseases. The mechanosensitivity of muscle resident stem cells may play an important role in the altered differentiation and activation seen in cerebral palsy and other fibrotic diseases. Targeting this mechanosensitivity is a potential therapeutic avenue to attenuate fibrosis and improve muscle function.

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