mTOR inhibition and BMP signaling act synergistically to reduce muscle fibrosis and improve myofiber regeneration
Shailesh Agarwal, David Cholok, Shawn Loder, John Li, Christopher Breuler, Michael T. Chung, Hsiao Hsin Sung, Kavitha Ranganathan, Joe Habbouche, James Drake, Joshua Peterson, Caitlin Priest, Shuli Li, Yuji Mishina, Benjamin Levi
Shailesh Agarwal, David Cholok, Shawn Loder, John Li, Christopher Breuler, Michael T. Chung, Hsiao Hsin Sung, Kavitha Ranganathan, Joe Habbouche, James Drake, Joshua Peterson, Caitlin Priest, Shuli Li, Yuji Mishina, Benjamin Levi
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Research Article Inflammation Therapeutics

mTOR inhibition and BMP signaling act synergistically to reduce muscle fibrosis and improve myofiber regeneration

Abstract

Muscle trauma is highly morbid due to intramuscular scarring, or fibrosis, and muscle atrophy. Studies have shown that bone morphogenetic proteins (BMPs) reduce muscle atrophy. However, increased BMP signaling at muscle injury sites causes heterotopic ossification, as seen in patients with fibrodysplasia ossificans progressiva (FOP), or patients with surgically placed BMP implants for bone healing. We use a genetic mouse model of hyperactive BMP signaling to show the development of intramuscular fibrosis surrounding areas of ectopic bone following muscle injury. Rapamycin, which we have previously shown to eliminate ectopic ossification in this model, also eliminates fibrosis without reducing osteogenic differentiation, suggesting clinical value for patients with FOP and with BMP implants. Finally, we use reporter mice to show that BMP signaling is positively associated with myofiber cross-sectional area. These findings underscore an approach in which 2 therapeutics (rapamycin and BMP ligand) can offset each other, leading to an improved outcome.

Authors

Shailesh Agarwal, David Cholok, Shawn Loder, John Li, Christopher Breuler, Michael T. Chung, Hsiao Hsin Sung, Kavitha Ranganathan, Joe Habbouche, James Drake, Joshua Peterson, Caitlin Priest, Shuli Li, Yuji Mishina, Benjamin Levi

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Figure 3

Rapamycin preserves SMAD 1/5 signaling and osteogenic differentiation.

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Rapamycin preserves SMAD 1/5 signaling and osteogenic differentiation. (...
(A) Rapamycin significantly increased osteogenic differentiation of mesenchymal cells with hyperactive BMP signaling (normalized ratio: 1.77 vs. 1.0, Student’s 2-tailed t test, n = 3, P < 0.05). (B) Rapamycin preserves SMAD 1/5 phosphorylation in mesenchymal cells with hyperactive BMP signaling in vitro (normalized ratio: 1.00 vs. 0.81, Student’s 2-tailed t test, n = 3, P = 0.15, not significant). (C) Representative p-SMAD 1/5 immunostaining in the hindlimbs of untreated and rapamycin-treated caAcvr1fl/fl mice after adenoviral Cre and cardiotoxin (Ad.cre/CTX) injury (white arrowheads indicate p-SMAD 1/5 expression). (D) Whole-tissue immunoblot for p-SMAD 1/5 expression at the injury site of untreated and rapamycin-treated caAcvr1fl/fl mice after Ad.cre/CTX injury (normalized ratio: 1.00 vs. 0.70, Student’s 2-tailed t test, n = 3, P = 0.13, not significant). (E) Experimental design for transplantation of mesenchymal cells with hyperactive BMP signaling. (F) Representative microCT reconstructions showing presence of ectopic bone in untreated and rapamycin-treated hindlimbs after transplantation of mesenchymal cells with hyperactive BMP signaling.(G) Representative H&E and alizarin red staining of hindlimbs from rapamycin-treated mice after cell transplantation. (H) Representative fluorescence imaging showing expression of GFP from transplanted cells at sites of alizarin red staining; dotted border represents corresponding areas of muscle damage. All scale bars: 200 μm. *P < 0.05.