University of Illinois researchers have uncovered a molecular mechanism that influences muscle weakness in a mouse model of Duchenne Muscular Dystrophy, the most common inherited neuromuscular disease and one of the most severe forms of inherited muscular dystrophies.

The genetic disorder causes progressive muscle degeneration and weakness due to loss of or alterations in dystrophin, a protein that is essential for muscle contraction and integrity. Symptoms of Duchenne Muscular Dystrophy, or DMD, typically appear in early childhood; the disorder usually only affects males. The loss of muscle quantity and quality ultimately leads to premature death before the age of 30 years old. Many research efforts have been focused on developing gene therapies to correct or replace the dystrophin gene or stem cell therapies to deliver healthy cells expressing a functional dystrophin to dystrophic muscles.

However, “this disease is very unpredictable with so many different mutations, manifestations as well as diverse responses in treatment,” said Jie Chen, a professor of cell and developmental biology in the School of Molecular & Cellular Biology and the study’s principal investigator. Identifying mechanisms underpinning muscle weakness common in DMD regardless of the specific mutations can lead to novel therapeutic strategies.

Autophagy: An important process for muscle quality
In the recently published article, “RhoA/ROCK signaling activated by ARHGEF3 promotes muscle weakness via autophagy in dystrophic mdx mice,” Jie Chen and Jae-Sung You, the lead authors of the article, explored the role of autophagy on muscle quality and its molecular mechanism in a mouse model of the disorder.

Autophagy is a process in which cells remove cellular waste and promote cell survival. One of the inhibitors for this process is ARHGEF3, an upstream activator of RhoA/ROCK signaling. Elevated RhoA/ROCK activity is found in skeletal muscles of DMD mouse models, and inhibition of this pathway promotes muscle regeneration and increases muscle quantity. However, the role of RhoA/ROCK signaling in muscle quality is unknown.

“Past research from our lab has shown that activation of autophagy is really important in maintaining muscle function, but the molecular pathway that regulates this process in [DMD] context has not been well understood,” said Jae-Sung You, a former postdoctoral researcher in Chen’s lab.

The group found that by genetically deleting ARHGEF3 or pharmacologically inhibiting ROCK in mdx mice, a mouse model used for studying DMD, the muscle quality of the mice is restored.

3D muscle in a dish: an in vitro model to study muscular dystrophy
In this paper, Jie Chen lab collaborated with Rashid Bashir, professor of bioengineering, affiliate professor of molecular and integrative physiology, and dean of the Grainger College of Engineering, to use a method pioneered by the Bashir lab to test muscle quality in 3D in vitro.

“We used a 3D printing approach to create a scaffold to help build muscles in vitro and to test out muscle contraction in normal as well as disease state,” noted Jae-Sung You.

The authors found that muscle cells from mdx mice have a faulty expression of ARHGEF3 which affects the autophagy mechanism hence decreasing muscle quality. 

“When comparing cells from wild-type and mdx mice, the difference in contraction is day and night and it’s clear that the cells have different muscle strength,” Chen said. Importantly, mdx muscle strength in the 3D culture was rescued by a small molecule inhibitor of ROCK.

The researchers’ observations in the 3D in-vitro experiments motivated them to perform animal experiments, which led to the conclusion that inhibiting the ARHGEF3-RhoA-ROCK pathway by genetically removing the ARHGEF3 gene or using the small-molecule inhibitor of ROCK can restore autophagy and improve muscle quality.

Autophagy-based therapies: The horizon might be near
Based on this novel finding, the research group is hopeful autophagy-based therapies, by strengthening dystrophic muscles, could in the future improve quality of life for patients with Duchenne muscular dystrophy.

“Every patient has a different type of mutation in DMD, and the way they respond to treatment is different. By targeting to restore autophagy, a common attribute in DMD, it [could become] a novel approach to improve overall health of all DMD patients,” You said.

This work is supported by the Muscular Dystrophy Association, National Institutes of Health, and National Science Foundation.

Photo: From left: Jae-Sung You, Jie Chen, Rashid Bashir.