Dystrophin and utrophin handle muscle stress in different ways, with implications for a leading Duchenne muscular dystrophy therapy

Fundamentally differing responses by dystrophin and utrophin to mechanical stress complicates treatment strategies.

A collaborative study led by Professors Murti Salapaka (Vincentine Hermes-Luh Chair in ECE) and James M. Ervasti (Department of Biochemistry, Molecular Biology and Biophysics) has found that utrophin, a protein being developed as a potential substitute therapy for Duchenne muscular dystrophy (DMD) handles mechanical stress in a fundamentally different way from dystrophin, the protein it is meant to replace. 

The details are presented in a paper titled, "Multiple modes of AFM reveal distinct mechanical properties for dystrophin and utrophin not manifest by small fragments," which appears in the PNAS.

Combining the Salapaka group’s expertise in developing single-molecule force spectroscopy methods using atomic force microscopy (AFM) and the Ervasti group’s leadership in the molecular biology of dystrophin and muscular dystrophy, the team measured, one molecule at a time, the mechanical behavior of proteins that have largely resisted full-length characterization.

First author Cailong Hua addressed the significance of the study: "Dystrophin and utrophin are closely related proteins, and that similarity has motivated interest in whether utrophin could help compensate for the loss of dystrophin. Our results suggest that their full-length mechanical responses are not identical. Dystrophin shows behavior consistent with shock absorption, while utrophin appears to respond more like a force-transmitting element. That distinction is important to consider when thinking about how these proteins protect muscle."

Emphasizing the collaborative interdisciplinary research, Salapaka says, “What made this study possible was the combination of two things: full-length proteins of a quality you can actually do single-molecule mechanics on, which came out of Jim Ervasti's group, and an AFM approach that lets us interrogate the same molecule in two independent operational modes and confirm we are seeing the same physics from both. Neither side could have done this work alone, and what emerged once we had both pieces in place was a clear mechanical contrast between dystrophin and utrophin that simply was not visible in earlier studies of smaller fragments.”

DMD affects roughly one in 5,000 boys born in the United States. It is caused by the absence of dystrophin, a protein that lines the muscle cell membrane, or sarcolemma, and is thought to act as a molecular shock absorber protecting the membrane from damage during muscle stretching and contraction. Without dystrophin, muscle fibers progressively degenerate.

One leading therapeutic strategy under active investigation is to upregulate utrophin, a closely related protein that is abundant during fetal development but largely disappears from adult skeletal muscle, as a stand-in for missing dystrophin. Whether utrophin can substitute for dystrophin in a mechanical sense, however, has remained unresolved.

To address the question, the team studied full-length dystrophin, full-length utrophin, DysN-R10, a large dystrophin fragment, and Dp260, a naturally occurring isoform of dystrophin. Each construct was stretched one molecule at a time using AFM. 

The results revealed two distinct mechanical signatures. Dystrophin, DysN-R10, and Dp260 all displayed brittle unfolding behavior: each successive structural domain came apart at roughly the same force, regardless of how many domains had already unfolded. Utrophin, by contrast, behaved more like a stiffening spring. Each unfolding event required more force than the one before it.

A key finding is that this divergence is essentially invisible in small fragments of either protein. The stiffening-spring character of utrophin emerges only when full-length proteins, or large multi-repeat constructs, are pulled and many sequential unfolding events can be tracked. The contrast also widens at lower forces and slower pulling speeds, closer to physiological conditions, suggesting that the difference may be even more pronounced in living muscle than in the AFM measurements themselves.

The team interprets these contrasting mechanics in light of where the two proteins normally function in muscle. Dystrophin lines the entire sarcolemma and repeated exposure to mechanical strain during contraction entails a brittle, energy-dissipating response. On the other hand, utrophin in adult skeletal muscle is localized primarily to the myotendinous and neuromuscular junctions, where forces are transferred between muscle and tendon rather than absorbed across a broad membrane. In this context, a stiff, force-transmitting spring may be better suited.

The findings suggest that utrophin and dystrophin perform distinct mechanical functions, and they question whether simply substituting utrophin for dystrophin at the sarcolemma as a DMD therapy can deliver equivalent mechanical protection.

The authors note that AFM probes higher forces than those typically encountered in vivo, and that mechanical behavior in living muscle will be shaped by interactions with actin, the plasma membrane, binding proteins, and post-translational modifications. Future work integrating single-molecule and cellular approaches will be needed to translate these mechanical signatures fully into physiological function.

"The next step is to connect these single-molecule measurements to what happens in cells and tissues," says Hua. "AFM lets us isolate the intrinsic mechanical response of each protein, but muscle is a much more complex environment. Understanding how these signatures change when the proteins interact with actin will be important for evaluating future therapeutic strategies."

Read the complete paper at the PNAS website

The research was supported by the National Institutes of Health (5R01AR042423). In addition to lead author Cailong Hua and senior authors Professors Salapaka and Ervasti, the team included Joseph Vavra, Jacob Powers, and Joseph M. Muretta, all from the Department of Biochemistry, Molecular Biology and Biophysics. 

Cailong Hua received his doctoral degree under the supervision of Professor Salapaka and has been working as an applied scientist at Amazon since October 2025.

Feature image by Anirudh on Unsplash