DMD is a rare inherited neuromuscular disease caused by mutations in the largest gene known in the human genome, DMD, located on the X chromosome, that produces the dystrophin protein. The condition affects all muscles, including cardiac, skeletal, and smooth muscles, severely affecting cardiac and motor functions, with patients commonly succumbing to cardiorespiratory failures. Conventional drug treatments, such as steroids, combined with physical therapy remain the standard course of treatment despite merely delaying disease progression.

Recently, much attention has been focused on next-generation therapies, including gene therapy and antisense oligonucleotides, as potential treatments or even possibly cures for this devastating disorder. Nonetheless, safety concerns and the limited generalizability of these new therapeutic options have hindered their advancement to the clinic. Cell transplantation represents an alternative approach applicable to a broader range of DMD patients, thus potentially offering a more favorable outlook for translation to the bedside.

In this new study, the researchers examined the therapeutic effects of supplementing dystrophin by implanting myoblasts—a type of muscle cell—into the gastrocnemius, one of the calf muscles of DMD model mice. Because commonly used clinical assessments of muscle function not only measure muscular strength but also endurance tolerance, the team first devised a new method to measure motor functions that consider both aspects. As the treatment group, myoblast cells were injected into the gastrocnemius muscle of DMD model mice between 1-2 months of age and compared to healthy mice and untreated DMD model mice in terms of motor functions periodically over 2 to 6 months, with some analyses performed up to 8 months. Measurements of muscular strength at rest throughout the study revealed a decline in motor functions in DMD model mice. Notably, cell transplantation did not improve this motor measurement. Conversely, when muscle fatigue endurance was assessed by measuring the decline of muscular strength following extended treadmill running, DMD model mice receiving cell transplantation showed significant improvement compared to non-treated DMD model mice. Indeed, dystrophin-supplemented DMD model mice showed no difference in muscle fatigue endurance compared to healthy mice, thus demonstrating that cell transplantation ameliorated defects in muscle fatigue tolerance in DMD model mice.

Next, a detailed examination of the gastrocnemius muscle revealed that over 10% of muscle fibers in transplanted-DMD model mice contained the dystrophin protein, whose levels correlated positively with muscular strength at rest. Further analysis of this data indicated 10% as the lowest level of dystrophin supplementation necessary to exhibit non-diseased muscular strength, thus providing valuable insights into the target level of dystrophin supplementation for effective cell transplantation. Additional measurements of muscle fiber numbers and diameters revealed that although dystrophin supplementation by cell transplantation resulted in the highest number of muscle fibers, they remained smaller than healthy mice, like untreated DMD model mice, thus explaining the absence of muscular strength improvement described above.

The researchers next examined the correlation between the number of dystrophin-supplemented muscle fibers and muscle fatigue endurance. Unlike muscular strength at rest, they observed that muscle fatigue tolerance was ameliorated in transplanted-DMD model mice with no changes to the number of muscle fibers by dystrophin supplementation. Nonetheless, histological analyses revealed remarkable protection by dystrophin against muscular damage and degeneration and reduced fibrosis after cell transplantation. Consistently, the research team observed a clear correlation between muscle damage and fatigue tolerance.

DMD preferentially affects fast-twitch muscle fibers, which the researchers confirmed upon detailed examination. Notably, the number of slow and fast oxidative muscle fibers was increased in DMD model mice receiving cell transplantation compared to untreated DMD model mice. Further analyses indicated that most dystrophin-supplemented muscle fibers are of the oxidative type, meaning oxidative metabolism (as opposed to anaerobic glycolysis) is preferentially used to generate ATP, the principal cellular energy source. Therefore, the researchers focused on ATP levels and mitochondria, the primary site of ATP production in cells, to examine how they are impacted in DMD mice and how cell transplantation may rescue such deficits. By generating transgenic DMD model mice with an ATP-visualizing probe, they monitored ATP levels in the gastrocnemius muscle of live healthy and DMD model mice with and without cell transplantation during muscle fatigue induced by electrical stimulation. Remarkably, whereas DMD model mice showed lower ATP levels following extended electrical stimulation, DMD model mice with dystrophin supplementation showed a time-dependent increase in ATP levels (although not to the same level) as healthy mice. Although not all measurements were rescued to the same degree as healthy mice, biochemical and ultrastructural analyses detected higher levels of mitochondrial proteins and healthy mitochondria in DMD model mice receiving cell transplantation than in untreated ones.

Altogether, these experiments demonstrated the therapeutic potential of cell transplantation as a viable DMD treatment and revealed a mechanism by which dystrophin supplementation via cell transplantation restores motor functions. With new insights gained from this study, the research team may be able to improve this therapeutic strategy for clinical applications in the future.

• doi: 10.1186/s13287-024-03922-x