Revertant Fibers in the <i>mdx</i> Murine Model of Duchenne Muscular Dystrophy: An Age- and Muscle-Related Reappraisal

<div><p>Muscles in Duchenne dystrophy patients are characterized by the absence of dystrophin, yet transverse sections show a small percentage of fibers (termed “revertant fibers”) positive for dystrophin expression. This phenomenon, whose biological bases have not been fully elucidated, is present also in the murine and canine models of DMD and can confound the evaluation of therapeutic approaches. We analyzed 11 different muscles in a cohort of 40 <i>mdx</i> mice, the most commonly model used in pre-clinical studies, belonging to four age groups; such number of animals allowed us to perform solid ANOVA statistical analysis. We assessed the average number of dystrophin-positive fibers, both absolute and normalized for muscle size, and the correlation between their formation and the ageing process. Our results indicate that various muscles develop different numbers of revertant fibers, with different time trends; besides, they suggest that the biological mechanism(s) behind dystrophin re-expression might not be limited to the early development phases but could actually continue during adulthood. Importantly, such finding was seen also in cardiac muscle, a fact that does not fit into the current hypothesis of the clonal origin of “revertant” myonuclei from satellite cells. This work represents the largest, statistically significant analysis of revertant fibers in <i>mdx</i> mice so far, which can now be used as a reference point for improving the evaluation of therapeutic approaches for DMD. At the same time, it provides new clues about the formation of revertant fibers/cardiomyocytes in dystrophic skeletal and cardiac muscle.</p></div

Average (± stdev) of RFs/mm² in different muscles, at each age group.

<p>Average (± stdev) of RFs/mm² in different muscles, at each age group.</p

Interaction between the number of isolated RFs and the age of the animals.

<p>Chart is organized as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072147#pone-0072147-g001" target="_blank">Figure 1B</a>; for each muscle, values were obtained by averaging the absolute number of single dystrophin-positive fibers (i.e., separated by at least two negative fibers from neighboring RFs) found in each section.</p

Interaction between the percentage of clustered revertant fibers per section and the age of the animals.

<p>Chart is organized as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072147#pone-0072147-g001" target="_blank">Figure 1B</a>; for each muscle, values were obtained by averaging the percentage of clustered fibers over the total for each section.</p

Number of revertant fibers (or of cardiomyocytes, in the case of heart) per square millimeter in different muscles.

<p>(A) Values calculated without differentiating between age groups; numbers were obtained by averaging the single RF/mm² value of each of the 10–12 sections per muscle, for all forty mice. Abbreviations are as follows: TA, tibialis anterior; EDL, extensor digitorum longus; Q, quadriceps; SOL, soleus; GC, gastrocnemius; PL, plantaris; D, diaphragm; H, heart; IC intercostal; PT, pectoralis; TRIC, triceps. (B) Interaction between the number of revertant fibers per square millimeter (Y-axis) and the age of the animals (X-axis). For each muscle, the segmented line joins the four values of RFs/mm², one for each age group; data were obtained by averaging the counts of all the sections, 10–12 per muscle, for ten animals. Note that the order of the muscles’ names at the right side of the chart reflects the order of the values found for group 4.</p

Maximum number of RFs/mm2 per cluster, at each age group.

<p>Maximum number of RFs/mm2 per cluster, at each age group.</p