We previously reported that the body weight of DMD rats decreased progressively after the age of 6 months [22]. Suspecting that eating disorder occurs in DMD rats, we measured the amount of food intake at the ages of 3 months (before the onset of body weight decrease) and 7 months (after the onset of bodyweight decrease). The amount of food intake at 3-month-old was comparable between WT and DMD rats (Fig. 1). However, at 7-month-old, significantly decreased food intake was observed in DMD rats, indicating the occurrence of eating disorder (Fig. 1).
The above results suggest that muscles involved in food intake are affected by the progress of the disease. We then performed morphometric analyses of muscles involved in food intake. We chose masseter and tongue muscles because these two muscles are to be approached easily and distinguishable from other muscles. As shown in Fig. 1A, the area corresponding to the masseter muscle was considered as an ellipse, and its long and short diameters were measured with calipers to calculate the area. The area of masseter muscle was measured from 1-month to 8-month-old. At 1-month-old and after 6-month-old, the area of masseter muscle was significantly reduced in DMD rats compared to WT rats. Especially, the greater reduction was observed at 6- and 8-month-old (Fig. 2B).
The appearance of the tongue at 8-month-old is shown in Fig. 3A. The cross-section of the posterior part of the tongue was larger in DMD rats than in WT rats (Fig. 3A). Then, we measured the area and width of the upper half of the tongue sections. Quantitative analysis showed that the width of the tongue of DMD rats was significantly larger than that of WT rats after 3-month-old (Fig. 3B). In addition, the area of the tongue was significantly larger in DMD rats at the age of 8 months than in WT rats (Fig. 3C). These results indicate that macroglossia is occurring in DMD rats.
To further gain insight into the age-related changes of masseter and tongue muscles of DMD rats, histological analyses were performed. The masseter muscle of DMD rats showed myofiber degeneration and necrosis with infiltration of neutrophils and macrophages at 1-month-old (Fig. 4B), and regenerative myofibers with enlarged centralized nuclei and basophilic cytoplasm at 3-month-old (Fig. 4D). At 8-month-old, in addition to inflammatory and degenerative changes, severe fibrosis and fatty infiltration were observed (Fig. 4F and H). No significant pathological changes were observed in the masseter muscle of WT rats at all ages (Fig. 4A, C, E, and G).
In contrast, in the tongue muscle of DMD rats, regenerative myofibers with enlarged centralized nuclei and basophilic cytoplasm were observed at 1-month-old (Fig. 5B), and these regenerative changes were accompanied by mild fibrosis at 3-month-old (Fig. 5D). At 8-month-old, mild fibrosis, also confirmed by Masson's trichrome stain (Fig. 5H), was persisted (Fig. 5F). No significant pathological changes were observed in the tongue muscle of WT rats at all ages (Fig. 5A, C, E, and G).
To further examine whether myofiber necrosis and muscle regeneration are ongoing at 6-month-old when macroglossia is occurring in the tongue muscle of DMD rats, immunohistochemical analyses of endogenous IgG (myofiber necrosis) and embryonic MHC (muscle regeneration) were performed. The masseter muscle of DMD rats, though there is variation among individuals, showed marked myofiber necrosis (Fig. 6A and E) and muscle regeneration (Fig. 6C and F), while the tongue muscle showed none of these, (Fig. 6B, D, E and F). These results indicate that, although the masseter and tongue muscles of DMD rats undergo pathological changes that are not seen in WT rats, the changes seen in the tongue muscle are much milder than in the masseter muscle of DMD rats.
To investigate the factors that are responsible for macroglossia seen in DMD rats, we measured the myofiber diameters of the tongue muscle of WT and DMD rats at 6-month-old. The myofiber diameters of DMD rat were larger than those of WT rats (Fig. 7A and B; p < 0.01 by the Wilcoxon rank sum test). The ratio of myofibers with central nuclei was significantly higher in DMD rats than in WT rats, indicating that muscle regeneration had occurred in the past (Fig. 7C). In addition, quantitative analysis of Masson's trichrome stained sections as shown in Fig. 5 revealed that the fibrotic area in the tongue muscle of DMD rats is about twice that of WT rats (Fig. 7D). Thus, these results suggest that both the hypertrophic response of myofibers and the accumulation of collagen between myofibers were combined to produce macroglossia.
We reported previously that cellular senescence with increased expression of p16 occurs in the hindlimb muscles of DMD rats, and that the proliferation of muscle satellite cells is markedly suppressed in culture [22]. Therefore, we examined the expression of the p16 gene in masseter and tongue muscles at 9-month-old and found that it was markedly up-regulated in the masseter muscle of DMD rats compared to WT rats, whereas it was only slightly up-regulated in the tongue muscle (Fig. 8A). There was no significant difference in the number of Pax7-positive satellite cells in the sections, although there was a large variation between individuals (Fig. 8B). On the other hand, when cultured in vitro for 4 days, the number of Pax7- or MyoD-positive satellite cells derived from the masseter muscle of DMD rats was significantly lower than that of WT rats (Fig. 8C and D, Supplementary Fig. 1A and B), while the number of satellite cells derived from the tongue muscle of DMD rats was only slightly lower than that of WT rats (Fig. 8C and D, Supplementary Fig. 1A and B). These results indicate that cellular senescence occurs in the masseter muscle of DMD rats as was seen in their hindlimb muscles [22], while it is significantly less severe in the tongue muscle.
The above results suggest that the tongue muscle of DMD rats is protected from degenerative changes, which occur in other muscles due to the absence of dystrophin. Utrophin, known as dystrophin-related protein, had been shown to compensate for the function of dystrophin [4, 8, 13, 17]. In a mouse model of DMD, mdx mouse, the skeletal muscle phenotype is milder than that seen in DMD patients [13], and this had been once thought to be due to an up-regulation of utrophin [13]. Thus, we examined the expression of utrophin in the masseter and tongue muscles of WT and DMD rats at 9-month-old. In masseter muscle, utrophin expression was significantly upregulated by the absence of dystrophin (Fig. 8E). Interestingly, in tongue muscle, utrophin expression was also upregulated but its basal expression in the presence of dystrophin (WT rats) was comparable to that in the masseter muscle of DMD rats (Fig. 8E). This result suggests that the tongue muscle is intrinsically expressing a higher level of utrophin even in the presence of dystrophin, and this might be related to the resistance of this tissue against degenerative changes in the absence of dystrophin.