This work aimed to validate biomarkers for diagnosis and progression of DMD through the analysis of metabolites, protein, and histology of the tongue muscles of dystrophic mdx mice and non-dystrophic control mice, aged 30 days (young) and 21–25 months (old).

Surprisingly, our histological results demonstrated that the mid-belly tongue of old mdx remains partially spared from fibrosis and myonecrosis. Previous studies from 23- and 26-month-old mdx mice described the tongue muscles as displaying significant histological abnormalities, including foci of inflammation and fibrosis, and localized loss of muscle fibers [10, 18]. However, in these studies, the analysis of the tongue muscle was approached qualitatively, yet without specific delineation of the particular region of the tongue analyzed. The region analyzed may interfere with the results, given the complex composition of the tongue muscle, the presence of intrinsic and extrinsic muscles by region, coupled with age-related shifts in MyHC isoform composition and differences in fiber size from the anterior, middle, and posterior regions of the tongue [33,34,35]. In contrast, here, the qualitative and quantitative analysis of mid-belly tongue did not show such severity. This protection was evidenced by metabolomics and Western blotting experiments performed with whole muscle of the tongue, analyzing the biomarker proteins for inflammation and fibrosis, and the concentrations of the metabolites 3-methylhistidine, acetic acid, glutamic acid, alanine, creatine, phosphocreatine, glycerol, glycine, isoleucine, methionine, succinic acid, and taurine already used for dystrophic muscle differentiation and that in this work presented similar results between wild type and mdx. This similarity between the chosen biomarkers was observed in young and old animals, corroborating the histological result.

Kunert-Keil and colleagues [36] report that dystrophic masseter and temporalis muscles are histologically similar to other skeletal muscles involved in the degeneration process, whereas the tongue remains with a milder phenotype. However, from the findings stated in the article, masseter and temporalis are more resistant to the calcium (Ca2+) overload when compared to tongue muscles of 100-day-old mdx. Hence, histological findings assert that inflammatory foci are hardly detectable, and dystrophic tongue contains only 11.2% of regenerated muscle, when compared to masseter, temporalis, and even soleus [36]. In DMD rats, the tongue showed hypertrophy of myofibers with less advanced dystrophic changes until 9 months old compared to masseter. This resistance against degeneration might be related to a higher transcription level of utrophin in tongue of wild-type and DMD rats compared to masseter of wild-type rats [37]. Increased expression of utrophin in the skeletal muscles of mdx mice results in a functional improvement in muscle function [38,39,40]. However, in mdx muscles spared from degeneration, as extraocular and intrinsic laryngeal muscles, the utrophin upregulation is unlikely to be a protective mechanism [41,42,43]. The extraocular muscles remain unaffected even in dystrophin-deficient utrophin double knockout mice [42]. In the intrinsic laryngeal muscles, the reduction of utrophin at 20 months in mdx mice did not instigate any degeneration in muscle fibers [43]. Further investigation of utrophin in partially spared tongue muscles throughout life span would be interesting, as utrophin and concomitant rescue of dystrophin-associated glycoproteins are irregularly correlated with other protected muscles.

The study of biomarkers in a muscle partially spared from the absence of dystrophin allows us to identify dystrophinopathy markers that change with age, regardless of muscle degeneration. Among the possible biomarkers analyzed, the metabolites alanine, methionine, 3-methylhistidine, taurine, and glycerol change with aging, but not between control and dystrophic whole tongue muscles. Thus, it is suggested that these biomarkers may be efficient for specific assessments, but care should be taken when using them to monitor the progression of the disease, since they change throughout life.

The analysis of the metabolic profile of tongue suggests that the muscle aging process has a high impact on its metabolism, regardless of the lineage, since it was possible to distinguish the different ages of animals of the same lineage (Fig. 3). High-resolution 1H NMR spectroscopy has been shown to differentiate skeletal muscle from adult and old mice. In addition, there is a general difference in composition between younger and older muscles in mice [44], and these results are confirmed in the scenario of dystrophinopathy.

It was not possible to distinguish the metabolic profile of the whole tongue between the wild-type and mdx strains. This result corroborates our histological analysis, which showed protection against fibrosis and myonecrosis in the mid-belly tongue of young and old dystrophic animals. The chosen metabolites (3-methylhistidine, acetate, alanine, creatine, glutamic acid, glycerol, glycine, isoleucine, methionine, phosphocreatine, succinic acid, and taurine) were altered between the wild-type and mdx animals in muscles that suffer degeneration, such as the diaphragm, quadriceps, and soleus [13, 20, 21]. Since these metabolites were not different between the strains in this study, it suggests that the protection previously observed in the tongue of 3-month-old adult animals [17] is also observed in young (1 month old) and old (21–25 months old) animals.

Taurine is considered a biomarker for the aging of skeletal muscle in mice [44], corroborating the data from this project, since its concentration was higher in old dystrophic and non-dystrophic mice. Its decrease in young mdx mice, compared to wild type, has already been seen in other muscles, indicating the possibility of a deficient taurine synthesis by the mdx muscles [21]. In the tongue, this decrease occurs both in the mdx and in the wild type compared to older age, corroborating the protection of the tongue muscle in dystrophic pathology. Taurine has an osmoregulatory function that helps to balance intracellular Ca2+ levels, helping with cell integrity and membrane stability [13]. Kunert-Keil et al. (2014) [34] studied the differential expressions of genes involved in Ca2+ homeostasis in dystrophic masticatory muscles and found uneven expressions in the studied muscles. Despite being a priori protected muscle, in its study, the tongue of 100-day-old mice presented levels of expression of Ca2+ regulatory proteins typical of dystrophic muscles. Analyzing Ca2+ regulatory proteins at the age of 21 to 25 months old would provide a better view on the relationship of increased taurine, found in this study, with Ca2+ homeostasis in old mice. In addition, in muscle regeneration, there is an increase in taurine, regardless of the type of muscle or genetic etiology of the damage to the fiber, suggesting that metabolic changes are significant indicators of muscle status [45]. The level of taurine diminishes with advancing age, and reinstating this decline via taurine supplementation protects against age-related muscle mass loss and decreased functionality and also improves both health and lifespan across various species [46,47,48]. Its greater concentration in the old group tongue, in this project, suggests the activation of fiber metabolism to prevent muscle degeneration.

Glycerol is a component of triglycerides (fats and oils) and phospholipids [30]. In muscles affected by DMD, the glycerol concentration was increased in relation to the wild type, at 6 months of age, being one of the metabolites responsible for the differentiation between the dystrophic and non-dystrophic muscles [13]. The results of this work showed that glycerol is more concentrated in the tongue of old animals, both in wild-type and mdx mice, with no difference between strains, suggesting its relationship with the aging process in the tongue mice muscle.

Alanine is a nonessential amino acid resulting from the conversion of pyruvate or the breakdown of DNA and carnosine and anserine dipeptides [30]. It can be used as a fuel for gluconeogenesis directly from muscle tissue and therefore plays an important role in glucose homeostasis [49, 50]. With the progression of dystrophinopathy in affected muscles, there is an increase in energy expenditure [10, 51], for the incorporation of amino acids into new proteins [52]. Therefore, the lower concentration of alanine in the tongue muscle of old animals may indicate incorporation of alanine into proteins for processes of muscle maintenance and regeneration related to age, since the decrease is seen in old dystrophic and non-dystrophic mice.

Methionine is an essential amino acid, substrate for protein synthesis, necessary for normal mammalian growth and development [30]. Martins-Bach et al. (2012) [13] showed that methionine increased with aging in quadriceps muscle samples from control mice from 3 months to 6 months of age. Besides, it showed that methionine concentrations did not change for mdx samples. However, in the whole tongue muscles, with a greater interval between the ages studied, methionine changed for both strains, being more concentrated in younger animals. The growth of the animals can elucidate this difference between the concentrations of methionine throughout the aging of the mice. From the 26th day of life until the 150th–200th, approximately 7 months of age, the mice’s growth is observed, then remaining on a plateau until the end of life [53]. The high concentration of methionine at a young age in this study suggests growth in young animals. In older animals, which are not in the growth phase, the metabolite is decreased. Furthermore, this similarity of methionine concentrations between strains suggests that the tongue muscle in mdx is metabolically closer to wild type, since this metabolite participates in the regulation of the immune system, lipid metabolism, oxidative stress, and other metabolic regulation processes (for review, see [54]). New studies should be carried out to characterize the involvement of this metabolite in the metabolism of DMD.

3-Methylhistidine (3-MeH) is an amino acid present in actin and myosin. It has been determined that more than 90% of body 3-MeH is located in the skeletal muscle. When skeletal muscle is degraded, 3-MeH is released but is not reused for protein synthesis [55]; therefore, the urinary excretion of 3-MeH can be used to indicate muscle protein degradation [56]. Furthermore, there is a progressive loss of skeletal muscle mass and muscle strength with aging [57, 58]. The higher concentration of 3-MeH observed in the tongue of the young groups may reflect the decrease in muscle mass and the consequent reduction in 3-MeH concentrations seen in older muscles.

Muscle degeneration biomarkers, which in other studies proved to be efficient (acetic acid, phosphocreatine, isoleucine, succinic acid, creatine, TNF-α, and TGF-β protein) [13, 19, 23], did not show any difference with aging and in the analysis between strains. The TGF-β protein is actively involved in the proliferation of fibrous connective tissue in the skeletal muscles of patients and mdx mice [26, 27]. In previous studies, the levels of TGF-β have already been related to the presence of fibrous tissue in mdx’s respiratory muscles [23]. Spassov et al. [17] studied changes in the expression of collagen in the masticatory muscles in 100-day-old animals and concluded that there was no difference between the tongue of mdx and control groups. Our results corroborate theirs, due to the equality in the quantification of TGF-β in young animals indicating protection against tissue fibrosis of this muscle in the initial phase of the disease. In old animals, the levels of TGF-β were also equal between strains and suggest that this muscle protection against fibrosis continues until advanced stages of dystrophy in the mice, corroborating the histological results for fibrosis found in our study, for both ages.

TNF-α is a protein mediator of inflammatory response produced by activated macrophages, mast cells, endothelial cells, fibroblasts, myoblasts and myotubes, and injured myofibers [59, 60]. It stimulates the expression of adhesion molecules in endothelial cells, increasing leukocyte recruitment and adhesion. TNF-α is secreted at the site of inflammation and can enter the bloodstream [59]. The TNF-α is related to the progression of DMD, and its absence is related to muscle protection for the disease. Maranhão et al. [23] evidenced the progressive increase in the concentration of TNF-α in mdx diaphragm muscles, in comparison with the control and over age, with 1, 4, and 9 months. The intrinsic muscles of the larynx were identified as muscles spared from dystrophinopathy in the mdx mice, as they did not show signs of muscle damage, degeneration, or regeneration, during the course of the disease [24]. The concentrations of TNF-α in intrinsic muscles of the larynx were comparable to those of the control animal, during the progression of the disease, even in later stages, at 20 months of age [23]. These results corroborate with this work, since the concentrations of TNF-α in the tongue of the mdx animals did not differ from the wild-type animals, in young and old. Messina et al. (2011) [25] analyzed vastus lateralis muscle samples from DMD patients and showed an increase in the concentration of TNF-α in relation to the control group, besides pointing out the increase in its expression with age and disease progression, from 2 to 9 years of age. In this work, the concentration of TNF-α did not increase with aging in tongue in both strains, corroborating with the protection previously mentioned, including with disease progression and aging.

The metabolites glycine and glutamic acid were different only in dystrophic animals, being more concentrated in young mdx mice. Glycine is a nonessential amino acid involved in the production of DNA, phospholipids, and collagen, in addition to being involved in the release of energy [30]. Ham, Gardner, Kennedy, Trieu, Naim, Chee, Alves, Caldow, Lynch, and Koopman [61] demonstrated that glycine supplementation in mdx mice and dystrophin/utrophin double knockout mice can attenuate the progression of dystrophic pathology, as well as improve the effectiveness of prednisolone, the current gold standard treatment for DMD. Anderson and Skrabek [62] studied the heart of mdx mice treated with deflazacort, from a metabolomic perspective. They showed that the concentration of glycine in the mdx heart decreased with disease progression, but with a 2-week high-dose treatment with deflazacort, glycine levels increased above the levels of the mdx placebo group. Glycine supplementation has already been shown to protect against loss of myotubes in nutrient restricted/growth factor restriction models with C2C12 muscle cells in vitro. This in vitro protection has been shown to be dependent on mammalian target of rapamycin complex 1 (mTORC1) signaling [63] and also a specific glycine activation of mTORC1 involved in muscle regeneration in dystrophic mice [48, 64]. Thus, the protection observed in the 1-month-old tongue of the mdx muscle, close to the 20th day degeneration peak, may be related to the high levels of glycine presented in our study. In mdx and control mice, a gradual decline in muscle mass becomes apparent around 18 months, marking the onset of sarcopenia [65]. Mouisel and colleagues (2010) [66] have reported decreased levels of recovery of maximum force after myotoxic injury and of phosphorylated Akt and S6 (i.e., participants in the mTOR signaling pathway) in the tibialis anterior of 18–24-month-old mdx mice in comparison to 5-month-old mdx mice [64]. The decrease in regeneration of skeletal muscles from 18 months of age would explain the lower glycine levels in the tongue of old mdx found in this study, since a specific glycine activation of mTORC1 pathway is linked to muscle regeneration in dystrophic mice.

Glutamic acid, also known as glutamate (the anion), is a nonessential amino acid, one of the 20 proteinogenic amino acids and the most abundant rapid excitatory neurotransmitter in the nervous system [30]. Glutathione is the main cellular antioxidant and regulates free radical homeostasis. Glutamate plays an essential regulatory role in the synthesis of glutathione and is also related to its functions (for review, see [67, 68]). It is proven the relationship between decreased intracellular glutathione and stress states, such as chronic diseases [68], besides demonstrating benefits in the use of different antioxidant drugs for preclinical studies in mdx mice, such as improvement in dystrophinopathy with decreased necrosis [69]. In a metabolomic study of the biceps femoris muscle of the canine model for DMD (golden retriever muscular dystrophy), there was an increase in the concentration of glutamic acid when compared to the control [70]. Laferte, Rosenkrantz, and Berlinguet (1963) [71] observed an increase in weight loss and acceleration of the beginning of the terminal phase of the disease in mice that received exogenous glutamic acid. The increased glutamate concentration in the dystrophic muscles of the quadriceps and diaphragm suggests its connection with muscle regeneration and its requirement of protein replacement [13] and its relationship with the pathology of DMD. In this study, the amounts of glutamic acid in the tongue mdx muscles were equivalent to those of control muscles of the same age, indicating protection against myonecrosis on the tongue. As previously mentioned, skeletal muscle of older mdx mice (18-24-month-old) present sarcopenia with reduced regeneration, including reduced mTORC1 activation for muscle protein synthesis [66]. Thus, lower glutamate levels in old versus young mdx mice are expected. The observed decrease of glycine and glutamate levels suggests that mTOR-related pathways may not contribute to the protective mechanisms against the dystropathology seen in the old mdx tongue.

These results corroborate the protection observed in histology and the use of these as muscle biomarkers of degeneration and regeneration. Other studies on affected muscles can elucidate their specificities as biomarkers for the progression of DMD, without the interference of aging.