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10.21203/rs.3.rs-3038923/v1
10.21203/rs.3.rs-3038923
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Article
Biomarkers for Duchenne muscular dystrophy progression: impact of age in the mdx tongue spared muscle
Lorena Marcelo dos Santos Voltani Department of Structural and Functional Biology, Institute of Biosciences of Botucatu, São Paulo State University (UNESP)

Santos Estela Kato Department of Structural and Functional Biology, Institute of Biosciences of Botucatu, São Paulo State University (UNESP)

Ferretti Renato Department of Structural and Functional Biology, Institute of Biosciences of Botucatu, São Paulo State University (UNESP)

Gowda G.A. Nagana Northwest Metabolomics Research Center; Mitochondria and Metabolism Center, Anesthesiology and Pain Medicine, University of Washington

Odom Guy L. Department of Neurology, Wellstone Muscular Dystrophy Specialized Research Center, University of Washington School of Medicine

Chamberlain Jeffrey S. Department of Neurology, Wellstone Muscular Dystrophy Specialized Research Center, University of Washington School of Medicine

Matsumura Cintia Yuri Department of Structural and Functional Biology, Institute of Biosciences of Botucatu, São Paulo State University (UNESP)

Authors’ contributions

MSVL JSC and CYM conceived and designed experiments; MSVL, EKS, NGGA and CYM performed experiments; MSVL, RF, NGGA, GLO, JSC and CYM analyzed the data; MSVL, EKS, GLO, JSC and CYM interpreted the results of the experiments and wrote the manuscript with editing from MSVL. The authors read and approved the final manuscript.

✉ cintia.matsumura@unesp.br
13 6 2023
rs.3.rs-3038923https://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License, which allows reusers to distribute, remix, adapt, and build upon the material in any medium or format, so long as attribution is given to the creator. The license allows for commercial use.
nihpp-rs3038923v1.pdf
Background:

Duchenne muscular dystrophy (DMD) is a severe form of muscular dystrophy without an effective treatment, caused by mutations in the DMD gene, leading to the absence of dystrophin. DMD results in muscle weakness, loss of ambulation and death at an early age. Metabolomics studies in mdx mice, the most used model for DMD, reveal changes in metabolites associated with muscle degeneration and aging. In DMD, the tongue muscles exhibit unique behavior, initially showing partial protection against inflammation but later experiencing fibrosis and loss of muscle fibers. Certain metabolites and proteins, like TNF-α and TGF-β, are potential biomarkers for dystrophic muscle characterization.

Methods:

To investigate disease progression and aging, we utilized young (1-month old) and old (21–25 months old) mdx and wild-type mice. Metabolite changes were analyzed using 1-H Nuclear Magnetic Resonance, while TNF-α and TGF-β were assessed using Western blotting to examine inflammation, and fibrosis. Morphometric analysis was conducted to assess the extent of myofiber damage between groups.

Results:

The histological analysis of the tongue showed no differences between groups. No differences were found between the concentrations of metabolites from wild type or mdx animals of the same age. The metabolites alanine, methionine, 3-methylhistidine were higher, and taurine and glycerol were lower in young animals in both wild type and mdx (p < 0.001). The metabolites glycine (p < 0.001) and glutamic acid (p = 0.0018) were different only in the mdx groups, being higher in young mdx mice. Acetic acid, phosphocreatine, isoleucine, succinic acid, creatine and the proteins TNF-α and TGF-β had no difference in the analysis between groups (p > 0.05).

Conclusions:

Surprisingly, histological and protein analysis reveals that the tongue of young and old mdx animals is protected from severe myonecrosis observed in other muscles. The metabolites alanine, methionine, 3-methylhistidine, taurine, and glycerol may be effective for specific assessments, although their use for disease progression monitoring should be cautious due to age-related changes. Acetic acid, phosphocreatine, isoleucine, succinate, creatine, TNF-α, and TGF-β do not vary with aging and remain constant in spared muscles, suggesting their potential as specific biomarkers for DMD progression independent of aging.

São Paulo Research Foundation (FAPESP)2018/25598-0 2019/20162-1 2013/00312-2 CNPq456357/2014-9 NIHR01AR040864 DODW81XWH-18-1-0624
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pmcIntroduction

Duchenne muscular dystrophy (DMD) affects approximately 1 in 3600–6000 male live births [1] and is a severe form of muscular dystrophy without an effective treatment [2]. It is an X-linked recessive disease [2] caused by mutations in the DMD gene [3]) which leads to the absence of dystrophin [4].

The lack of dystrophin causes continuous loss of muscle strength, myofiber damage, chronic inflammation, progressive fibrosis, and muscle stem cell dysfunction. This dystrophic scenario leads to a loss of ambulation in the early teens to twenties. Although patients’ life expectancy has improved with current standards of cardioprotective care and respiratory support, they often die around the third or fourth decade of life, mainly due to cardiac and respiratory complications [2][5][6].

The mdx mice is the most used animal model for research on DMD [7][8]. Around 20 days old, these animals begin to show their first signs of muscle degeneration and regeneration [9]. The mdx mice have reduced life spans, by about 17 to 19% compared to wild-type. At 26 months, average lifespans, the muscles present typical dystrophic characteristics: loss of muscle fibers with increased fibrosis, fat infiltration, necrotic fibers and regenerated fibers [10].

Metabolomics is the quantitative analysis of metabolites produced by an organism under certain conditions. Metabolomics provides an integrated view of biochemical pathways in complex organisms, thus producing a more detailed and systematic overview of the cellular processes and its response to diseases. Therefore, this approach is essential for the definition of personalized medicine, through the establishment of metabolite profiles and biomarkers for certain pathological states [11]. Studies in mdx mice show changes in metabolites related to the progression of muscle degeneration and aging [12][13] [14]

In DMD, the muscles of the oral cavity are also affected, causing dysphagia in late stages of the disease, which worsens with advancing age and disease progression [15] [16]. In mdx mice, the tongue muscles (TON) show an unusual behavior as the disease progresses compared to other muscles. In 3-month mdx mice, inflammatory cells were hardly found in the TON, unlike other masticatory muscles. In addition, the expression of collagen did not change in the TON of these mdx, while it was three times higher in masseter muscle (MAS), when compared to the control [17]. These results indicate partial protection of this muscle against myonecrosis and inflammation at 3 months of age. However, at 26 months of age, Chamberlain et al. (2007) [10] described the TON of the mdx as the second most affected muscle, due to fibrosis and the loss of fibers in the central portion of the muscle, just after the diaphragm.

Some metabolites from dystrophic mice analyzed by Nuclear Magnetic Resonance (NMR) have been suggested to determine biomarkers for the state of muscle fibers [18], such as: increase of glutamate, glutamine, succinate, isoleucine, acetate, alanine and glycerol, in contrast to decreased of carnosine, taurine, glycine, methionine and creatine in the mdx’s diaphragm and quadriceps muscles, compared to the wild-type [13].

Besides metabolites, there are some proteins that are well established in the current literature related to inflammation and fibrosis, like Tumor Necrosis Factor alpha (TNF-α) and Transforming Growth Factor beta (TGF-β), respectively. These proteins are very promising biomarkers for the dystrophic muscle characterization, for their relation to the myonecrosis as seen in the mdx animal model and human patients [19].

The present study aimed to validate biomarkers for diagnosis and progression of DMD through the analysis of metabolites, proteins related to inflammation and fibrosis, and histology of the tongue of mdx mice over time. We evaluated metabolites and proteins in the tongue muscles, within young (30 days old) and old (21 to 25 months old) mdx and wild type mice. Surprisingly, our histological results found that the tongue remains protected in older mdx mice (21 to 25 months). Our results allowed us to identify biomarkers that change with aging, regardless of the absence of dystrophin. Other biomarkers seem to be excellent candidates to indicate the progression of dystrophinopathy over time, as they do not change with aging and remain constant in the spared muscles. Furthermore, analyzing these possible biomarkers in TON at different ages can help to understand the protection mechanisms involved in the pathological process and support the development of future approaches for the diagnosis and monitoring progression of DMD.

Materials and Methods

Animals

Male and female, young and old mdx (C57BL/10-DMDmdx/PasUnib) and age-match wild type mice (C57BL/10ScCr/PasUnib) were obtained and maintained by our institutional animal care facility of Institute of Bioscience (Botucatu) - UNESP. All mouse experimentation was approved by our Institution committee and done in accordance with the guidelines of the Brazilian College for Animal Experimentation (protocol n° 1095-CEUA). The animals were divided into four groups: young mdx (1-month old), old mdx (21–25 months old), young wild type (1 month-old) and old wild type (21–25 months old).

Tissue harvesting

The animals were euthanized with an overdose of intraperitoneal anesthesia of xylazine hydrochloride (30 mg / kg) and ketamine hydrochloride (300 mg / kg). The tongue (TON) was dissected and fixed for histological techniques or frozen in liquid nitrogen for western blotting and metabolome assays.

Histology

The TON muscles of the young wild type (n = 5), young mdx (n = 5), old wild type (n = 4) and old mdx (n = 6) groups were sectioned and stained with Masson’s Trichrome, to distinguish and quantify the areas of fibrosis (FIB), areas of muscle fibers with peripheral nuclei (PN) and central nuclei (CN). The analyzes were performed blindly, and the areas were expressed in relation to the area of total transverse fibers of the section.

Proteins analysis

Proteins related to the mechanisms of inflammation (TNF-α) and fibrosis (TGF-β) were quantified with the Western Blotting assay, as described previously [20]. The values were normalized with the glyceraldehyde 3-phosphate dehydrogenase protein (GAPDH), incubated on the same membrane after routine stripping methods.

3.8 Metabolomics by nuclear magnetic resonance (NMR) spectroscopy

In order to verify possible changes in the metabolic profile, metabolomics analysis was performed by NMR spectroscopy of the tongue muscles of young (n = 10) and old (n = 5) wild type mice and young (n = 11) and old (n = 5) mdx mice.

Data acquisition was performed on a Varian INOVA spectrometer operating at a resonance frequency of 1 H of 600 MHz. The samples were homogenized in a methanol / chloroform solution (2:1). After fifteen minutes, a solution of chloroform / milli Q water (1:1) was added to the pellet. Then, the samples were centrifuged at 4000 RPM for 20 minutes. The supernatant was collected and lyophilized. The obtained powder was resuspended in deuterated water (D2O) with trimethylsilyl tetradeuteropropionic acid (TSP, 10 mM). Subsequently, the samples were transferred to a standard NMR tube for spectral analysis. The D2O allowed the device to be monitored and blocked by the device’s resonant frequency. The TSP reference signals were used to assess the quality of the spectra as well as to quantify the identified substances. Proton spectra in one dimension, using pulse sequences optimized for suppression of the water signal and were collected at 25 ° C [21].

Spectrum treatment and identification and quantification of metabolites were performed using the Bruker Topspin 3.1 and Chenomx NMR Suite (Version 7.1; Chenomx Inc., Edmonton, Canada) application packages, in conjunction with the Human Metabolome Database [22] and literature already published. All the chemical shift intervals (ppm) are listed in Table 1. In parallel with the chemometric analysis using the separation of the spectrum in small intervals, the statistical analysis was performed directly with the concentrations of the identified metabolites, through the targeted profiling methodology, developed by the creators of the Chenomx NMR Suite application. In this methodology the groups of peaks corresponding to each metabolite are identified and quantified using a database of pure substance spectra. We also used the Spectral binning methodology, in which the spectra are divided into predefined frequency intervals, while the integrals of the signals within each interval are used in the statistical analysis [23].

Statistical analysis

Statistical analysis was performed through analysis of variance (ANOVA, p ≤ 0.05) with Tukey’s post hoc test for histological and western blotting data.

For the metabolomics analysis, Principal Component Analysis (PCA) unsupervised was used in order to visualize the complex sample space and multivariate after identification and quantification of metabolites. These discriminant analyses were performed through standard procedures implemented in Pirouette 4.0 (Infometrix, Washington, USA) [24]. The input variables will consist of the integral of the area intervals (spectral binning) and / or the concentration values obtained with Targeted Profiling. Analysis of Variance (ANOVA, p ≤ 0.05) with Tukey’s post hoc test was performed directly with the concentrations of the identified metabolites, using the MetaboAnalyst platform.

Results

Tongue muscles remain spared in old mdx

In the qualitative histopathological analysis of the sections, we observed different histological aspects in the muscle fibers. Fibers with peripheral nuclei (PN) were observed, indicating normal muscle tissue status; fibers with central nuclei (CN), indicating regenerated muscle fibers; and areas of fibrosis (FIB), shown in blue by Masson’s Trichrome stain (Fig. 1). In the quantitative histological analysis of the TON of the groups described (Table 2), there was no difference (p > 0.05) in the areas of PN, CN, or FIB, both between ages, young and old, and between lineages, wild type and mdx.

Changes in proteins related to inflammation and fibrosis

The quantification of TNF-α and TGF-β in the TON muscle was performed by Western blotting to verify the presence of inflammation and fibrosis, respectively. There was no significant difference in the concentrations of both proteins in the TON muscle between the groups (two-way ANOVA, TGF-β p = 9.968; TNF-α p = 7.558), as shown in Fig. 2.

Changes in the metabolomic profile

The tongues of the mice are grouped according to their metabolic profile through the PCA. It was possible to distinguish animals of the same lineage at different ages (Fig. 3, C and D), however it was not possible to distinguish mdx mice from wild type mice, in both young or old for the tongue muscle (Fig. 3, A and B).

No differences were found in the comparison between the concentrations of metabolites from wild type or mdx animals of the same age, whether young or old (Fig. 4), suggesting that the protection of the dystrophic tongue muscle is observed in both ages. Table 3 summarizes the metabolites responsible for the differentiation of the tongue muscle of same strain mice at different ages. The metabolites alanine, methionine, 3-methylhistidine were higher in young animals in both wild type and mdx (p < 0.001), and the metabolites taurine and glycerol were lower in both the young wild type and mdx groups (p < 0.001). The metabolites glycine (p < 0.001) and glutamic acid (p = 0.0018) were different only in the mdx groups, being more concentrated in young mdx mice. The metabolites acetic acid, phosphocreatine, isoleucine, succinic acid, creatine had no difference in the analysis between groups (p > 0.05).

Discussion

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 and non-dystrophic animals, young and old.

Surprisingly, our histological results demonstrated protection of TON against fibrosis and myonecrosis in both young (30 days) and old mdx animals (21 to 25 months). Chamberlain et al. (2007) studied the 26-month mdx TON and described those muscles as displaying significant histological abnormalities such as fibrosis and focal loss of muscle fibers. However, the muscle was analyzed qualitatively and comparisons were not made to TON from younger animals [10]. In contrast, here the qualitative and quantitative analysis of mid-belly TON did not show such severity. This protection was evidenced by the biomarker proteins for inflammation and fibrosis, and by 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 [25] believe that dystrophic MAS and temporalis (TEM) 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, MAS and TEM are more resistant to the calcium (Ca2+) overload when compared to TON muscles of 100 days old mdx. Hence, histological findings assert that inflammatory foci is hardly detectable and dystrophic TON contains only 11.2% of regenerated muscle in the calcium-regulating genes, when compared to MAS, TEM and even soleus [25]. In DMD rats, the TON showed hypertrophy of myofibers with less advanced dystrophic changes until 8 months old compared to MAS. This resistance against degeneration might be related to a higher level of utrophin transcription in TON of wild-type and DMD rats compared to MAS of wild-type rats (Yamanouchi 2022).

The study of biomarkers in a muscle protected 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 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 TON 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, C and D). 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 [30], and these results are confirmed in the scenario of dystrophinopathy.

It was not possible to distinguish the metabolic profile of the tongue between the wild type and mdx strains. This result corroborates our histological analysis, which showed protection against fibrosis and myonecrosis in the 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][31][32]. Since these metabolites were not different between the strains in this study, it suggests that the protection previously observed in the TON 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 [30], 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 [31]. 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) [25] 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 days-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 [33]. Its greater concentration in the old group TON, in this project, suggests the activation of fiber metabolism to prevent muscle degeneration in the advanced stage of dystrophinopathy.

Glycerol is a component of triglycerides (fats and oils) and phospholipids [22]. 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 TON 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 non-essential amino acid resulting from the conversion of pyruvate or the breakdown of DNA and carnosine and anserine dipeptides [22]. It can be used as a fuel for gluconeogenesis directly from muscle tissue and therefore plays an important role in glucose homeostasis [34][35]. With the progression of dystrophinopathy in affected muscles, there is an increase in energy expenditure [10][36], for the incorporation of amino acids into new proteins [37]. 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 [22]. 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 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 [38]. 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 TON 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 Martínez et al 2017). 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 [39], therefore the urinary excretion of 3-MeH can be used to indicate muscle protein degradation [40]. Furthermore, there is a progressive loss of skeletal muscle mass and muscle strength with aging [41][42]. 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][18][27] 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 [43][44]. In previous studies, the levels of TGF-β have already been related to the presence of fibrous tissue in mdx’s respiratory muscles [27]. Spassov et al. [17] studied changes in the expression of collagen in the masticatory muscles in 100 days old animals and concluded that there was no difference between the tongue of mdx and control groups. Our results corroborate yours, 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 pro-inflammatory protein produced by activated macrophages, mast cells, endothelial cells and some other cell types. 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 [26]. The TNF-α is related to the progression of DMD and its absence is related to muscle protection for the disease. Maranhão et al. [27] 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 (ILM) were identified as muscles protected from dystrophinopathy in the mdx mice, as they did not show signs of muscle damage, degeneration or regeneration, during the course of the disease [28]. The concentrations of TNF-α in ILM were comparable to those of the control animal, during the progression of the disease, even in later stages, at 20 months of age [27]. These results corroborate with this work, since the concentrations of TNF-α in the TON of the mdx animals did not differ from the wild type animals, in young and old. Messina et al. (2011) [29] 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 TON 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 non-essential amino acid involved in the production of DNA, phospholipids and collagen, in addition to being involved in the release of energy [22]. D.J. Ham et al. [45] 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 [46] 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 [47] and also a specific glycine activation of mTORC1 involved in muscle regeneration in dystrophic mice [48]. 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.

Glutamic acid, also known as glutamate (the anion), is a non-essential amino acid, one of the 20 proteinogenic amino acids and the most abundant rapid excitatory neurotransmitter in the nervous system [22]. 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 [49][50]. It is proven the relationship between decreased intracellular glutathione and stress states, such as chronic diseases [50], besides demonstrating benefits in the use of different antioxidant drugs for preclinical studies in mdx mice, such as improvement in dystrophinopathy with decreased necrosis [51]. 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 [52]. Laferte, Rosenkrantz and Berlinguet [53] 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 [13] and its relationship with the pathology of DMD. In this study, the amounts of glutamic acid in the mdx were equivalent to those of control animals of the same age, indicating protection against myonecrosis on the tongue.

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

Conclusions

The tongue of young (1 month) and old (21 to 25 months) mdx animals remains protected from the intense and progressive myonecrosis described in other muscles, as evidenced by histological analysis. This protection was verified by the biomarker proteins for inflammation and fibrosis, and by 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 in this work presented similar results between wild type and mdx.

Among the possible biomarkers analyzed, the metabolites alanine, methionine, 3-methylhistidine, taurine and glycerol 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.

Muscle degeneration biomarkers, which in other studies proved to be efficient (acetic acid, phosphocreatine, isoleucine, succinate, creatine, TNF-α and TGF-β) did not show any difference with aging and in the spared muscles. Other studies on affected muscles can elucidate their specificities as biomarkers for the progression of DMD, without the interference of aging.

Funding

This work was supported by São Paulo Research Foundation (FAPESP) grant#2018/25598-0, grant#2019/20162-1 and grant#2013/00312-2 (to MSVL and CYM). CYM was supported by CNPq grant 456357/2014-9. JSC is supported by NIH grant R01AR040864. GLO is supported by DOD grant award#W81XWH-18-1-0624.

Availability of data and materials

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Abbreviations

3-MeH 3-Methylhistidine

ANOVA analysis of variance

CEUA Brazilian College for Animal Experimentation

CN central nuclei

D2O deuterated water

DMD Duchenne muscular dystrophy

FIB areas of fibrosis

GAPDH glyceraldehyde 3-phosphate dehydrogenase protein

ILM intrinsic muscles of the larynx

MAS masseter muscle

mdx X chromosome-linked muscular dystrophy

mTORC1 mammalian target of rapamycin complex 1

NMR Nuclear Magnetic Resonance

PN areas of muscle fibers with peripheral nucle

PCA Principal Component Analysis

TEM temporalis

TON tongue muscles

TNF-α Tumor Necrosis Factor alpha

TGF-β Transforming Growth Factor beta

TSP trimethylsilyl tetradeuteropropionic acid

UNESP São Paulo State University

Figure 1 Cross-sections of mid-belly TON muscle.

Young wild type (A), young mdx (B), old wild type (C) and old mdx (D) groups. In B, areas of fibrosis highlighted in blue by Masson’s Trichrome (*). In C, muscle fibers with a central nucleus (arrow) and muscle fibers with a peripheral nucleus (arrowhead). Scale: 50 μm

Figure 2 Quantification of TNF-α and TGF-β.

The quantification of TNF-α and TGF-β was performed by Western blotting analysis in crude extracts of TON muscles from young wild type, young mdx, old wild type and old mdx groups. Same blot reprobed for GAPDH as a loading control. Graphs represent the level of proteins expressed in arbitrary units and normalized to GAPDH levels. Bars represent standard deviation. No significant difference was observed between the groups (p>0.05, ANOVA).

Figure 3 Principal component analysis (PCA) of TON with the scores plot between groups.

mdx animals (mdx) vs. wild type (wt) young (A) and old (B); tongue of young vs. old mdx animals (C); tongue of young vs. old wild type animals (D). Each point on the graph represents the spectrum of an animal.

Figure 4 Differences in the concentrations of metabolites between groups.

Difference between metabolite concentrations between young and old mdxanimals (a). Difference between metabolite concentrations between young and old wild type animals (b), ANOVA, p< 0.001. The metabolites acetic acid, creatine, phosphocreatine, isoleucine and succinic acid showed no differences between groups (p> 0.05).

Table 1 Assignments of resonance peaks from 1H Nuclear Magnetic Resonance (NMR) data.

Areas were calculated considering the indicated chemical shift intervals.

Chemical shifts interval (ppm)	Compound	
1.0056–1.0283	Isoleucine	
1.4639–1.5053	Alanine	
1.9155–1.9262	Acetic acid	
2.4002–2.4134	Succinic acid	
3.0253–3.0459	Creatine	
3.0458–3.0533	Phosphocreatine	
3.3964–3.4469	Taurine	
3.5587–3.5648	Glycine	
2.6372–2.6426	Methionine	
2.3322–2.3754	Glutamic acid	
7.0091–7.0516	3-Methylhistidine	
3.639–3.6507	Glycerol	
−0.1362–0.1198	TSP	

Table 2 Quantitative histological analysis of TON.

	%PN	%CN	%FIB	
young wild type	91.33 ± 0.98	1.24 ± 0.63	7.42 ± 1.37	
youngmdx	80.94 ± 13.62	2.40 ± 1.17	16.67 ± 13.30	
old wild type	83.43 ± 10.68	4.99 ± 2.77	11.58 ± 9.67	
oldmdx	75.67 ± 5.49	6.18 ± 3.87	18.16 ± 7.29	
Mean ± SD of fiber percentage areas with peripheral nucleus (%PN), central nucleus (%CN) and areas of fibrosis (%FIB) in the TON of the young and old mdx and wild type groups. There were no differences found for areas of PN, CN and FIB between the four groups (ANOVA, p > 0,05).

Table 3 Different metabolites between animals of the same lineage at different ages in tongue samples.

	Higher in young	Lower in young	
wild type (young vs. old)	alanine, methionine, 3-methylhistidine	taurine, glycerol	
mdx (young vs. old)	alanine, methionine, 3-methylhistidine, glutamic acid, glycine	taurine, glycerol	

Competing interests

The authors declare that they have no competing interests

Declarations

Ethics approval and consent to participate

All experiments were carried out in accordance with the guidelines for animal experimentation of the Ethics Committee on Animal Use - CEUA-IBB/UNESP, protocol#1095.

Consent for publication

NOT APPLICABLE
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References

1. Mendell JR , Shilling C , Leslie ND , Flanigan KM , al-Dahhak R , Gastier-Foster J , Evidence-based path to newborn screening for duchenne muscular dystrophy. Annals of Neurology. 2012 Mar;71 (3 ):304–13. DOI: 10.1002/ana.23528 22451200
2. Hoffman EP , Brown RH , Kunkel LM . Dystrophin: The protein product of the duchenne muscular dystrophy locus. Cell. 1987 Dec; 51 (6 ):919–28. DOI: 10.1016/0092-8674(87)90579-4 3319190
3. Tuffery-Giraud S , Béroud C , Leturcq F , Yaou RB , Hamroun D , Michel-Calemard L , Genotype-phenotype analysis in 2,405 patients with a dystrophinopathy using the UMD-DMD database: a model of nationwide knowledgebase. Human Mutation. 2009 Jun;30 (6 ):934–45. DOI: 10.1002/humu.20976 19367636
4. Tinsley JM , Blake DJ , Zuellig RA , Davies KE . Increasing complexity of the dystrophin-associated protein complex. Proceedings of the National Academy of Sciences. 1994 Aug 30];91 (18 ):8307–13. DOI: 10.1073/pnas.91.18.8307
5. Bogdanovich S , Perkins KJ , Krag TOB , Khurana TS . Therapeutics for Duchenne muscular dystrophy: current approaches and future directions. Journal of Molecular Medicine. 2004 Feb 1;82 (2 ):102–15. DOI: 10.1007/s00109-003-0484-1 14673527
6. Landfeldt E , Thompson R , Sejersen T , McMillan HJ , Kirschner J , Lochmüller H . Life expectancy at birth in Duchenne muscular dystrophy: a systematic review and meta-analysis. European Journal of Epidemiology. 2020 Feb 27;35 (7 ):643–53. DOI: 10.1007/s00109-003-0484-1 32107739
7. Bulfield G , Siller WG , Wight PA , Moore KJ . X chromosome-linked muscular dystrophy (mdx) in the mouse. Proceedings of the National Academy of Sciences. 1984 Feb];81 (4 ):1189–92. DOI: 10.1073/pnas.81.4.1189
8. McGreevy JW , Hakim CH , McIntosh MA , Duan D . Animal models of Duchenne muscular dystrophy: from basic mechanisms to gene therapy. Disease Models & Mechanisms. 2015 Mar 1;8 (3 ):195–213. DOI:10.1242/dmm.018424 25740330
9. Coulton GR , Morgan JE , Partridge TA , Sloper JC . The mdx mouse skeletal muscle myopathy: I. A histological, morphometric and biochemical investigation. Neuropathology and Applied Neurobiology. 1988 Feb;14 (1 ):53–70. DOI: 10.1111/j.1365-2990.1988.tb00866.x 2967442
10. Chamberlain JS , Metzger J , Reyes M , Townsend D , Faulkner JA . Dystrophin-deficient mdx mice display a reduced life span and are susceptible to spontaneous rhabdomyosarcoma. The FASEB Journal. 2007 Mar 14;21 (9 ):2195–204. DOI: 10.1096/fj.06-7353com 17360850
11. Donald S , Chen R , Snyder M . Systems biology approaches to disease marker discovery. Disease markers. 2013;28 (4 ). DOI: 10.3233/DMA-2010-0707
12. Gulston MK , Rubtsov DV , Atherton HJ , Clarke K , Davies KE , Lilley KS , A Combined Metabolomic and Proteomic Investigation of the Effects of a Failure to Express Dystrophin in the Mouse Heart. Journal of Proteome Research [Internet]. 2008 Apr 3;7 (5 ):2069–77. DOI: 10.1021/pr800070p 18386883
13. Martins-Bach AB , Bloise AC , Vainzof M , Rahnamaye Rabbani S . Metabolic profile of dystrophic mdx mouse muscles analyzed with in vitro magnetic resonance spectroscopy (MRS). Magnetic Resonance Imaging. 2012 Oct;30 (8 ):1167–76. DOI: 10.1016/j.mri.2012.04.003 22673895
14. Maurício AF , de Carvalho SC , Santo Neto H , Marques MJ . Effects of dietary omega-3 on dystrophic cardiac and diaphragm muscles as evaluated by 1H magnetic resonance spectroscopy: Metabolic profile and calcium-related proteins. Clinical Nutrition ESPEN. 2017 Aug;20 :60–7. DOI: 10.1016/j.clnesp.2017.03.005 29072171
15. Archer SK , Garrod R , Hart N , Miller S . Dysphagia in Duchenne muscular dystrophy assessed by validated questionnaire. International Journal of Language & Communication Disorders. 2013 Mar;48 (2 ):240–6. DOI: 10.1111/j.1460-6984.2012.00197.x 23472962
16. van den Engel-Hoek L , de Groot IJM , Sie LT , van Bruggen HW , de Groot SAF , Erasmus CE , Dystrophic changes in masticatory muscles related chewing problems and malocclusions in Duchenne muscular dystrophy. Neuromuscular Disorders. 2016 Jun;26 (6 ):354–60. DOI: 10.1016/j.nmd.2016.03.008 27132120
17. Spassov A , Gredes T , Gedrange T , Lucke S , Pavlovic D , Kunert-Keil C . Histological changes in masticatory muscles of mdx mice. Archives of Oral Biology. 2010 Apr];55 (4 ):318–24. DOI: 10.1016/j.archoralbio.2010.02.005 20207343
18. Al-Khalili Szigyarto C , Spitali P . Biomarkers of Duchenne muscular dystrophy: current findings. Degenerative Neurological and Neuromuscular Disease. 2018 Jan;Volume 8 :1–13. DOI: 10.2147/DNND.S121099 30050384
19. Grounds MD , Terrill JR , Al-Mshhdani BA , Duong MN , Radley-Crabb HG , Arthur PG . Biomarkers for Duchenne muscular dystrophy: myonecrosis, inflammation and oxidative stress. Disease Models & Mechanisms. 2020 Mar;Volume 13 (2 ). DOI:10.1242/dmm.043638
20. Ferretti R. , Marques M. J. , Pertille A. , & Santo Neto H. (2009). Sarcoplasmic-endoplasmic-reticulum Ca2+-ATPase and calsequestrin are overexpressed in spared intrinsic laryngeal muscles of dystrophin-deficient mdx mice. Muscle & Nerve. 2019 May;39 (5 ), 609–615. DOI: 10.1002/mus.21154
21. Sukumaran DK , Garcia E , Hua J , Tabaczynski W , Odunsi K , Andrews C , Standard operating procedure for metabonomics studies of blood serum and plasma samples using a 1H-NMR micro-flow probe. Magnetic Resonance in Chemistry. 2009 Aug 18;47 (S1 ):S81–5. DOI: 10.1002/mrc.2469 19688872
22. Wishart D. S. , Guo A. , Oler E. , Wang F. , Anjum A. , Peters H. , Dizon R. , Sayeeda Z. , Tian S. , Lee B. L. , Berjanskii M. , Mah R. , Yamamoto M. , Jovel J. , Torres-Calzada C. , Hiebert-Giesbrecht M. , Lui V. W. , Varshavi D. , Varshavi D. , Allen D. , … Gautam V. (2022). HMDB 5.0: the Human Metabolome Database for 2022. Nucleic acids research, 50 (D1 ), D622–D631. 10.1093/nar/gkab1062 34986597
23. Weljie AM , Newton J , Mercier P , Carlson E , Slupsky CM . Targeted Profiling: Quantitative Analysis of 1H NMR Metabolomics Data. Analytical Chemistry [Internet]. 2006 May 17;78 (13 ):4430–42. DOI: 10.1021/ac060209g 16808451
24. Beckonert O , Keun HC , Ebbels TMD , Bundy J , Holmes E , Lindon JC , Metabolic profiling, metabolomic and metabonomic procedures for NMR spectroscopy of urine, plasma, serum and tissue extracts. Nature Protocol. 2007 Oct 25.];2 (11 ):2692–703. DOI: 10.1038/nprot.2007.376
25. Kunert-Keil CH ; Gredes T ; Lucke S ; Botzenhart U ; Dominiak M ; Gedrange T . Differential expression of genes involved in the calcium homeostasis in masticatory muscles of MDX mice. Journal of physiology and pharmacology: an official journal of the Polish Physiological Society. 2014;65 (2 ).
26. Kumar V. ; Abbas A. ; Fausto N. Robbins e Cotran Pathologic Basis of Disease. 8th. ed. Rio de Janeiro: Elsevier, 2010;
27. Maranhão J , de Oliveira Moreira D , Maurício AF , de Carvalho SC , Ferretti R , Pereira JA , Changes in calsequestrin, TNF-α, TGF-β and MyoD levels during the progression of skeletal muscle dystrophy in mdx mice: a comparative analysis of the quadriceps, diaphragm and intrinsic laryngeal muscles. International Journal of Experimental Pathology. 2015 Oct;96 (5 ):285–93. DOI: 10.1111/iep.12142 26515458
28. Marques MJ , Ferretti R , Vomero VU , Minatel E , Neto HS . Intrinsic laryngeal muscles are spared from myonecrosis in the mdx mouse model of Duchenne muscular dystrophy. Muscle & Nerve. 2007;35 (3 ):349–53. DOI: 10.1002/mus.20697 17143878
29. Messina S , Vita GL , Aguennouz M , Sframeli M , Romeo S , Rodolico C , Activation of NF-kappaB pathway in Duchenne muscular dystrophy: relation to age. Acta myologica: myopathies and cardiomyopathies : official journal of the Mediterranean Society of Myology. 2011;30 (1 ):16–23.21842588
30. Sobolev A. P. , Mannina L. , Costanzo M. , Cisterna B. , Malatesta M. , & Zancanaro C. (2017). Age-related changes in skeletal muscle composition: A pilot nuclear magnetic resonance spectroscopy study in mice. Experimental Gerontology. 2017 Jun;92 , 23–27. DOI: 10.1016/j.exger.2017.03.005 28286172
31. Terrill JR , Grounds MD , Arthur PG . Taurine deficiency, synthesis and transport in the mdx mouse model for Duchenne Muscular Dystrophy. The International Journal of Biochemistry & Cell Biology 2015 Sep;66 :141–8. DOI: 10.1016/j.biocel.2015.07.016 26239309
32. Griffin JL , Williams HJ , Sang E , Clarke K , Rae C , Nicholson JK . Metabolic Profiling of Genetic Disorders: A Multitissue 1H Nuclear Magnetic Resonance Spectroscopic and Pattern Recognition Study into Dystrophic Tissue. Analytical Biochemistry. 2001 Jun;293 (1 ):16–21. DOI: 10.1006/abio.2001.5096 11373073
33. McIntosh LM , Garrett KL , Megeney L , Rudnicki MA , Anderson JE . Regeneration and myogenic cell proliferation correlate with taurine levels in dystrophin- and MyoD-Deficient muscles. The Anatomical Record. 1998 Oct];252 (2 ):311–24. DOI: 10.1002/(SICI)1097-0185(199810)252:2<311::AID-AR17>3.0.CO;2-Q 9776086
34. Nishio H , Wada H , Matsuo T , Horikawa H , Takahashi K , Nakajima T , Glucose, free fatty acid and ketone body metabolism in duchenne muscular dystrophy. Brain and Development. 1990 Jan;12 (4 ):390–402. DOI: 10.1016/s0387-7604(12)80071-4 2240459
35. Srivastava NK , Yadav R , Mukherjee S , Sinha N . Perturbation of muscle metabolism in patients with muscular dystrophy in early or acute phase of disease: In vitro, high resolution NMR spectroscopy based analysis. Clinica Chimica Acta. 2018 Mar;478 :171–81. DOI: 10.1016/j.cca.2017.12.036
36. Radley-Crabb HG , Marini JC , Sosa HA , Castillo LI , Grounds MD , Fiorotto ML . Dystropathology Increases Energy Expenditure and Protein Turnover in the Mdx Mouse Model of Duchenne Muscular Dystrophy. Fraidenraich D, editor. PLoS ONE. 2014 Feb 19;9 (2 ):e89277. DOI:10.1371/journal.pone.0089277 24586653
37. Strakova J , Kamdar F , Kulhanek D , Razzoli M , Garry DJ , Ervasti JM , Integrative effects of dystrophin loss on metabolic function of the mdx mouse. Scientific Reports. 2018 Sep 11.;8 (1 ). DOI: 10.1038/s41598-018-31753-3
38. Schäfer R , Zweyer M , Knauf U , Mundegar RR , Wernig A . The ontogeny of soleus muscles in mdx and wild type mice. Neuromuscular Disorders. 2005 Jan;15 (1 ):57–64. DOI: 10.1016/j.nmd.2004.09.011 15639122
39. Williams A.P. AMINO ACIDS | Determination. Encyclopedia Of Food Sciences And Nutrition, [s.l.], p.192–197, 2003. Elsevier. DOI:
40. Keller U. Nutritional Laboratory Markers in Malnutrition. Journal of Clinical Medicine. 2019 May 31;8 (6 ):775. DOI: 10.3390/jcm8060775 31159248
41. Janssen I , Heymsfield SB , Wang Z , Ross R . Skeletal muscle mass and distribution in 468 men and women aged 18–88 year. Journal of Applied Physiology. 2000 Jul 1;89 (1 ):81–8. DOI: 10.1152/jappl.2000.89.1.81 10904038
42. Nilwik R , Snijders T , Leenders M , Groen BBL , van Kranenburg J , Verdijk LB , The decline in skeletal muscle mass with aging is mainly attributed to a reduction in type II muscle fiber size. Experimental Gerontology. 2013 May;48 (5 ):492–8. DOI: 10.1016/j.exger.2013.02.012 23425621
43. Bernasconi P , Torchiana E , Confalonieri P , Brugnoni R , Barresi R , Mora M , Expression of transforming growth factor-beta 1 in dystrophic patient muscles correlates with fibrosis. Pathogenetic role of a fibrogenic cytokine. Journal of Clinical Investigation. 1995 Aug 1;96 (2 ):1137–44. DOI: 10.1172/JCI118101 7635950
44. Carvalho SC , Matsumura CY , Santo Neto H , Marques MJ . Identification of plasma interleukins as biomarkers for deflazacort and omega-3 based Duchenne muscular dystrophy therapy. Cytokine. 2018 Feb;102 :55–61. DOI: 10.1016/j.cyto.2017.12.006 29276972
45. Ham DJ , Gardner A , Kennedy TL , Trieu J , Naim T , Chee A , Glycine administration attenuates progression of dystrophic pathology in prednisolone-treated dystrophin/utrophin null mice. Scientific Reports [Internet]. 2019 Sep 10;9 (1 ). DOI: 10.1038/s41598-019-49140-x
46. Skrabek RQ ; Anderson JE . Metabolic shifts and myocyte hypertrophy in deflazacort treatment of mdx mouse cardiomyopathy. Muscle & Nerve. 2016;24 (2 ). DOI: 10.1002/1097-4598(200102)24:2<192::aid-mus40>3.0.co;2-s
47. Caldow MK , Ham DJ , Trieu J , Chung JD , Lynch GS , Koopman R . Glycine Protects Muscle Cells From Wasting in vitro via mTORC1 Signaling. Frontiers in Nutrition. 2019 Nov 13. DOI: 10.3389/fnut.2019.00172
48. Lin C , Han G , Ning H , Song J , Ran N , Yi X , Glycine Enhances Satellite Cell Proliferation, Cell Transplantation, and Oligonucleotide Efficacy in Dystrophic Muscle. Molecular Therapy. 2020 May;28 (5 ):1339–58. DOI:32209436
49. Wu G , Fang YZ ., Yang S , Lupton JR , Turner ND . Glutathione Metabolism and Its Implications for Health. The Journal of Nutrition. 2004 Mar 1.;134 (3 ):489–92. DOI: 10.1093/jn/134.3.489 14988435
50. Rutten EP , Engelen MP , Schols AM , Deutz NE . Skeletal muscle glutamate metabolism in health and disease: state of the art. Current Opinion in Clinical Nutrition and Metabolic Care. 2005 Jan;8 (1 ):41–51. DOI: 10.1097/00075197-200501000-00007 15585999
51. Petrillo S , Pelosi L , Piemonte F , Travaglini L , Forcina L , Catteruccia M , Oxidative stress in Duchenne muscular dystrophy: focus on the NRF2 redox pathway. Human Molecular Genetics. 2017 May 3;26 (14 ):2781–90. DOI:10.1093/hmg/ddx173 28472288
52. Abdullah M , Kornegay JN , Honcoop A , Parry TL , Balog-Alvarez CJ , O’Neal SK , Non-Targeted Metabolomics Analysis of Golden Retriever Muscular Dystrophy-Affected Muscles Reveals Alterations in Arginine and Proline Metabolism, and Elevations in Glutamic and Oleic Acid In Vivo. Metabolites. 2017 Jul 29;7 (3 ):E38. DOI: 10.3390/metabo7030038
53. Laferté RO , Rosenkrantz H , Berlinguet L . TRANSAMINATION IN MUSCULAR DYSTROPHY AND THE EFFECT OF EXOGENOUS GLUTAMATE: A STUDY ON VITAMIN E DEFICIENT RABBITS, AND MICE WITH HEREDITARY DYSTROPHY. Canadian Journal of Biochemistry and Physiology. 1963 Jan 1;41 (1 ):1423–32. DOI: 10.1139/y63-162 13928008