Identification and characterization of dystrophin-locus-derived testis-specific protein: A testis-specific gene within the intronic region of the rat dystrophin gene.

The mammalian X chromosome exhibits enrichment in genes associated with germ cell development. Previously, we generated a rat model of Becker muscular dystrophy (BMD) characterized by an in-frame mutation in the dystrophin gene, situated on the X chromosome and responsible for encoding a protein crucial for muscle integrity. Male BMD rats are infertile owing to the absence of normal spermatids in the epididymis. Within the seminiferous tubules of BMD rats, elongated spermatids displayed abnormal morphology. To elucidate the cause of infertility, we identified a putative gene containing an open reading frame situated in the intronic region between exons 6 and 7 of the dystrophin gene, specifically deleted in male BMD rats. This identified gene, along with its encoded protein, exhibited specific detection within the testes, exclusively localized in round to elongated spermatids during spermiogenesis. Consequently, we designated the encoded protein as dystrophin-locus-derived testis-specific protein (DTSP). Given the absence of DTSP in the testes of BMD rats, we hypothesized that the loss of DTSP contributes to the infertility observed in male BMD rats.

Dietary lysine restriction induces lipid accumulation in skeletal muscle through an increase in serum threonine levels in rats.

We previously reported that dietary amino acid restriction induces the accumulation of triglycerides (TAG) in the liver of growing rats. However, differences in TAG accumulation in individual cell types or other tissues were not examined. In this study, we show that TAG also accumulates in the muscle and adipose tissues of rats fed a low amino acid (low-AA) diet. In addition, dietary lysine restriction (low-Lys) induces lipid accumulation in muscle and adipose tissues. In adjusting the nitrogen content to that of the control diet, we found that glutamic acid supplementation to the low-AA diet blocked lipid accumulation, but supplementation with the low-Lys diet did not, suggesting that a shortage of nitrogen caused lipids to accumulate in the skeletal muscle in the rats fed a low-AA diet. Serum amino acid measurement revealed that, in rats fed a low-Lys diet, serum lysine levels were decreased, while serum threonine levels were significantly increased compared with the control rats. When the threonine content was restricted in the low-Lys diet, TAG accumulation induced by the low-Lys diet was completely abolished in skeletal muscle. Moreover, in L6 myotubes cultured in medium containing high threonine and low lysine, fatty acid uptake was enhanced compared with that in cells cultured in control medium. These findings suggest that the increased serum threonine in rats fed a low-Lys diet resulted in lipid incorporation into skeletal muscle, leading to the formation of fatty muscle tissue. Collectively, we propose conceptual hypothesis that "amino-acid signal" based on lysine and threonine regulates lipid metabolism.

Ketogenic diet with medium-chain triglycerides restores skeletal muscle function and pathology in a rat model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is an intractable genetic disease associated with progressive skeletal muscle weakness and degeneration. Recently, it was reported that intraperitoneal injections of ketone bodies partially ameliorated muscular dystrophy by increasing satellite cell (SC) proliferation. Here, we evaluated whether a ketogenic diet (KD) with medium-chain triglycerides (MCT-KD) could alter genetically mutated DMD in model rats. We found that the MCT-KD significantly increased muscle strength and fiber diameter in these rats. The MCT-KD significantly suppressed the key features of DMD, namely, muscle necrosis, inflammation, and subsequent fibrosis. Immunocytochemical analysis revealed that the MCT-KD promoted the proliferation of muscle SCs, suggesting enhanced muscle regeneration. The muscle strength of DMD model rats fed with MCT-KD was significantly improved even at the age of 9 months. Our findings suggested that the MCT-KD ameliorates muscular dystrophy by inhibiting myonecrosis and promoting the proliferation of muscle SCs. As far as we can ascertain, this is the first study to apply a functional diet as therapy for DMD in experimental animals. Further studies are needed to elucidate the underlying mechanisms of the MCT-KD-induced improvement of DMD.

Disruption of insulin receptor substrate-2 impairs growth but not insulin function in rats.

Insulin receptor substrate (IRS)-2, along with IRS-1, is a key signaling molecule that mediates the action of insulin and insulin-like growth factor (IGF)-I. The activated insulin and IGF-I receptors phosphorylate IRSs on tyrosine residues, leading to the activation of downstream signaling pathways and the induction of various physiological functions of insulin and IGF-I. Studies using IRS-2 knockout (KO) mice showed that the deletion of IRS-2 causes type 2 diabetes due to peripheral insulin resistance and impaired ß-cell function. However, little is known about the roles of IRS-2 in other animal models. Here, we created IRS-2 KO rats to elucidate the physiological functions of IRS-2 in rats. The body weights of IRS-2 KO rats at birth were lower compared with those of their WT littermates. The postnatal growth of both male and female IRS-2 KO rats was also suppressed. Compared with male WT rats, the glucose and insulin tolerance of male IRS-2 KO rats were slightly enhanced, whereas a similar difference was not observed between female WT and IRS-2 KO rats. Besides the modestly increased insulin sensitivity, male IRS-2 KO rats displayed the enhanced insulin-induced activation of the mTOR complex 1 pathway in the liver compared with WT rats. Taken together, these results indicate that in rats, IRS-2 plays important roles in the regulation of growth but is not essential for the glucose-lowering effects of insulin.

Age-Dependent Echocardiographic and Pathologic Findings in a Rat Model with Duchenne Muscular Dystrophy Generated by CRISPR/Cas9 Genome Editing.

Duchenne muscular dystrophy (DMD) is X-linked recessive myopathy caused by mutations in the dystrophin gene. Although conventional treatments have improved their prognosis, inevitable progressive cardiomyopathy is still the leading cause of death in patients with DMD. To explore novel therapeutic options, a suitable animal model with heart involvement has been warranted.We have generated a rat model with an out-of-frame mutation in the dystrophin gene using CRISPR/Cas9 genome editing (DMD rats). The aim of this study was to evaluate their cardiac functions and pathologies to provide baseline data for future experiments developing treatment options for DMD.In comparison with age-matched wild rats, 6-month-old DMD rats showed no significant differences by echocardiographic evaluations. However, 10-month-old DMD rats showed significant deterioration in left ventricular (LV) fractional shortening (P = 0.024), and in tissue Doppler peak systolic velocity (Sa) at the LV lateral wall (P = 0.041) as well as at the right ventricular (RV) free-wall (P = 0.004). These functional findings were consistent with the fibrotic distributions by histological analysis.Although the cardiac phenotype was milder than anticipated, DMD rats showed similar distributions and progression of heart involvement to those of patients with DMD. This animal may be a useful model with which to develop effective drugs and to understand the underlying mechanisms of progressive heart failure in patients with DMD.

Progranulin regulates lysosomal function and biogenesis through acidification of lysosomes.

Progranulin (PGRN) haploinsufficiency resulting from loss-of-function mutations in the PGRN gene causes frontotemporal lobar degeneration accompanied by TDP-43 accumulation, and patients with homozygous mutations in the PGRN gene present with neuronal ceroid lipofuscinosis. Although it remains unknown why PGRN deficiency causes neurodegenerative diseases, there is increasing evidence that PGRN is implicated in lysosomal functions. Here, we show PGRN is a secretory lysosomal protein that regulates lysosomal function and biogenesis by controlling the acidification of lysosomes. PGRN gene expression and protein levels increased concomitantly with the increase of lysosomal biogenesis induced by lysosome alkalizers or serum starvation. Down-regulation or insufficiency of PGRN led to the increased lysosomal gene expression and protein levels, while PGRN overexpression led to the decreased lysosomal gene expression and protein levels. In particular, the level of mature cathepsin D (CTSDmat) dramatically changed depending upon PGRN levels. The acidification of lysosomes was facilitated in cells transfected with PGRN. Then, this caused degradation of CTSDmat by cathepsin B. Secreted PGRN is incorporated into cells via sortilin or cation-independent mannose 6-phosphate receptor, and facilitated the acidification of lysosomes and degradation of CTSDmat. Moreover, the change of PGRN levels led to a cell-type-specific increase of insoluble TDP-43. In the brain tissue of FTLD-TDP patients with PGRN deficiency, CTSD and phosphorylated TDP-43 accumulated in neurons. Our study provides new insights into the physiological function of PGRN and the role of PGRN insufficiency in the pathogenesis of neurodegenerative diseases.

Identification of CCL5/RANTES as a novel contraction-reducible myokine in mouse skeletal muscle.

Skeletal muscle is an endocrine organ that secretes several proteins, which are collectively termed myokines. Although many studies suggest that exercise regulates myokine secretion, the underlying mechanisms remain unclear and all the exercise-dependent myokines have not yet been identified. Therefore, in this study, we attempted to identify novel exercise-dependent myokines by using our recently developed in vitro contractile model. Differentiated C2C12 myotubes were cultured with or without electrical pulse stimulation (EPS) for 24 h to induce cell contraction, and the myokines secreted in conditioned medium were analyzed using a cytokine array. Although most myokine secretions were not affected by EPS, the secretion of Chemokine (C-C motif) ligand 5 (CCL5) (regulated on activation, normal T cell expressed and secreted (RANTES)) was significantly reduced by EPS. This was further confirmed by ELISA and quantitative PCR. Contraction-dependent calcium transients and activation of 5'-AMP activating protein kinase (AMPK) appears to be involved in this decrease, as the chelating Ca2+ by EGTA blocked contraction-dependent CCL5 reduction, whereas the pharmacological activation of AMPK significantly reduced it. However, Ccl5 gene expression was increased by AMPK activation, suggesting that AMPK-dependent CCL5 decrease occurred via post-transcriptional regulation. Finally, mouse experiments revealed that voluntary wheel-running exercise reduced serum CCL5 levels and Ccl5 gene expression in the fast-twitch muscles. Overall, our study provides the first evidence of an exercise-reducible myokine, CCL5, in the mouse skeletal muscle. Although further studies are required to understand the precise roles of the skeletal muscle cell contraction-induced decrease in CCL5, this decrease may explain some exercise-dependent physiological changes such as those in immune responses.

Skeletal muscle cell contraction reduces a novel myokine, chemokine (C-X-C motif) ligand 10 (CXCL10): potential roles in exercise-regulated angiogenesis.

Accumulating evidence indicates that skeletal muscle secrets proteins referred to as myokines and that exercise contributes to their regulation. In this study, we propose that chemokine (C-X-C motif) ligand 10 (CXCL10) functions as a novel myokine. Initially, we stimulated differentiated C2C12 myotubes with or without electrical pulse stimulation (EPS) to identify novel myokines. Cytokine array analysis revealed that CXCL10 secretion was significantly reduced by EPS, which was further confirmed by enzyme-linked immunosorbent assay and quantitative polymerase chain reaction analysis. Treadmill experiments in mice identified significant reduction of Cxcl10 gene expression in the soleus muscle. Additionally, contraction-dependent p38 MAPK activation appeared to be involved in this reduction. Furthermore, C2C12 conditioned medium obtained after applying EPS could induce survival of MSS31, a vascular endothelial cell model, which was partially attenuated by the addition of recombinant CXCL10. Overall, our findings suggest CXCL10 as a novel exercise-reducible myokine, to control endothelial cell viability.

Roles of chondroitin sulfate proteoglycan 4 in fibrogenic/adipogenic differentiation in skeletal muscle tissues.

Intramuscular adipose tissue and fibrous tissue are observed in some skeletal muscle pathologies such as Duchenne muscular dystrophy and sarcopenia, and affect muscle strength and myogenesis. They originate from common fibrogenic/adipogenic cells in the skeletal muscle. Thus, elucidating the regulatory mechanisms underlying fibrogenic/adipogenic cell differentiation is an important step toward the mediation of these disorders. Previously, we established a highly adipogenic progenitor clone, 2G11, from rat skeletal muscle and showed that basic fibroblast growth factor (bFGF) is pro-adipogenic in these cells. Here, we demonstrated that 2G11 cells give rise to fibroblasts upon transforming growth factor (TGF)-ß1 stimulation, indicating that they possess mesenchymal progenitor cells (MPC)-like characteristics. The previously reported MPC marker PDGFRα is expressed in other cell populations. Accordingly, we produced monoclonal antibodies that specifically bind to 2G11 cell surface antigens and identified chondroitin sulfate proteoglycan 4 (CSPG4) as a potential MPC marker. Based on an RNA interference analysis, we found that CSPG4 is involved in both the pro-adipogenic effect of bFGF and in TGF-ß-induced alpha smooth muscle actin expression and stress fiber formation. By establishing an additional marker for MPC detection and characterizing its role in fibrogenic/adipogenic differentiation, these results will facilitate the development of effective treatments for skeletal muscle pathologies.

Lack of estrogen receptor α in astrocytes of progranulin-deficient mice.

Progranulin (PGRN) is a multifunctional growth factor with functions in neuroprotection, anti-inflammation, and neural progenitor cell proliferation. These functions largely overlap with the actions of estrogen in the brain. Indeed, we have previously shown that PGRN mediates the functions of estrogen, such as masculinizing the rodent brain and promoting adult neurogenesis. To evaluate the underlying mechanism of PGRN in mediating the actions of estrogen, the localization of estrogen receptor α (ERα) in the brains of wild-type (WT) and PGRN-deficient (KO) mice was investigated. First, double-labeling immunofluorescence was performed for ERα with neuronal nuclei (NeuN), ionized calcium-binding adaptor molecule 1 (Iba1), and glial fibrillary acidic protein (GFAP), as markers for neurons, microglia, and astrocytes, respectively, in female mice in diestrous and estrous stages. ERα-immunoreactive (IR) cells were widespread and co-localized with NeuN in brain sections analyzed (bregma -1.06 to -3.16 mm) of both WT and KO mice. In contrast, expression of ERα was not observed in Iba1-IR cells from both genotypes. Interestingly, although ERα was co-localized with GFAP in WT mice, virtually no ERα expression was discernible in GFAP-IR cells in KO mice. Next, the brains of ovariectomized adult female, adult male, and immature female mice were subjected to immunostaining for ERα and GFAP. Again, co-localization of ERα with GFAP was observed in WT mice, whereas this co-localization was not detected in KO mice. These results suggest that PGRN plays a crucial role in the expression of ERα in astrocytes regardless of the estrous cycle stage, sex, and maturity.

Myotube formation is affected by adipogenic lineage cells in a cell-to-cell contact-independent manner.

Intramuscular adipose tissue (IMAT) formation is observed in some pathological conditions such as Duchenne muscular dystrophy (DMD) and sarcopenia. Several studies have suggested that IMAT formation is not only negatively correlated with skeletal muscle mass but also causes decreased muscle contraction in sarcopenia. In the present study, we examined w hether adipocytes affect myogenesis. For this purpose, skeletal muscle progenitor cells were transfected with siRNA of PPARγ (siPPARγ) in an attempt to inhibit adipogenesis. Myosin heavy chain (MHC)-positive myotube formation was promoted in cells transfected with siPPARγ compared to that of cells transfected with control siRNA. To determine whether direct cell-to-cell contact between adipocytes and myoblasts is a prerequisite for adipocytes to affect myogenesis, skeletal muscle progenitor cells were cocultured with pre- or mature adipocytes in a Transwell coculture system. MHC-positive myotube formation was inhibited when skeletal muscle progenitor cells were cocultured with mature adipocytes, but was promoted when they were cocultured with preadipocytes. Similar effects were observed when pre- or mature adipocyte-conditioned medium was used. These results indicate that preadipocytes play an important role in maintaining skeletal muscle mass by promoting myogenesis; once differentiated, the resulting mature adipocytes negatively affect myogenesis, leading to the muscle deterioration observed in skeletal muscle pathologies.

[Transdisciplinary Approach for Sarcopenia. Molecular mechanism of sarcopenia : The role of skeletal muscle niche component SPARC in the regulation of myogenesis and adipogenesis and its alteration with age].

Sarcopenia, an age-related decline in skeletal muscle mass and strength, causes the decline of the quality of life in the elderly. The age-related alteration in the differentiation potency of satellite cells, myogenic tissue specific stem cells in skeletal muscle, and preadipocytes in skeletal muscle is possibly involved in the disruption of homeostasis in skeletal muscle. The differentiation of the cells is affected by the microenvironment surrounding the cells, called niche. Here, we focused on SPARC (secreted protein acidic and rich in cysteine) as a secreted glycoprotein existing in the niche. We review the roles of SPARC on the differentiation of satellite cells and preadipocytes in the muscle and their alteration with age.

Loss of SPARC in mouse skeletal muscle causes myofiber atrophy.

INTRODUCTION: The expression of secreted protein acidic and rich in cysteine (SPARC) in skeletal muscle decreases with age. Here, we examined the role of SPARC in skeletal muscle by reducing its expression. METHODS: SPARC expression was suppressed by introducing short interfering RNA (siRNA) into mouse tibialis anterior muscle. Myofiber diameter, atrogin1, and muscle RING-finger protein 1 (MuRF1) expression, and tumor necrosis factor-α (TNFα) and transforming growth factor-ß (TGFß) signaling were then analyzed. RESULTS: Reduced SPARC expression caused decreases in the diameter of myofibers, especially fast-type ones, accompanied by upregulation of atrogin1, but not MuRF1, at 10 days after siRNA transfection. The expression of TNFα and TGFß and the phosphorylation status of p38 were not affected by SPARC knockdown, whereas Smad3 phosphorylation was increased at 2 days after siRNA transfection. CONCLUSIONS: The loss of SPARC not only upregulates atrogin1 expression but also enhances TGFß signaling, which may in turn cause muscle atrophy.

Taurine counteracts the suppressive effect of lipopolysaccharide on neurogenesis in the hippocampus of rats.

Neurogenesis has been generally accepted to happen in the subventricular zone lining the lateral ventricular and subgranular zone (SGZ) in the hippocampus of adult mammalian brain. Recent studies have reported that inflammatory stimuli, such as injection of lipopolysaccharide (LPS), impair neurogenesis in the SGZ. Taurine, a sulfur-containing ß-amino acid, is a major free intracellular amino acid in many tissues of mammals and having various supplementary effects on the mammalian body functions including the brain. Recently, it has been also reported that taurine levels in the brain significantly increase under stressful conditions. The present study was aimed to evaluate the possible beneficial effects of taurine on the neurogenesis in the SGZ under the condition of acute inflammatory stimuli by LPS. Adult male rats were intraperitoneally injected with taurine once a day for 39 days. Twenty-four hours before the animals were sacrificed on the last day of taurine treatment, LPS was injected simultaneously with bromodeoxyuridine (BrdU). Immunohistochemistry for BrdU, Ki67, and Iba-1 in the brain was performed, and serum levels of TNF-α and IL-1ß 2 h after LPS injection were determined. The results showed that LPS significantly decreased the number of immunoreactive cells for both BrdU and Ki67 in the SGZ, while increased that for Iba-1, all of which were restored by taurine administration. Meanwhile, the serum concentrations of TNF-α and IL-1ß were significantly increased, which were significantly attenuated by taurine administration. These results suggest that taurine effectively maintains neurogenesis in the SGZ under the acute infectious condition by attenuating the increase of microgliosis in the hippocampus as well as proinflammatory cytokines in the peripheral circulation.

In Vivo Modeling of Skeletal Muscle Diseases Using the CRISPR/Cas9 System in Rats.

The CRISPR/Cas9 system is a powerful gene editing tool that can be used to modify a target gene in almost all species. It unlocks the possibility of generating knockout or knock-in genes in laboratory animals other than mice. The Dystrophin gene is implicated in human Duchenne muscular dystrophy; however, Dystrophin gene mutant mice do not show severe muscle degenerating phenotypes when compared to humans. On the other hand, Dystrophin gene mutant rats made with the CRISPR/Cas9 system show more severe phenotypes than those seen in mice. The phenotypes seen in dystrophin mutant rats are more representative of the features of human DMD. This implies that rats are better models of human skeletal muscle diseases than mice. In this chapter, we present a detailed protocol for the generation of gene-modified rats by microinjection into embryos using the CRISPR/Cas9 system.

A new immunodeficient Duchenne muscular dystrophy rat model to evaluate engraftment after human cell transplantation.

Duchenne muscular dystrophy (DMD) is an X-linked fatal muscular disease, affecting one in 3,500 live male births worldwide. Currently, there is no cure for this disease, except for steroid-based treatment to attenuate disease progression. Cell transplantation therapy is a promising therapeutic approach, however, there is a lack of appropriate animal models to conduct large-scale preclinical studies using human cells, including biochemical and functional tests. Here, we established an immunodeficient DMD rat model and performed exhaustive pathological analysis and transplantation efficiency evaluation to assess its suitability to study DMD. Our DMD rat model exhibited histopathological characteristics similar to those observed in human patients with DMD. Human myoblasts demonstrated successful engraftment following transplantation into these rats. Therefore, this immunodeficient DMD rat model would be useful in preclinical studies to develop cellular transplantation therapies for DMD.

Characterization of pathogenic factors for premenstrual dysphoric disorder using machine learning algorithms in rats.

We established a methodology using machine learning algorithms for determining the pathogenic factors for premenstrual dysphoric disorder (PMDD). PMDD is a disease characterized by emotional and physical symptoms that occurs before menstruation in women of childbearing age. Owing to the diverse manifestations and various pathogenic factors associated with this disease, the diagnosis of PMDD is time-consuming and challenging. In the present study, we aimed to establish a methodology for diagnosing PMDD. Using an unsupervised machine-learning algorithm, we divided pseudopregnant rats into three clusters (C1 to C3), depending on the level of anxiety- and depression-like behaviors. From the results of RNA-seq and subsequent qPCR of the hippocampus in each cluster, we identified 17 key genes for building a PMDD diagnostic model using our original two-step feature selection with supervised machine learning. By inputting the expression levels of these 17 genes into the machine learning classifier, the PMDD symptoms of another group of rats were successfully classified as C1-C3 with an accuracy of 96%, corresponding to the classification by behavior. The present methodology would be applicable for the clinical diagnosis of PMDD using blood samples instead of samples from the hippocampus in the future.

Ivabradine ameliorates cardiomyopathy progression in a Duchenne muscular dystrophy model rat.

Duchenne muscular dystrophy (DMD) is an X-linked recessive myopathy caused by dystrophin mutations. Inevitable progressive cardiomyopathy is a current leading cause of premature death although respiratory management has improved the prognosis of patients with DMD. Recent evidence shows that reducing the heart rate is expected as one of the promising strategies for heart failure treatment, but administering a sufficient dose of ß-blocker for patients with DMD with tachycardia is difficult because of their low blood pressure (BP). Thus, this study aimed to clarify the role of ivabradine, which suppresses cardiac sinus node pacemakers without decreasing BP, in ameliorating cardiomyopathy progression in a rat model with DMD. A trans-oral single ivabradine administration demonstrated a declined dose-dependent heart rate without any significant BP reduction. Trans-gastric repeated administrations of 5 mg/kg of ivabradine twice a day for 3 months showed ameliorated cardiomyopathy in DMD rats based on echocardiography and histopathological observations (left ventricular dysfunction, right ventricular dysfunction, and myocardial fibrosis) as compared with vehicle administration. Our finding indicates that ivabradine is expected as another treatment choice for patients with DMD having tachycardia.

Induction of Ski protein expression upon luteinization in rat granulosa cells without a change in its mRNA expression.

The Ski protein is implicated in the proliferation/differentiation of a variety of cells. We previously reported that the Ski protein is present in granulosa cells of atretic follicles, but not in preovulatory follicles, suggesting that Ski has a role in apoptosis of granulosa cells. However, granulosa cells cannot only undergo apoptosis but can alternatively differentiate into luteal cells. It is unknown whether Ski is expressed and has a role in granulosa cells undergoing luteinization. Thus, the aim of the present study was to determine the localization of the Ski protein in the rat ovary during luteinization to examine if Ski might play a role in this process. In order to examine the Ski protein expression during the progression of luteinization, follicular growth was induced in immature female rats by administration of equine chorionic gonadotropin, and luteinization was induced by human chorionic gonadotropin treatment to mimic the luteinizing hormone (LH) surge. While no Ski-positive granulosa cells were present in the preovulatory follicle, Ski protein expression was induced in response to the LH surge and was maintained after formation of the corpus luteum (CL). Although the Ski protein is absent from the granulosa cells of the preovulatory follicle, its mRNA (c-ski) was expressed, and the level of c-ski mRNA was unchanged even after the LH surge. The combined results demonstrated that Ski protein expression is induced in granulosa cells upon luteinization, and suggested that its expression is regulated posttranscriptionally.

Ketogenic diet containing medium-chain triglyceride ameliorates transcriptome disruption in skeletal muscles of rat models of duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a myopathy characterized by progressive muscle weakness caused by a mutation in the dystrophin gene on the X chromosome. We recently showed that a medium-chain triglyceride-containing ketogenic diet (MCTKD) improves skeletal muscle myopathy in a CRISPR/Cas9 gene-edited rat model of DMD. We examined the effects of the MCTKD on transcription profiles in skeletal muscles of the model rats to assess the underlying mechanism of the MCTKD-induced improvement in DMD. DMD rats were fed MCTKD or normal diet (ND) from weaning to 9 months, and wild-type rats were fed with the ND, then tibialis anterior muscles were sampled for mRNA-seq analysis. Pearson correlation heatmaps revealed a one-node transition in the expression profile between DMD and wild-type rats. A total of 10,440, 11,555 and 11,348 genes were expressed in the skeletal muscles of wild-type and ND-fed DMD rats the MCTKD-fed DMD rats, respectively. The MCTKD reduced the number of DMD-specific mRNAs from 1624 to 1350 and increased the number of mRNAs in common with wild-type rats from 9931 to 9998. Among 2660 genes were differentially expressed in response to MCTKD intake, the mRNA expression of 1411 and 1249 of them was respectively increased and decreased. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses suggested that the MCTKD significantly suppressed the mRNA expression of genes associated with extracellular matrix organization and inflammation. This suggestion was consistent with our previous findings that the MCTKD significantly suppressed fibrosis and inflammation in DMD rats. In contrast, the MCTKD significantly increased the mRNA expression of genes associated with oxidative phosphorylation and ATP production pathways, suggesting altered energy metabolism. The decreased and increased mRNA expression of Sln and Atp2a1 respectively suggested that Sarco/endoplasmic reticulum Ca2+-ATPase activation is involved in the MCTKD-induced improvement of skeletal muscle myopathy in DMD rats. This is the first report to examine transcription profiles in the skeletal muscle of CRISPR/Cas9 gene-edited DMD model rats and the effect of MCTKD feeding on it.