Pharmacological Inhibition of N-terminal methyltransferase 1 promotes myogenic differentiation and muscle regeneration in a mouse model of Duchenne muscular dystrophy

Abstract

Background

Duchenne muscular dystrophy (DMD), caused by mutations of the DMD gene, is a lethal degenerative disease with no cure. Stimulating myogenesis of muscle stem cells (MuSCs) represents a promising strategy to ameliorate muscle pathology in DMD patients. Although previous work has revealed a role of N-terminal methyltransferase 1 (NTMT1) in myogenesis, its potential as a therapeutic target to ameliorate muscular dystrophy remains unexplored.

Method

We employed GD433, a highly specific and potent pharmacological inhibitor of NTMT1, and evaluated its role in myogenesis using cell and animal models (mdx mice). C2C12 and primary myoblasts, and MuSCs were treated with GD433, and cell proliferation, differentiation, fusion, and gene expression were examined. Mdx mice were treated by intraperitoneal injection of GD433 (25 mg/kg, 5 days/week), and muscle pathology was evaluated. We finally employed co-immunoprecipitation (IP) and methylation-specific antibodies to identify NTMT1 substrates in myoblasts.

Results

A low dose of GD433 (300 nM) significantly enhances the differentiation and fusion of cultured C2C12 and primary myoblasts without affecting their proliferation. Similar effects are observed in myofiber-associated MuSCs. GD433 further enhances myogenic differentiation and muscle regeneration in mdx mice. Mechanistically, GD433 elevates expression of myogenic differentiation and fusion factors (Myog, Mymk, Mymx) through affecting KLHK31 methylation. Specifically, GD433 reduces the methylation level of KLHL31, promoting its interaction with MYOD and upregulating the expression of Myog, Mymk and Mymx.

Conclusion

These results show that pharmacological inhibition of NTMT1 by GD433 promotes myogenic differentiation and enhances muscle regeneration in mdx mice. The pro-myogenic effect of GD433 is associated with reduced KLHL31 methylation, which strengthens KLHL31–MYOD interactions to upregulate genes related to myogenic differentiation. This study highlights NTMT1 inhibition as a potential therapeutic target to improve muscle repair in DMD patients.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13287-026-04934-5.

Background

MuSCs, or satellite cells, are responsible for postnatal repair and regeneration of injured muscles [1]. These cells remain quiescent under normal physiological conditions in the adult, but become activated in response to injury, exercise, or other stimuli. MuSC activation and myogenic differentiation are intricately regulated by a complex network of signaling factors, including growth factors, inflammatory mediators, and mechanical stress [2]. Activated MuSCs undergo proliferation, differentiation into myoblasts, and fusion with existing muscle fibers [3]. This process contributes to muscle repair, regeneration, and the maintenance of muscle homeostasis. Myogenesis of MuSCs is regulated by intrinsic factors such as PAX7, MYOD, and MYOG. PAX7 is expressed in all quiescent and proliferating MuSCs to underly their quiescence and stem cell status [4]. MYOD is a myogenic transcriptional activator that promotes the commitment of MuSC during embryonic development and postnatal regeneration [5]. MYOG is a critical regulatory factor that modulates early myogenic differentiation [6].

NTMT1 introduces a methyl group on the a-N-terminus of proteins with “X-Pro-Lys /Arg” motifs, where X can be any amino acid except for Asp or Glu [7, 8]. The a-N-terminal (Na) methylation can modulate interaction, distribution, and stability of substrates [9, 10]. Previous studies have shown that NTMT1 knockdown increases breast cancer cell proliferation but suppresses the growth of retinoblastoma and cervical cancers [11]. NTMT1 knockout (KO) mice exhibit mitochondrial dysfunction and deficiency in DNA repair, resulting in premature aging [12, 13]. NTMT1 KO in murine C2C12 myoblasts abolishes PAX7 expression, and the KO cells fail to produce myosin heavy chain (MHC) positive myotubes [14]. These studies establish the diverse biological functions of NTMT1, including its function in myogenesis.

Our group has recently developed a small-molecule inhibitor of NTMT1, namely GD433 [15]. GD433 exhibits a potent activity with an IC₅₀ value of 30 nM in HEK293 cells, with over 100-fold selectivity for NTMT1 over other methyltransferases [15]. We have also developed a structure-related inactive analog, YD2160 (GD433N), which has no effect on NTMT1 at concentrations of 10 µM, representing a valuable control molecule. Here, we examined the effects of GD433 and its inactive analog GD433N on myogenesis in myogenic cells and mdx mouse models. We found that NTMT1 inhibition by GD433 promotes myogenic differentiation of myoblasts and improves muscle regeneration in mdx mice. These results establish GD433 as a potential agent for the treatment of muscular dystrophy diseases.

Materials and methods

Animals

Dmd^mdx mice purchased from Jackson Laboratory (#013141). The mice were housed in a 12 h light/12 h dark cycle with free access to water and food. The Purdue Animal Care and Use Committee approved all animal experimental protocols. Generally, 7-to 9-week-old mice were used for all experiments. No formal randomisation was performed; animals were allocated directly into treatment and control groups. Both male and female mice were included, and animals were sex-matched across groups to minimise sex-related confounding.

Primary myoblast isolation and culture

Primary myoblasts were isolated from hind limb skeletal muscles of wild-type or mdx mice at 4–6 weeks. Muscles were minced and digested in type I collagenase and Dispase B (Roche Applied Science). The digestions were stopped with F-10 Ham’s medium containing 20% FBS. Cells were then filtered from debris, centrifuged, and cultured in growth medium (F-10 Ham’s medium supplemented with 20% FBS, 4 ng ml^− 1 basic fibroblast growth factor, and 1% penicillin-streptomycin) on collagen-coated cell culture plates at 37 °C, 5% CO₂. The treatment group samples were treated with the above medium containing 300 nM inhibitor.

Cell differentiation

For differentiation, C2C12 and primary myoblasts were seeded on BD Matrigel-coated cell culture plates and induced to differentiate in the differentiation medium (DMEM supplemented with 2% horse serum and 1% penicillin-streptomycin). The treatment group samples were treated with the above medium containing 300 nM inhibitor.

NTMT1 inhibitor treatment

The NTMT1 inhibitor (GD433) synthesized by the Huang lab was dissolved in DMSO to a stock concentration of 10 mM. This stock solution was diluted using the relevant culture media to final working concentrations.

EdU labeling to mark cell proliferation

Cells were incubated with 10 µM EdU in culture medium for 2 h, cells were then washed to remove non-incorporated free EdU. Cells were fixed in 4% paraformaldehyde in PBS for 10 min. EdU was visualized by the Click-iT method with red fluorescent dye tetramethylrhodamine azide (Invitrogen, cat#T10182).

Total RNA extraction and real-time PCR

Total RNA was extracted from tissues using TRIzol reagent according to the manufacturer’s instructions. RNA was treated with RNase-free DNase I to remove contaminating genomic DNA. The purity and concentration of the total RNA were determined by a spectrophotometer Nanodrop 2000c (Thermo Fisher). 1 µg of total RNA was reverse transcribed using random primers with M-MLV reverse transcriptase (Invitrogen). Real-time PCR was performed using a Roche LightCycler 480 PCR System with SYBR Green Master Mix, and gene-specific primers are listed in Supplementary Table 1. The 2 ^−∆∆Ct method was used to analyze the relative changes in each gene’s expression normalized against 18 S rRNA expression.

Single myofiber isolation and culture

Single myofibers were isolated from EDL muscles by 40–45 min of digestion in 0.2% collagenase type I (Worthington Biochemical) in Dulbecco’s modified Eagle’s medium (DMEM) at 37 °C. The digested muscle was transferred to horse serum-coated culture dishes (60 mm) containing pre-warmed DMEM with 1% penicillin-streptomycin. Single myofibers were isolated by gently flushing muscles with a fire-polished glass pipette. Individual myofibers were then collected by pipette to remove dead myofibers and cellular debris and subjected to immunofluorescence staining and culture. Single myofiber was isolated and cultured in DMEM medium containing 20% serum for 48 h. The treatment group samples were treated with the above medium containing 300 nM inhibitor.

Muscle injury and regeneration

Muscle regeneration was induced by BaCl₂ injection. 10 mM of the NTMT1 inhibitor (GD433) was dissolved in saline at a final concentration of 2 mM. The compound solution was intraperitoneally injected into 8-week-old mdx mice at 25 mg/kg body weight for 5 days consecutively, with the littermate mdx mice injected with saline as the control. Adult mice were anesthetized using a ketamine–xylazine cocktail, and BaCl₂ was injected (50 µL of 1.2%(w/v)BaCl₂, Sigma) into the TA muscle. Muscles were then harvested at the stated time to assess the completion of regeneration and repair.

Histology and immunofluorescence staining

Tibialis anterior muscles (TA) from mdx mice were dissected and frozen immediately in an OCT (optimum cutting temperature) compound. Frozen muscles were sectioned using a Leica CM1850 cryostat at 10 μm thickness. Slides were subjected to H&E and immunofluorescence staining.

For H&E staining, the slides were stained in a hematoxylin solution for 10 min and rinsed in running water three times. Slides were then stained in an eosin solution for 1–2 min, dehydrated in a graded ethanol and xylene series, and covered with Permount for further imaging. For quantification, H&E-stained sections were segmented using Cellpose (v2.2) with the “cyto2” model to delineate individual myofibers. The resulting segmentation masks were imported into ImageJ for measurement of fiber cross-sectional area (CSA). At least 500 fibers per sample were analyzed to generate CSA distribution histograms.

For immunofluorescence staining, cross-sections were fixed in 4% paraformaldehyde (PFA) in PBS for 5 min and incubated in blocking buffer (5% goat serum, 2% BSA, 0.1% Triton X-100, and 0.1% sodium azide in PBS) for 1 h at room temperature. Samples were then incubated with primary antibodies at 4 °C overnight. After incubation with corresponding second antibodies and DAPI at room temperature for 1 h, samples were covered with the fluorescent mounting medium for further imaging. For F4/80 Immunofluorescence, F4/80 and laminin co-stained sections were analyzed using ImageJ, and mean F4/80 fluorescence intensity per field was quantified to assess macrophage infiltration.

Protein extraction and western blot analysis

Total protein was isolated from muscle tissues with radioimmunoprecipitation assay (RIPA) buffer containing 25 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 0.5% nonyl phenoxypolyethoxylethanol (NP-40), 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate (SDS). Sample protein concentrations were determined using Pierce bicinchoninic acid (BCA) protein assay reagent (Pierce Biotechnology). SDS-PAGE resolved the Total protein extract and transferred it onto polyvinylidene fluoride membranes (Millipore). The membranes were incubated in blocking buffer (5% fat-free milk) for 1 h at room temperature. Then, they blotted with primary antibodies at 4 °C overnight. After incubation with corresponding second antibodies at room temperature for 1 h, membranes were incubated with chemiluminescence substrate (Santa Cruz Biotechnology) and detected using the FluorChem R system (ProteinSimple).

Cell lysates were precleared with protein A/G agarose at 4 °C for 2 h. Then, 4 µg of primary antibody was added to the cell lysate containing 500 µg of total protein, and the mixture was rotated at 4 °C overnight. Protein A/G agarose was added to the cell lysate and rotated for 2 h at 4 °C. The samples were washed extensively six times and subjected to Western blot analysis.

Statistical analysis

All experiments involving mice were conducted with a minimum of three biological replicates. Muscle histological analyses were quantified using Fiji-ImageJ or Adobe Photoshop software. Although investigators responsible for in vivo treatments were aware of group allocation, image collection was performed randomly, and subsequent quantification of histological sections was conducted in a blinded manner. Statistical analyses were carried out using two-tailed Student’s t-tests. Data are presented as mean ± SEM, and a P value of < 0.05 was considered statistically significant.

Results

NTMT1 Inhibition promotes differentiation and fusion of C2C12 myoblasts

We have recently reported the development and pharmacokinetics of GD433, a potent and highly selective inhibitor of NTMT1 [15]. To establish the safety dosages of GD433 in myogenic cells, we first assessed the survival of C2C12 myoblasts in response to different concentrations of GD433 (Fig. S1A). Cell viability assays indicate that GD433 had no effects on C2C12 cell survival at concentrations below 3µM (Fig. S1B). Based on the results, we selected a safe and effective dose range of 30 nM to 300 nM for the next assay, as 30 nM is the IC₅₀ concentration for NTMT1 inhibition in HEK293 cells [15].

To examine the effect of GD433 on cell proliferation, we treated C2C12 with GD433 or GD433N in growth medium (GM) for 48 h, in the presence of 5-ethynyl-20-deoxyuridine (EdU) to label newly synthesized DNA (Fig. 1A). Quantitative analysis show that 30–300 nM of GD433 or its non-functional analog (GD433N) had no effects on proliferation of C2C12 cells, based on percentages of EdU⁺ cells (Fig. 1B). We then treated C2C12 with different concentrations of GD433 or GD433N in the differentiation medium (DM) for 72 h, followed by MYOG and MHC staining (Fig. 1C). We observed more and larger MHC⁺ myotubes in the GD433 groups than in vehicle control and GD433N groups (Fig. 1C). Quantitative analyses reveal that GD433 significantly elevated the differentiation (MyoG⁺ cells %) at 300 nM (Fig. 1D). We also caculated fusion index by dividing MyoG⁺ nuclei located in myotubes containing two or more nuclei by total MyoG⁺ nuclei. This analysis reveal that GD433 significantly increased the fusion index at all concentrations examined (Fig. 1E). These data collectively demonstrate that GD433 promotes myogenic differentiation and myoblast fusion without affecting the proliferation of C2C12 cells.

Fig. 1.

NTMT inhibition by GD433 promotes differentiation and fusion of C2C12 myoblasts without affecting proliferation. A EdU immunofluorescence of cells grown in growth media and treated with DMSO control or various concentrations of GD433 and non-active analog GD433N for 48 h in the presence of EdU to mark cell proliferation. DAPI (blue) marks nuclei. Scale bar, 100 μm. B Quantification of % EdU+ cells based on staining in A. N=5. C MYOG and Myosin Heavy Chain (MHC, stained with MF20 antibody) staining of cells grown in differentiation media for 72 h, treated with DMSO control or various concentrations of GD433/ GD433N. DAPI (blue) marks nuclei. Scale bar, 100 μm. D Quantification of % MYOG+ cells among all DAPI-marked nuclei. N=3. E Fusion index, calculated as the proportion of MYOG-positive nuclei within multinucleated myotubes (≥ 2 nuclei) relative to all MYOG-positive nuclei. N = 3. P-value was determined using unpaired two-tailed t-tests; bars without p-values indicate p > 0.05]

GD433 promotes differentiation of MuSCs in myofiber explants

A myofiber explant culture system was used to mimic proliferation, self-renewal, and differentiation of MuSCs in vivo. We isolated single myofibers from mouse extensor digitorum longus (EDL) and cultured them with or without 300 nM GD433 culture medium for 72 h (Fig. 2A). We used 300 nM as the dosage in the rest of the study as this concentration effectively increases differentiation of C2C12 myoblasts (Fig. 1D). After treatment we fixed the myofibers and associated MuSCs, and stained the cells with PAX7 and MYOD (Fig. 2B). Previous studies have established that PAX7⁺MYOD^–, PAX7⁺MYOD⁺, and PAX7^–MYOD⁺ cells represent self-renewal, proliferating, and differentiating MuSCs, respectively [16]. We found that GD433 significantly reduced the self-renewal (Fig. 2C) but promoted the differentiation of MuSCs (Fig. 2D). In contrast, GD433 had no significant effect on the proliferation of MuSCs on single myofibers (Fig. 2E). Consistently, the total number of cells per cluster was similar among the three treatment groups (Fig. 2F). These results indicate that GD433 promotes differentiation of MuSCs at the expense of self-renewal.

Fig. 2.

GD433 promotes differentiation but inhibits self-renewal of myofiber-associated satellite cells (MuSCs). A Diagram showing myofiber isolation from extensor digitorum longus (EDL) muscles and subsequent treatment with DMSO control, GD433N (300 nM), and GD433 (300 nM) in myofiber culture media for 72 h. B Representative immunofluorescence staining with PAX7 (red), MYOD (Green), and DAPI (blue) on clusters of MuSCs-derived progenies. Scale bar, 50 μm. C–F Quantitative results showing percentages of cells that are differentiating (PAX7–/MYOD+, C), self-renewing (PAX7+/MYOD–,D), and proliferating (PAX7+/MYOD+, E), and average numbers of cells per cluster (F). N=5. P-value was determined using unpaired two-tailed t-tests; bars without p-values indicate p > 0.05

GD433 promotes differentiation of MuSCs-derived primary myoblasts

To further validate the role of NTMT1 in myogenesis, we treated primary myoblasts fresh-isolated from limb muscles of mice with GD433 or control compounds in growth media (GM) for 48 h (Fig. 3A). Cells were then stained with PAX7 and MYOD (Fig. 3B). The results revealed that GD433 significantly increased the percentage of PAX7^–MYOD⁺ differentiating cells, but reducing both PAX7⁺MYOD^– and PAX7⁺MYOD⁺ cells (Fig. 3C). Quantitative qPCR analysis showed that mRNA levels of Myod1 was increased significantly by GD433, without significant effects on Pax7 (Fig. 3D). These data suggest that GD433 promote myogenic differentiation through upregulating MYOD in primary myoblasts.

Fig. 3.

GD433 induces premature differentiation of primary myoblasts under growth conditions. A Experimental design showing treatment with 300nM GD433/ GD433N for 48h. B Immunofluorescence staining with PAX7 (red), MYOD (green), and DAPI (blue). Scale bar, 50 μm. C Quantitative results showing percentages of cells that are self-renewing (PAX7+/MYOD–), differentiating (PAX7–/MYOD+), and proliferating (PAX7+/MYOD+). N=5. D Relative expression of Pax7 and MyoD genes determined by qPCR. N=3. P-value was determined using unpaired two-tailed t-tests; bars without p-values indicate p > 0.05

We also performed EdU assay to examine the effects of GD433, GD433N, and vehicle control on proliferation of primary myoblasts (Fig. S2A). We found that GD433 had no effects on the percentage of EdU⁺ cells (Fig. S2B). Additionally, the mRNA levels of cell cycle-related genes (Cdk2, Ki-67, p27, and p21) were similar among the three treatment groups (Fig. S2C). These results suggest that GD433 has no effect on the proliferation of primary myoblasts under growth conditions.

We next examined if GD433-induced MYOD expression affect early differentiation of the primary myoblasts by switching the GM to differentiation media (DM), with GD433 present in both media (Fig. 4A). At 24 h after switching to differentiation media, the cells were stained with MYOD and MYOG (Fig. 4B). We observed a significantly higher proportion of MYOG⁺ cells in the GD433-treated group (Fig. 4C). Consistently, the mRNA levels of Myod1 and Myog were dramatically increased (Fig. 4D). These results affirm that GD433 facilitates differentiation of myoblasts through upregulating MYOD and MYOG.

Fig. 4.

NTMT inhibition by GD433 enhances myogenic differentiation and gene expression in primary myoblasts. A Overview of experimental design for continuous treatment of primary myoblasts with 300 nM GD433/GD433N for 48 h during proliferation and 24 h during differentiation. B Immunofluorescence staining with MYOG (red), MYOD (green), and DAPI (blue). Scale bar, 50 μm. C Quantification of % MYOG+ cells in different groups. N = 5. D Relative expression of MyoG and MyoD gene determined by qPCR. N = 3. E Overview of NTMT1 inhibitor (300 nM GD433 or GD433N) treatment after initiation of differentiation for 72 h. F Immunofluorescence of MHC (red), MYOG( green), and DAPI (blue). Scale bar, 50 μm. G Quantification of the percentage of non-fused mononuclear MyoG+ cells, fusion index (fraction of MyoG+ cells in multinucleated myotubes). N = 3. H Relative expression of myogenic differentiation and fusion-related genes. N = 3. P values are based on unpaired two-tailed t-tests, and bar graphs without a labeled p value indicate p > 0.05

To directly examine the effect of GD433 on myoblast differentiation and fusion without the confounding effect of MYOD expression during proliferation, we treated freshly isolated primary myoblasts with GD433 only after they were induced to differentiate (Fig. 4E). At 72 h, MHC⁺ myotubes emerged in all groups. However, the size and density of myotubes were substantially larger and higher in the GD433-treated groups compared to the controls (Fig. 4F). Quantification indicated that the non-fused MYOG⁺ cells were lowest in the GD433 group (Fig. 4G), and the fusion index was highest in the GD433 group (Fig. 4G). Additionally, mRNA levels of fusogens Mymk and Mymx, as well as Myog, were significantly upregulated by GD433 (Fig. 4H). These data demonstrate that GD433 promotes differentiation and fusion of primary myoblasts.

GD433 ameliorates pathology and improves muscle regeneration in Mdx mice

To investigate the in vivo effects of GD433 on myogenesis, we utilized mdx mice, a model characterized by chronic muscle degeneration. Adult mdx mice were intraperitoneally injected with GD433 (25 mg/kg) daily for 5 days (Fig. S3A).We examined embryonic myosin heavy chain (eMHC) expression and observed the emergence of many small eMHC⁺ myofibers in response to GD433 treatment (Fig. S3B). We next evaluated MYOG, a marker of differentiated myoblasts, and found that GD433 treatment significantly increased the proportion of MyoG⁺ cells in mdx muscle sections (Fig. S3C-D). Collectively, these results demonstrate that GD433 promotes myogenic differentiation of DMD MuSCs in vivo.

To further investigate the effect of GD433 on muscle regeneration of mdx mice, we first induced muscle injury and then treated mice with GD433 via 5 daily intraperitoneal (I. P.) injections (Fig. 5A). H&E staining of injured TA muscle revealed markedly improved morphology in the GD433-treated group compared to control groups, characterized by a striking reduction in mononuclear cell infiltration (Fig. 5B). Quantitative analysis of fiber cross-sectional area (CSA) distribution revealed that GD433-treated muscles contained a higher proportion of large fibers, indicative of more successful regeneration (Fig. 5C). Immunofluorescence staining of MyoG (Fig. 5D) results indicated that more MyoG⁺ cells were observed in injured muscle tissue from the GD433 treatment group (Fig. 5E). This was linked to a trend of reduced Pax7⁺ cells (Fig. S3E-F). We also stained the newly regenerated myofiber with eMHC (Fig. 5F). The results showed that GD433 significantly increased the abundance of eMHC⁺ myofibers (Fig. 5G). In addition, F4/80 immunostaining was performed to label macrophages as indication of inflammation. The results revealed markedly reduced macrophage infiltration in the GD433-treated muscles compared with control and GD433N groups (Fig. 5H–I). These results demonstrate that GD433 not only promotes myogenic differentiation and myofiber regeneration but also mitigates inflammation in dystrophic muscles.

Fig. 5.

GD433-mediated NTMT inhibition ameliorates pathology and improves muscle repair in mdx mice. A Experimental design to examine the effects of GD433 (25 mg/kg/day, for 5 days) in muscle regeneration following an acute injury. B Representative H&E staining of TA muscles from control (vehicle), GD433N-, and GD433-treated mdx mice showing improved fiber morphology in the GD433 group. Scale bar, 100 µm. C Cross-sectional area (CSA) distribution of regenerated myofibers in control, GD433N-, and GD433-treated mice, showing a rightward shift (larger fibers) with GD433 treatment. D Representative immunofluorescence staining of MYOG (red), LAMININ (green), and DAPI (blue) in TA muscle at 5 dpi from control (no treatment), GD433N, and GD433 mdx mice. Scale bar, 100 μm. E Quantification of MYOG-positive cells in TA muscle at 5 dpi from control (no treatment), GD433N, and GD433 mdx mice. N=6. Scale bar, 100 μm. F Representative immunofluorescence staining of embryonic myosin heavy chain (eMHC, green) in TA muscle at 5 dpi from control (no treatment), GD433N, and GD433 mdx mice. Scale bar, 100 μm. G Quantification of the proportion of eMHC-positive area in TA muscle at 5 dpi from control (no treatment), GD433N, and GD433 mdx mice, N=4. H Representative immunofluorescence staining of macrophage marker F4/80 (green), LAMININ (red), and DAPI (blue) in TA muscles at 7 dpi. Scale bar, 100 µm. I Quantification of F4/80 fluorescence intensity per area (N = 4), showing reduced inflammatory response after GD433 treatment. P-value was determined using unpaired two-tailed t-tests; bars without p-values indicate p > 0.05

To directly examine the effect of GD433 on dystrophin-deficient MuSCs, we isolated primary myoblasts from DMD mice and treated them with GD433 or control media. We observed a significantly higher proportion of PAX7^–MYOD⁺ cells in the GD433-treated group under the growth condition (Fig. S4A-B). Furthermore, the proportion of the MYOG⁺ cells was dramatically increased by the GD433 treatment group in differentiation media at 24 h (Fig. S4C-D). GD433 further increased the differentiation and fusion of DMD myoblasts at 72 h after induced differentiation (Fig. S4E-F). These results provide compelling evidence that GD433 enhances differentiation and fusion of dystrophic MuSCs in mdx mice.

NTMT1 mediates methylation of KLHL31 to regulate myogenesis

To understand how GD433-mediated inhibition of NTMT1 regulates myogenesis, we screened NTMT1 substrates that we had previously identified by mass spectrometry [17]. KLHL31 caught our attention as it is highly expressed in skeletal muscles [18], and its loss-of-function leads to congenital myopathy [19]. Analysis of single-cell RNA-seq data [20] revealed that while Ntmt1 is expressed throughout myogenesis (Fig. 6A), Klhl31 expression markedly increases during differentiation (Fig. 6B), in parallel with MyoG (Fig. 6C), suggesting a role for KLHL31 in this process. To further assess KLHL31 in the dystrophic context, we analyzed the publicly available GSE213925 dataset and found that Klhl31⁺ MuSC and Ntmt1⁺ MuSCs were increased in mdx-D2 MuSCs rather than myonuclei (Fig. S5A, B). More importantly, single-cell RNA-seq analysis showed that Klhl31, Myod1, and Ntmt1 genes are significantly elevated in mdx-D2 MuSCs compared with WT (Fig. 6D). Consistent with this transcriptional difference, Western blot analysis of primary MuSCs revealed significantly increased KLHL31 methylation in mdx MuSCs compared with WT (Fig. 6E), indicating heightened NTMT1 activity in dystrophic MuSCs. Furthermore, this elevation in methylated KLHL31 persisted and was more pronounced after 72 h of differentiation (Fig. 6F), reinforcing the sustained dysregulation of KLHL31 methylation during myogenesis in the dystrophic background.

Fig. 6.

NTMT inhibitor GD433 demethylates KLHL31 and promotes its binding with MYOD. A–C Violin plot showing relative expression of Ntmt1 (A), Klhl31 (B), and Myog (C) genes in quiescent satellite cells (QSC), activated satellite cells (ASC), and differentiating satellite cells (DSC). Based on data from GSE150366. (D) Single-cell RNA-seq violin plots showing the log-normalized expression of Klhl31, Myod1, Myog, and Ntmt1 in MuSCs and myonuclei from WT and mdx-D2 mice. E Western blot analysis of MET3–KLHL31 and GAPDH in isolated WT and mdx MuSCs at 0 hours of induced differentiation (proliferation stage), followed by quantification of relative protein levels. F Western blot analysis of MET3–KLHL31 and GAPDH in WT and mdx MuSCs after 72 hours of induced differentiation, followed by quantification of relative protein levels. G Representative Western blot analysis of KLHL31 and MYOG protein levels in C2C12 myoblasts differentiated for 24 h and 72 h under GD433N or GD433 treatment. GAPDH served as a loading control. H Quantification of KLHL31 and MYOG protein levels from (D), normalized to GAPDH. N=3. P-value was determined using unpaired two-tailed t-tests; bars without p-values indicate p > 0.05. I Representative Western blot showing N-terminal methylation states, including monomethylation (Me1), dimethylation (Me2), and trimethylation (Me3), of proteins at the KLHL31 position in differentiated MuSCs under different treatments. Tubulin served as a loading control. J Co-immunoprecipitation using KLHL31 antibody, followed by Western blotting with KLHL31, MYOD and MYOG in differentiated myoblasts treated with GD433, GD433N, or DMSO (Ctrl). Input and IgG controls are also shown

We next investigated how GD433 affects KLHL31 abundance and methylation during myogenic differentiation (Fig. 6G-J) of KLHL31 in C2C12 myoblasts at 24 h and 72 h during differentiation (Fig. 6G). GD433 treatment did not alter levels of KLHL31 at either time point (Fig. 6G). Consistent with the mRNA expression pattern (Fig. 6B), the protein levels of KLHL31 was higher at 72 h compared to 24 h, regardless of GD433 treatment (Fig. 6H). Consistent with earlier observation that GD433 increases mRNA levels of Myog, the protein level of MYOG was elevated by GD433 (Fig. 6H). In addition, we also observed an increase in the level of MYOG at 72 h compared to 24 h (Fig. 6H). We further examined methylation of KLHL31 using antibodies detecting monomethylated (Me1), dimethylated (Me2) and trimethylation(Me3) proteins at 72 h. We found that levels of Me1, Me2, and Me3 were all reduced by GD433 at the same position corresponding to KLHL31 bands (Fig. 6I). Consistently, KLHL31 immunoprecipitation followed by Me2/Me3 immunoblotting confirmed that KLHL31 methylation was readily detected in the GD433N control but was abolished upon GD433 treatment (Fig. S5C). These results demonstrate that GD433 inhibits methylation of KLHL31 without affecting the total protein level.

We further used co-immunoprecipitation to determine how KLHL31 methylation affects its interaction with MYOD and MYOG. We used the KLHL31 antibody for IP of C2C12 cell lysates at 72 h under control and GD433 treatments, and performed Western blotting to detect MYOD and MYOG. We detected MYOD band only in the presence of GD433 (Fig. 6J). However, we did not detect any MYOG in control or GD433 groups (Fig. 6J), suggesting KLHL31 does not interact directly with MYOG. These results indicate that GD433 mediates the demethylation of KLHL31, promoting the formation of a KLHL31-MyoD transcriptional complex that regulates the expression of Myog.

Discussion

Using a highly selective NTMT1 inhibitor, we demonstrate a critical role of NTMT1 in myogenic differentiation in vitro and in vivo, and show that GD433-mediated NTMT1 inhibition reduces methylation levels of KLHL31, enhances its binding with MYOD to ultimately affect myogenesis. Our in vivo work demonstrating the efficacy of GD433 in promoting muscle differentiation in mdx mice identifies a potential compound for improving muscle regeneration in DMD patients.

A previous knockout (KO) study has shown that Ntmt1 KO mice either die shortly after birth or don’t thrive postnatally [13]. As a muscle-specific KO line is not available, the role of NTMT1 in skeletal myogenesis remains unknown. A cell culture study showed that Ntmt1 knockout C2C12 cells are unable to produce myosin heavy chain (MHC) positive myotubes but instead exhibit an osteogenic phenotype [14], establishing a requirement for NTMT1 in the myogenesis of C2C12 cells. Our data showed that inhibition of NTMT1 by GD433 promoted myogenic differentiation. The discrepancy may be due to the different modes of action. While the KO approach completely ablates the NTMT1 protein, our NTMT1 inhibitor (GD433) only blocks the methyltransferase activity of NTMT1 without affecting its level (Fig. 6G, I). NTMT1 is highly expressed in several cancers, and its defective expression results in mitotic defects [17, 21], indicating that NTMT1 is essential for cell proliferation. However, our data revealed that NTMT1 inhibition did not affect myoblast proliferation but promoted myogenic differentiation. These data collectively suggest that the NTMT1 protein may have additional roles beyond its methyltransferase function, possibly through its non-catalytic domains.

Consistent with its pro-differentiation effect, GD433 treatment in mdx mice led to a right-shift in the fiber cross-sectional area (CSA) distribution, reflecting an increased proportion of larger myofibers (Fig. 5C). This finding suggests that NTMT1 inhibition facilitates not only the initiation of myogenic differentiation but also subsequent myofiber growth and maturation. Moreover, GD433 markedly reduced F4/80⁺ macrophage infiltration in injured muscles (Fig. 5H–I), indicating attenuation of inflammation and restoration of a pro-regenerative environment. These results together imply that GD433 enhances muscle regeneration in dystrophic mice through dual mechanisms—accelerating MuSC differentiation and fusion while alleviating chronic inflammatory responses that otherwise hinder tissue repair.

NTMT1 mediates alpha N-terminal (Nα) methylation at canonical “X-P-K/R” motifs (X stands for any amino acids except for D/E) to modulate substrate structure and function [22]. The Kelch family protein KLHL31 has been identified as a potential substrate of NTMT1 and a key regulator of myogenesis [23]. Klhl31-knockout mice exhibit congenital muscle lesions such as postnatal skeletal muscle growth retardation and central nuclear myopathy [19]. Our results revealed that KLHL31 Nα-methylation, without affecting its total protein level, regulates myogenic differentiation. Comparative transcriptomic and Western blot analyses of normal and dystrophic myoblasts further showed an increase in KLHL31 Nα-methylation in the dystrophic context, which remained significantly elevated even after 72 h of differentiation (Fig. 6D–F). Consistently, mdx MuSCs showed a higher proportion of Ntmt1⁺ cells compared with WT (Fig. S5A). These findings indicate that NTMT1 regulates myogenesis through KLHL31 Nα-methylation, a process that becomes markedly amplified in the dystrophic context and provides mechanistic support for our model. To our knowledge, this is the first study to report the Nα-methylation of KLHL31 in myogenic regulation.

Consistent with NTMT1 substrate specificity, KLHL31 carries an N-terminal APK sequence, and its Nα-methylation has been mapped to the initiator alanine in prior work. In differentiated myoblasts, GD433 reduced the N-terminal methylation signal at the KLHL31 band (Fig. 6I). Moreover, immunoprecipitation of endogenous KLHL31 followed by immunoblotting with Me3- and Me2-specific antibodies showed that KLHL31 methylation was readily detected in the GD433N condition but was abolished upon GD433 treatment (Fig. S5C). GD433 also concomitantly enhanced KLHL31–MYOD co-immunoprecipitation (Fig. 6J). This strengthened association was accompanied by increased expression of MYOD target genes (Myog, Mymk, Mymx), supporting a model in which KLHL31 Nα-methylation restrains KLHL31–MYOD complex assembly and downstream MYOD-driven transcription; pharmacological inhibition of NTMT1 relieves this constraint to promote myogenic differentiation and regeneration.

MyoD is a master transcriptional regulator of myogenic cell lineage specification during development and stem cell differentiation [24–26]. MYOD regulates the expression of myogenesis-specific genes by acting as a transcriptional factor that binds to a cis-regulatory element containing a consensus E-box (CANNTG) and as a genome organizer that governs the three-dimensional genomic landscape necessary for myogenesis [27, 28]. Consistent with our model, we observed enhanced MYOD–KLHL31 complex assembly in myoblasts when KLHL31 Nα-methylation was reduced. This interaction is consistent with the increased expression of MYOD-target genes, including Myog. Our data further suggest that the MYOD–KLHL31 complex may contribute to induction of Mymk and Mymx, thereby promoting myoblast fusion. Myomaker (MYMK) and Myomixer (MYMX) are muscle-specific membrane proteins that directly regulate myofusion [29]. More importantly, MyoD is critical for initiating myogenic fusion and is both required and sufficient for Mymx and Mymk expression [30]. These results suggest that NTMT1 regulates the interaction between KLHL31 and MyoD through Nα-methylation, which may be crucial for the differentiation and fusion of skeletal muscle during myogenesis.

We used the new D2-mdx mouse strain as an injury model. D2-mdx skeletal muscles exhibit more prominent muscle weakness and atrophy, more rapid fibrosis and calcification of the diaphragm, and progressive accumulation of muscle fibrosis in the limbs compared to the C57BL/10-Dmd^mdx/J (B10-mdx) mouse model [31]. The GD433 treatment stimulated the formation of nascent myofibers within uninjured muscles of D2-mdx mice. The GD433 treatment also enhanced myofiber formation after acute injury in the mdx mice. Our data opens a new avenue towards therapeutic treatment of human DMD using a small molecular inhibitor.

Conclusion

Our data demonstrate that pharmacological inhibition of NTMT1 by GD433 enhances myogenic differentiation of progenitor cells and myofiber regeneration in mdx mice. This promyogenic effect is mediated by demethylation of KLHL31, which enhances KLHK31- MYOD interaction to upregulate Myog and myogenic fusion factors. These findings highlight the potential of NTMT1 inhibition as a therapeutic approach to enhance muscle regeneration in DMD.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

BCA

Bicinchoninic acid

C2C12

Mouse myoblast cell line C2C12

DMD

Duchenne muscular dystrophy

DM

Differentiation medium

DMEM

Dulbecco’s modified Eagle medium

eMHC

Embryonic myosin heavy chain

EdU

5-ethynyl-2′-deoxyuridine

GM

Growth medium

H&E

Hematoxylin and eosin

MHC

Myosin heavy chain

MuSCs

Muscle stem cells (satellite cells)

MYOD

Myogenic differentiation 1

MYOG

Myogenin

NTMT1

N-terminal methyltransferase 1

OCT

Optimum cutting temperature compound

PAX7

Paired box protein Pax-7

PBS

Phosphate-buffered saline

PFA

Paraformaldehyde

qPCR

Quantitative polymerase chain reaction

RIPA

Radioimmunoprecipitation assay

SDS-PAGE

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

TA

Tibialis anterior

Author contributions

Shihuan Kuang and Rong Huang: Conceptualization, supervision, project administration, and funding acquisition. Haoyuan Zhang, Min He, and Muhammad Asif: Methodology, Validation, and investigation (cell models). Youchao Deng: prepare and characterize GD433 and GD433N. Haoyuan Zhang and Muhammad Asif: Data curation and formal analysis (in vitro). Haoyuan Zhang: Investigation (animal models), Sample collection, formal analysis (in vivo), Visualization, and writing-original draft. All authors contributed to writing-review & editing and approved the final version.

Funding

The research was supported by NIH-R01AR078695, R01AR079235, R01DK132819 (to S. K.), and R01GM117275 (to R.H.).

Data availability

Single cell RNA-seq data have been deposited to GEO under accession number GSE150366.

Declarations

Ethics approval and consent to paticipate

All procedures involving animals were reviewed and approved by the Purdue University Institutional Animal Care and Use Committee (IACUC; protocol no. 1112000440 approved on 01/24/2023). Animals were housed and cared for in accordance with the Guide for the Care and Use of Laboratory Animals (NRC,2011). All methods were carried out in accordance with relevant institutional and federal regulations.

Consent for publication

The authors declare that they consent to publication.

Competing interests

The authors declare no competing interests.