Pbk positively regulates myoblast differentiation and muscle regeneration via enhancing AMPK/ULK1 mediated myogenic autophagy

Abstract

Background

The activity of normal myoblasts is essential for the regeneration of skeletal muscle following injury. Nevertheless, the intrinsic mechanisms governing myoblast functions and muscle regeneration remain inadequately elucidated. PDZ binding kinase (Pbk) is a serine–threonine kinase that plays critical roles in various cellular functions and pathologies. However, its role in skeletal muscle remains largely unexplored. In this study, we have identified Pbk as a novel positive regulator of myoblast functions in vitro and muscle regeneration in vivo.

Methods

Herein, We analyzed the effects of Pbk on myoblast function and muscle regeneration through in vitro and in vivo experiments. In vivo, we analyzed the effects of Pbk on skeletal muscle regeneration through bioinformatic analysis combined with a mouse skeletal muscle injury model. In vitro, we analyzed the effects of Pbk knockdown or overexpression on myoblast proliferation, survival, and differentiation through lentivirus-mediated cell infection. Also, we further studied the molecular mechanisms by which Pbk affects myoblast differentiation and muscle regeneration combined with the molecular biology and biochemistry, and drug rescue approaches.

Results

In vitro experiments demonstrated that knockdown of Pbk results in impaired cell proliferation and accelerated apoptosis of myoblasts. Unlike proliferating myoblasts, Pbk is upregulated and restricted from translocating from the cytoplasm to the nucleus during myoblast differentiation. Notably, the positive effect of Pbk on myoblast differentiation and fusion is contingent upon its kinase activity. Mechanistic investigations revealed that Pbk facilitates myogenic autophagy by enhancing AMPK-mediated phosphorylation of ULK1, which ultimately contributes to myogenic differentiation and fusion. In vivo, we observed that Pbk is upregulated in embryonic myosin heavy chain (eMyHC) positive regenerative myofibers in muscle specimens from patients with Duchenne muscular dystrophy (DMD) and immune-mediated necrotizing myopathy (IMNM). Furthermore, in a murine model of skeletal muscle injury, Pbk knockdown hinders the regeneration of myofibers.

Conclusions

Collectively, our findings suggest that Pbk plays a positive regulatory role in myoblast differentiation and muscle regeneration by modulating AMPK/ULK1-mediated autophagy signaling. This pathway may represent a novel target for the manipulation of myoblast functions and the development of myoblast-based therapies for skeletal muscle injuries.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12967-025-07173-z.

Background

Skeletal muscle possesses the capacity to recover following injury due to the presence of muscle stem cells known as satellite cells [1, 2]. During the process of skeletal muscle regeneration, satellite cells become activated, proliferate, and differentiate into myoblasts, which subsequently develop into myocytes and myotubes, ultimately fusing to form new myofibers [1, 2]. Previous research has highlighted the involvement of various genes and signaling pathways in myoblast differentiation and muscle regeneration. For instance, insulin-like growth factor (IGF-1), sonic hedgehog (Shh), and vascular endothelial growth factor (VEGF) have been shown to enhance muscle regeneration capacity and have been utilized in the treatment of muscle injuries [3–5]. Regarding signaling pathways, p38 MAPK signaling has been linked to the activation of satellite cells [6]. JNK/MAPK signaling inhibits, whereas Wnt signaling promotes myoblast differentiation [7, 8]. Additionally, JAK1/STAT1 signaling facilitates myoblast proliferation while inhibiting premature differentiation [9].

Autophagy is a critical cellular mechanism that facilitates the degradation of proteins and organelles to maintain cellular homeostasis. Under normal physiological conditions, autophagy functions at basal levels, protecting cells from the accumulation of misfolded proteins and damaged organelles, thereby mitigating the risk of certain diseases [10, 11]. However, during periods of starvation and nutrient deprivation, autophagy is upregulated to initiate the process of autophagic degradation, wherein cellular components are broken down and recycled to supply essential nutrients and energy for cellular activities [12]. In the context of muscle regeneration, autophagy plays a pivotal role in remodeling cellular structures to facilitate myoblast differentiation [13]. Despite its importance, the precise mechanisms governing myogenic autophagy during myoblast differentiation remain inadequately understood.

AMPK, a sensor of cellular energy status, is integral to the regulation of autophagy. In conditions of nutrient deprivation, AMPK facilitates autophagy by directly phosphorylating and activating the ULK1 kinase complex, a pivotal initiator of autophagic process [14]. The phosphorylation of ULK1 subsequently induces the phosphorylation of Becn1 and the VPS34 complex, which further enhances autophagosome fusion and formation [15]. The autophagosome then merges with the lysosome to form the autophagolysosome, where the encapsulated cargo is lysed and degraded. It is well-documented that the differentiation of myoblasts into myotubes activates AMPK, and the absence of AMPK impedes normal muscle regeneration following injury [16, 17]. Furthermore, skeletal muscle-specific Ulk1 deficiency results in compromised functional muscle regeneration [18]. Although both AMPK and ULK1 are implicated in muscle regeneration post injury, the mechanisms underlying AMPK/ULK1 mediated autophagic process in muscle differentiation and regeneration remains unexplored.

PDZ binding kinase (Pbk), also referred to as T-lymphokine-activated killer cell-originated protein kinase (TOPK), is a serine–threonine kinase belonging to the mitogen-activated protein kinase kinase (MAPKK) family. Pbk is known to activate multiple signaling pathways, including MAPK, Akt, and mTOR, through the phosphorylation of p38, JNK, Erk, and Akt [19]. Functionally, it has been well established that Pbk plays critical roles in various cellular functions and pathologies. Pbk is capable of regulating cell survival, growth, proliferation, and apoptosis. Pbk is aberrantly expressed or activated in various cancers and is essential for neuronal progenitor proliferation, self-renewal, and adult neurogenesis [20, 21]. However, whether Pbk is involved in skeletal muscle differentiation and regeneration remains largely unexplored. In this study, we demonstrate that Pbk positively regulates myoblast differentiation and muscle regeneration by enhancing AMPK/ULK1-mediated myogenic autophagy, indicating its potential as a therapeutic target for skeletal muscle injury.

Materials and methods

Bioinformatic analysis

Microarray datasets GSE45577 [22] and GSE103684 [23] were retrieved from the Gene Expression Omnibus (GEO) database. The dataset GSE45577, comprising data from tibialis anterior (TA) muscles of mice at three days post-cardiotoxin (CTX) induced injury, was compared against control samples. Similarly, dataset GSE103684, containing data from isolated satellite/myogenic progenitor cells at two days post-CTX induced injury, was analyzed against control samples using the official GEO2R tool. Differentially expressed genes (DEGs) were identified based on a fold change threshold of ≥ 2.0 and a p-value of ≤ 0.05. The expression levels of the Pbk gene in both TA muscles and isolated satellite/myogenic progenitor cells were quantified at each post-injury time point relative to the untreated control group. Subsequently, the identified DEGs were subjected to Gene Ontology (GO) analysis using the WEB-based Gene Set Analysis Toolkit (http://www.webgestalt.org) [24].

Reagents, lentivirus and antibodies

HI-TOPK-032 (HY-101550) and LYN-1604 (HY-101923) were sourced from MedChemExpress LLC (Shanghai, China). BaCl₂ (A602020) was bought from Sangon Biotech Co., Ltd (Shanghai, China). The target sequence of small interfering RNA targeting Pbk (siPbk) is as follow: 5′-ACG CCA AAC AAA TCT GAA A-3′. The lentivirus expressing siPbk, mouse Pbk (NCBI, NM_023209.3) fusion with Flag tag (Pbk-Flag) and siPbk together with RNAi-resistant Pbk fusion with Flag (siPbk + rPbk-Flag) were constructed by WZ Biosciences Inc. (Jian, China). The Flag tagged Pbk-resistant construct was generated using site-directed mutagenesis without changing the amino acid sequence and the Pbk siRNA target sequence was mutated to the following: 5′-ACA CCG AAT AAG TCA GAG A-3′. All antibodies used in this study are listed in Supplementary Table S1.

Cell culture and infection

C2C12 cells (CRL-1772, ATCC) were cultured in growth medium which containing 15% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin in high-glucose Dulbecco's Modified Eagle Medium (DMEM). Each cell experiment was independently repeated three times. For the Pbk knockdown and overexpression experiments, cells at the appropriate confluence were transduced with lentivirus carrying siPbk or Pbk-Flag at 1 × 10⁶ IU/ml. Two days later, Pbk silencing and overexpression efficiency were then assessed.

Cell viability assay

The viability of cells was assessed utilizing the CCK-8 kit (C0037, Beyotime, Shanghai, China). C2C12 cells were plated at a density of 2,000 cells per well in a 96-well cell culture plate. At 24, 48, 72, and 96 h post-seeding, 100 μl of medium supplemented with 10 μl of CCK-8 solution was added to each well. Following incubation at 37 °C for 1 h, optical densities at 450 nm were measured using a BioTek Synergy H1 microplate reader.

Cell proliferation and apoptosis assays

Cell proliferation was evaluated using the BeyoClick™ EdU-488 Cell Proliferation Kit (C0071, Beyotime, Shanghai, China). C2C12 cells were incubated with 10 μM EdU for 2 h, subsequently fixed, and apoptosis was assessed via TUNEL staining using the One Step TUNEL Apoptosis Assay Kit (C1088, Beyotime, Shanghai, China). Images were captured using an Olympus inverted fluorescence microscope. At least five random, non-overlapping fields per section were captured at 200 × magnification. EdU or TUNEL-positive cells were identified by bright green fluorescence signal in the nucleus, significantly above background and non-specific signals in control. The percentage of positive cells among DAPI-stained nuclei was calculated by two investigators blinded to the groups.

Induction of myoblast differentiation and quantification analysis

To induce differentiation of C2C12 myoblasts, the growth medium was replaced with a differentiation medium consisting of high-glucose DMEM, 2% horse serum, 100 U/ml penicillin, and 100 μg/ml streptomycin. To assess the impact of Pbk knockdown and overexpression on differentiation, C2C12 cells infected with lentivirus were exposed to differentiation medium for 3 days. Differentiation was observed using immunofluorescence staining of the myosin heavy chain (MyHC) marker, with images captured via an Olympus inverted fluorescence microscope. MyHC-positive cells were identified by their nuclei surrounded by MyHC signals with typical myofibrillar structures. Quantification involved capturing at least five non-overlapping fields at 200 × magnification. Differentiation was measured using the differentiation index (percentage of nuclei present within MyHC positive cells among the total DAPI positive nuclei) and the fusion index (percentage of nuclei within MyHC positive cells containing more than one nucleus).

Extraction of nuclear and cytoplasmic proteins

The extraction of nuclear and cytoplasmic proteins was conducted utilizing the Nuclear and Cytoplasmic Protein Extraction Kit (P0027, Beyotime, Shanghai, China). Cultured cells were harvested with 0.02% EDTA and subjected to centrifugation at 800 × g for 5 min at 4 °C. After washing with PBS, the cell pellet was mixed with ice-cold extraction reagent A and vortexed for 5 s. Following a 15-min incubation on ice, extraction reagent B was added, and the mixture was centrifuged at 12,000 × g for 5 min at 4 °C. Subsequently, the insoluble fraction was treated with ice-cold nuclear extraction reagent and vortexed every 3 min for 15 s. The resulting supernatants were prepared for Western blot analysis.

RNA extraction and real time quantitative RT-PCR

For RNA extraction and real-time quantitative RT-PCR, total RNA was extracted using the TRNzol Universal Reagent (GDP424) from Tiangen Biotech (Beijing) Co. LTD and reverse transcribed into cDNA with the HiScript II 1st Strand cDNA Synthesis Kit (R211-01, Vazyme, Nanjing, China). Quantitative real-time RT-PCR was performed using the ChamQ Universal SYBR qPCR Master Mix (Q711, Vazyme, Nanjing, China) on an Applied Biosystems QuantStudio 3 (Thermo Fisher, Singapore). PCR reactions were conducted using a two-step PCR amplification process: 95 °C for 15 s and 60 °C for 60 s. Primer sequences were listed in Supplementary Table S2.

Western blot analysis

Western blot analysis was conducted by homogenizing samples in RIPA lysis buffer, followed by subjecting the lysates to SDS-PAGE and transferring them onto a PVDF membrane (IPVH00010, Millipore, Shanghai, China). The membranes were blocked with 5% skimmed milk and subsequently incubated with primary antibodies at room temperature for 1 h or overnight at 4 °C. Thereafter, the membranes were exposed to HRP-conjugated secondary antibodies for 1 h. For membrane stripping, a stripping buffer (P0025, Beyotime, Shanghai, China) was employed. The membranes were then washed and re-incubated with the primary and secondary antibodies as previously described. Protein bands were visualized using the Tanon 5200 chemiluminescence imaging system and quantified using ImageJ software.

Immunofluorescence assay

In the immunofluorescence assay, cultured C2C12 cells were fixed with 4% paraformaldehyde, washed with PBS, and permeabilized using 0.5% Triton X-100. For skeletal muscle samples, sections of seven micrometers were fixed with acetone for 10 min at 4 °C. The sections were subsequently blocked with 10% goat serum and incubated with the specified primary antibodies overnight at 4 °C. The coverslips were subsequently washed with phosphate-buffered saline (PBS) and incubated with secondary antibodies conjugated to Alexa Fluor 488 or 594. Following incubation, the coverslips were washed again and mounted using an antifade mounting medium containing DAPI.

Muscle injury and regeneration in murine models

Male C57BL/6N mice aged 8–10 weeks were anesthetized with 0.3% pentobarbital sodium and injected with 0.9% barium chloride (BaCl₂) into their tibialis anterior (TA) muscles to establish a skeletal muscle injury and regeneration model. The hindlimbs were shaved before injury. To study Pbk expression changes during regeneration, three mice were used per time point post injury. For assessing Pbk knockdown effects, three mice were used for biochemical tests and four for histochemical test at each time point. TA muscles received lentivirus with siPbk or siNC along with 0.9% BaCl₂ as previously described [25]. The left TA was injected with BaCl₂ and lentivirus expressing siPbk, while the right TA received BaCl₂ and siNC lentivirus. A single 1 × 10⁷ IU of virus in 50 μl BaCl₂ was administered into the TA muscles using insulin syringes. The needle was inserted at a 45-degree angle above the TA tendon with the bevel upward, then advanced intramuscularly to the proximal TA muscle. The solution was injected while withdrawing the needle to ensure even distribution. TA muscles were subsequently harvested and frozen at 0, 3, 5, 7, 14, and 30 days post-injection. This study was approved by the Experimental Animal Ethics Board of Qilu Hospital, affiliated with Shandong University (Approval No. DWLL-2024-137).

Clinical specimens

Clinical muscle specimens were collected from patients with Duchenne muscular dystrophy (DMD) and immune-mediated necrotizing myopathy (IMNM), alongside age- and sex-matched healthy controls. The study involved six patients for each myopathy and six healthy controls, with specimens taken from the same site. Patients had no medication or electrophysiological tests before biopsy. Muscle samples were collected by surgeons and are rapidly frozen and stored at − 80 °C. Informed consent was obtained from all participants, and the study was approved by the Human Ethics Board of Qilu Hospital, Shandong University (KYLL-202011-135).

Masson’s trichrome staining

Masson's trichrome staining, using a kit (G1346, Solarbio, Beijing, China), was conducted to assess collagen content. Post-staining, slides were dehydrated, treated with xylene, and mounted with neutral balsam. Images were captured using an Olympus microscope with at least five random fields per section. Fibrosis was quantified using ImageJ software by calculating the percentage of collagen, identified by a color threshold, as the ratio of the 'blue' area to the total image area.

Immunohistochemical assay

Muscle tissue sections were initially fixed and subsequently blocked using 10% goat serum for a duration of one hour at ambient temperature. The sections were then incubated overnight at 4 °C with primary antibodies, specifically rabbit anti-Pbk or mouse anti-eMyHC. On the following day, post-washing with PBS, the sections were incubated for one hour with a polymer helper and HRP-conjugated goat anti-mouse and rabbit IgG polymer (PV-9000, ZSGB-BIO, Beijing, China). Visualization of the target proteins was achieved using the diaminobenzidine (DAB) substrate kit (ZLI-9018, ZSGB-BIO, Beijing, China). Finally, the slides were counterstained with hematoxylin and mounted using neutral balsam.

Statistics

Statistical evaluations were performed using IBM SPSS Statistics, version 26.0, Normal distributions were assessed with the Kolmogorov–Smirnov test, and comparisons were made using one-way ANOVA with LSD post hoc tests or unpaired Student’s t-tests. Results from cell experiments represent averages from multiple repeats, with “n” indicating biological replicates or individual counts. A p-value below 0.05 was deemed statistically significant.

Results

Pbk is upregulated after skeletal muscle injury

To identify novel genes involved in muscle regeneration, we analyzed the microarray expression profile from GSE45577 [22] and GSE103684 [23] to screen for differentially expressed genes (DEGs) in skeletal muscles after cardiotoxin (CTX) induced injury (Fig. 1A). From GSE45577 and GSE103684, 4310 and 1867 DEGs were identified respectively. Among these, 416 genes overlapped, comprising 307 upregulated and 109 downregulated genes (Fig. 1B). Gene Ontology (GO) enrichment analysis for biological processes identified 59 genes involved in protein phosphorylation among the 416 DEGs (Fig. 1C). Pbk was one of the most significantly upregulated protein kinase involved in modulating protein phosphorylation. As shown by the microarray data, the expression level of Pbk was obviously upregulated at day 3 after muscle injury and gradually returned to the level of the control during muscle regeneration (Fig. 1D, E). Notably, Pbk was also significantly upregulated in satellite / myogenic progenitor cells isolated from the injured muscles (Fig. 1F). Together, these data indicate the involvement of Pbk in muscle regeneration in response to muscle injury.

Fig. 1.

Pbk is upregulated after skeletal muscle injury. A Volcano plots of differentially expressed genes in TA muscles of mice on day 3 post injury versus control in GSE45577 and in isolated satellite/myogenic progenitor cells on day 2 post injury versus control in GSE103684. B Venn diagrams of the common differentially expressed genes in GSE45577 and GSE103684. C Gene ontology (GO) enrichment on biological process of common differentially expressed genes in GSE45577 and GSE103684. D–F Relative mRNA levels of Pbk in TA muscles and isolated satellite / myogenic progenitor cells of mice at indicated time points after injury in GSE45577 (n = 4–6 per group) and GSE103684 (n = 3 per group). Values are shown as mean ± SEM, **p < 0.01, ***p < 0.001, versus the D0 group. G, H Expression and quantification of the protein levels of Pbk in skeletal muscle specimens of patients with IMNM and DMD, n = 6 per group. All of the values are shown as mean ± SEM, *p < 0.05, ***p < 0.001, versus the healthy control group. I Representative images of H&E and immunohistochemical staining of Pbk and eMyHC in muscle sections of patients with DMD, IMNM and healthy controls. Scale bar = 100 μm

Subsequently, to verify the expression of Pbk during muscle regeneration, we conducted a detailed investigation of Pbk expression in skeletal muscle specimens from patients with Duchenne muscular dystrophy (DMD) and immune-mediated necrotizing myopathy (IMNM), which are characterized by significant myocyte regeneration [26, 27]. Western blot analysis was employed to assess Pbk protein levels in patient muscle samples, revealing a marked upregulation of Pbk in the muscles of DMD and IMNM patients compared to healthy controls (Fig. 1G, H). Furthermore, immunohistochemical staining was performed to directly observe the expression patterns of Pbk in the skeletal muscles of DMD and IMNM patients. Pbk was predominantly expressed in newly formed eMyHC-positive myofibers, which exhibited centrally located nuclei (Fig. 1I), thereby confirming its involvement in muscle regeneration. Collectively, these findings suggest that Pbk plays a role in the process of skeletal muscle regeneration.

Pbk knockdown impairs muscle regeneration in skeletal muscles in mice

To elucidate the role of Pbk in skeletal muscle regeneration in vivo, we applied a skeletal muscle injury model by administering BaCl₂ into the tibialis anterior (TA) muscles of mice [25]. Firstly, we constructed a lentivirus expressing siPbk, which could effectively reduce Pbk mRNA and protein levels in affected C2C12 cells in vitro (Supplementary Fig. S1A-C). Next, the TA muscles were injected with 0.9% BaCl₂, either in conjunction with or without lentivirus expressing siPbk or a negative control (siNC). Muscle tissues were harvested and rapidly frozen on days 0, 3, 5, 7, 14, and 30 post-injury (Fig. 2A). Western blot analysis indicated a significant upregulation of Pbk in TA muscle on days 3 and 7 following BaCl₂ injection, with expression levels returning to baseline by day 14 (Fig. 2B, C), corroborating the microarray expression profile data (Fig. 1D, E). Conversely, concurrent injection of lentivirus expressing siPbk and BaCl₂ into TA muscles resulted in a significant downregulation of Pbk expression on days 3, 7, 14, and 30 post-injury (Fig. 2D, E). Immunofluorescent assays demonstrated a marked reduction in Pax7-positive satellite cells on day 3 post-injury following Pbk knockdown (Fig. 2F, G). Furthermore, the number of newly formed eMyHC-positive regenerating myofibers in TA muscles expressing siPbk was substantially reduced on day 5 post-injury (Fig. 2H, I). Histological techniques were subsequently employed to investigate the effects of Pbk on myofiber morphology during muscle regeneration. The knockdown of Pbk resulted in impaired muscle regeneration in mice compared to the siNC group, as evidenced by increased fibrosis and a reduced cross-sectional area (CSA) of newly formed myofibers at 7 and 14 days post-injury (Fig. 2J–M). Notably, by day 30 post-injury, the muscles injected with lentivirus expressing siPbk displayed a myofiber CSA comparable to that of the siNC group (Fig. 2L, M). These findings indicate that Pbk deficiency hinders myogenic proliferation and differentiation, thereby impairing muscle regeneration.

Fig. 2.

Knockdown of Pbk impairs skeletal muscle regeneration in mice. A The scheme of BaCl₂ and lentivirus injection induced muscle injury and regeneration. B, C Detection and quantification of the protein levels of Pbk in TA muscles at day 0, 3, 7, 14 and 30 post injury, n = 3 per group. Values are shown as mean ± SEM, ***p < 0.001,versus the D0 group. D, E Detection and quantification of the protein levels of Pbk in TA muscles injected with lentivirus expressing siPbk or siNC at day 3, 7 and 14 post injury, n = 3 per group. Values are shown as mean ± SEM, *p < 0.05, **p < 0.01, versus the siNC group. F, G Immunostaining for Pax7 together with laminin was performed on the TA muscles at day 3 post injury. The number of Pax7⁺ cells per area was quantified. Scale bar = 100 μm, n = 4 per group. Values are shown as mean ± SEM, *p < 0.05, versus the siNC group. H, I Representative immunofluorescent images of TA muscle sections labeled with eMyHC (red) and laminin (green) at day 5 post BaCl₂ injection. The percentage of eMyHC⁺ myofibers within the laminin⁺ myofibers was quantified, n = 4 per group. Scale bar = 100 μm. Values are shown as mean ± SEM, *p < 0.05, versus the siNC group. J, K Masson’s trichrome staining of the TA muscles to visualize the degree of fibrosis at day 3, 7 and 14 post injury. Scale bar = 100 μm, n = 4 per group. Values are shown as mean ± SEM, *p < 0.05, ***p < 0.001, versus the siNC group. L H&E staining on siPbk and siNC treated TA muscle sections at day 0, 3, 7, 14 and 30 post injury. Scale bar = 50 μm. M Quantification of the CSA of myofibers in siPbk and siNC treated TA muscle sections at day 7, 14 and 30 post injury, n = 4 per group. Values are shown as mean ± SEM, *p < 0.05, **p < 0.01, versus the siNC group

Pbk is required for myoblast proliferation and survival in vitro

To further elucidate the role of Pbk in myoblast functions, lentivirus expressing either siPbk or Pbk-Flag was transduced into C2C12 cells to downregulate or upregulate Pbk expression, respectively. In contrary to lentivirus expressing siPbk (Supplementary Fig. S1A-C), lentivirus expressing Pbk-Flag could significantly increase Pbk protein levels in C2C12 myoblasts (Supplementary Fig. S1D-G). The CCK8 assay indicated that downregulation of Pbk led to a reduction in cell viability (Fig. 3A), whereas overexpression of Pbk enhanced cell viability over time (Fig. 3B). Reduced Pbk expression significantly decreased cell proliferation, as evidenced by a lower number of EdU-positive cells (Fig. 3C, D), while Pbk overexpression increased proliferation, as shown by more EdU-positive cells (Fig. 3E, F). Additionally, lower Pbk expression in C2C12 myoblasts increased TUNEL-positive cells, whereas higher Pbk expression significantly decreased them (Fig. 3G–J). Collectively, these findings demonstrate that Pbk is essential for the proliferation and survival of C2C12 myoblasts.

Fig. 3.

Pbk is required for myoblast proliferation and survival in vitro. A, B CCK-8 assays were performed to determine cell viability of C2C12 myoblasts in which Pbk was knockdown or overexpressed, n = 3 per group. C, D Representative immunofluorescent images of C2C12 cells transduced with siPbk or siNC labeled with EdU (green) and DAPI (blue) for nucleus and quantification of the percentage of EdU⁺ cells, n = 3 per group. E, F Representative immunofluorescent images of C2C12 cells transduced with Pbk-Flag or control labeled with EdU (green) and DAPI (blue) for nucleus and quantification of the percentage of EdU⁺ cells, n = 3 per group. G, H Representative immunofluorescent images of C2C12 cells transduced with siPbk or siNC labeled with TUNEL (green) and DAPI (blue) for nucleus and quantification of the percentage of TUNEL⁺ cells,, n = 3 per group. I, J Representative immunofluorescent images of C2C12 cells infected with Pbk-Flag or control labeled with TUNEL (green) and DAPI (blue) for nucleus and quantification of the percentage of TUNEL⁺ cells,, n = 3 per group. All of the values are shown as mean ± SEM, *p < 0.05, **p < 0.01, versus the siNC or Ctrl group. Scale bar = 50 μm

Pbk is upregulated and restricted from translocating from cytoplasm to nucleus during myoblast differentiation

To explore the link between Pbk and myoblast differentiation, we initially assessed Pbk expression in whole cell lysates throughout the C2C12 myoblast differentiation timeline. qPCR and Western blot analyses demonstrated a marked reduction in Pbk mRNA and protein levels during the initial two days of differentiation, followed by a gradual increase throughout the subsequent differentiation process (Fig. 4A–C). Subsequently, we examined the subcellular localization of Pbk in both proliferating and differentiated C2C12 cells. Immunofluorescence assays indicated that Pbk predominantly localized to the nucleus in proliferating C2C12 myoblasts, whereas it was primarily found in the cytoplasm post-differentiation (Fig. 4D). Subcellular fractionation assays revealed a significant decrease in cytosolic Pbk protein levels by day 2 following the initiation of differentiation, with a gradual upregulation returning to near-normal levels by day 6 of differentiation (Fig. 4E, F). In contrast, nuclear Pbk protein levels were immediately downregulated at the onset of differentiation and continued to decrease throughout the differentiation process (Fig. 4G, H). Collectively, these findings suggest that Pbk is upregulated and restricted from translocating from cytoplasm to nucleus during myoblast differentiation.

Fig. 4.

Pbk is upregulated and restricted from translocating from cytoplasm to nucleus during myoblast differentiation. A Relative Pbk mRNA levels during C2C12 myoblast differentiation, n = 3 per group. B, C Detection and quantification of Pbk protein levels during myoblast differentiation, n = 3 per group. D Immunofluorescent staining was performed to determine the subcellular localization of Pbk in proliferating C2C12 cells and differentiated myotubes on differentiating day 5. Scale bar = 50 μm. E–H Western blot was performed to determine the subcellular fractionation protein levels of Pbk in cytoplasm and nuclei during myogenic differentiation, n = 3 per group. All of the values are shown as mean ± SEM, ***p < 0.001, versus the DO group. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, versus the D2 group

Pbk positively regulates myoblast differentiation depending on its kinase activity

To assess the impact of Pbk on the differentiation of C2C12 myoblasts, cells transduced with either siPbk or Pbk-Flag were subjected to a differentiation protocol for a duration of three days. Knockdown of Pbk resulted in compromised differentiation and fusion of C2C12 myoblasts, as demonstrated by decreased differentiation and fusion indices in comparison to the siNC group (Supplementary Fig. S2A-C). Western blot analysis revealed a marked reduction in the protein levels of myogenin (MyoG) and myosin heavy chain (MyHC) in C2C12 cells undergoing differentiation following Pbk knockdown (Supplementary Fig. S2D-E). Conversely, overexpression of Pbk led to a significant upregulation of MyoG and MyHC expression in differentiating C2C12 myoblasts, thereby promoting enhanced differentiation and fusion, as evidenced by increased differentiation and fusion indices relative to the control group (Supplementary Fig. S2F-J). To further substantiate the role of Pbk in myoblast differentiation, C2C12 cells were transduced with a lentivirus co-expressing siPbk and an RNAi-resistant Pbk fusion protein tagged with Flag (siPbk + rPbk-Flag). The siPbk effectively silenced endogenous Pbk expression, which was subsequently restored by the introduction of rPbk-Flag. Correspondingly, the decrease in MyoG and MyHC expression levels, resulting from the diminished endogenous Pbk in differentiating C2C12 cells, was also reversed by rPbk-Flag (Fig. 5A, B). Immunofluorescence analysis demonstrated that C2C12 cells with Pbk knockdown exhibited significantly reduced differentiation and fusion indices compared to control cells, and this inhibitory effect was ameliorated by co-expression of rPbk-Flag (Fig. 5C, D). These data indicate that Pbk is necessary and sufficient for myoblast differentiation.

Fig. 5.

Pbk is sufficient for myoblast differentiation. C2C12 cells transduced with lentivirus expressing siNC, siPbk or siPbk + rPbk-Flag were induced to differentiation for 3 days. A, B The levels of Pbk, MyoG and MyHC on differentiating day 3 were detected and quantified by immunoblotting analysis, n = 3 per group, values are shown as mean ± SEM. *p < 0.05, ***p < 0.001,versus the siNC group. ^#p < 0.05, ^###p < 0.001,versus the siPbk group. C, D Cells were fixed and immunofluorescence stained for MyHC (green) and DAPI (blue) to detect and quantify the differentiation index and fusion index in differentiating C2C12. n = 3 per group, scale bar = 50 μm

To assess the necessity of the kinase activity of Pbk for myoblast differentiation, C2C12 myoblasts were exposed to the specific kinase inhibitor of Pbk HI-TOPK-032 during differentiation induction [28]. Western blot analysis demonstrated that HI-TOPK-032 significantly inhibited the expression of MyoD, MyoG and MyHC in differentiating C2C12 cells in a dose-dependent manner (Fig. 6A, B). Immunofluorescent assay demonstrated that inhibiting the kinase activity of Pbk impaired myoblast differentiation and fusion as evidenced by reduced differentiation and fusion indices compared with the vehicle control group (Fig. 6C–E). qPCR results indicated that inhibiting the kinase activity of Pbk during myoblast differentiation significantly decreased the mRNA expression of muscle specific genes (MyoD, MyoG, Myh3, Acta1, Ckm, Mylpf, Tnni2, Mymx and Mymk) associated with myogenic differentiation and fusion following HI-TOPK-032 treatment (Fig. 6F). These data indicate that the kinase activity of Pbk is essential for proper myogenic differentiation and fusion.

Fig. 6.

Pbk promotes myoblast differentiation depending on its kinase activity. A, B C2C12 cells were separately treated with 1 μM, 2 μM, 5 μM HI-TOPK-032 or vehicle control and induced to differentiation for 3 days. Cells were harvested and the protein levels of MyoD, MyoG and MyHC were assessed and quantified by western blot, n = 3 per group (C–E) C2C12 cells were treated with HI-TOPK-032 (2 μM) or vehicle and induced to differentiation for 3 days. After that, cells were fixed and stained for MyHC (green) and DAPI (blue) to detect and quantify the differentiation index and fusion index of differentiating C2C12 cells, n = 3 per group. Scale bar = 50 μm. F qPCR analysis of the muscle specific genes in differentiating C2C12 cells after HI-TOPK-032 (2 μM) treatment for 3 days, n = 3 per group. All of the values are shown as mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, versus the vehicle control group

Pbk promotes myogenic autophagy via enhancing AMPK mediated phosphorylation of ULK1 during myoblast differentiation

Subsequently, our objective was to explore whether Pbk facilitates myoblast differentiation by enhancing the myogenic autophagy. Initially, we verified the upregulation of autophagy during myoblast differentiation, as evidenced by increased LC3 expression and decreased p62 expression (Supplementary Fig. S3A-B). Notably, Pbk knockdown resulted in a significant reduction in the protein levels of LC3, Atg5 and Becn1 (Supplementary Fig. S4A), which are associated with autophagy activation and autophagosome formation. Additionally, Pbk knockdown led to a marked increase in p62 protein levels in differentiating C2C12 cells (Supplementary Fig. S4A), indicating a diminished degradation of autophagic substrates. Conversely, Pbk. overexpression enhanced the expression of LC3, Atg5 and Becn1, while decreasing p62 expression in differentiating C2C12 cells (Supplementary Fig. S4B). These findings imply that Pbk modulates myogenic autophagy during myoblast differentiation. Subsequently, we assessed the necessity of Pbk kinase activity in regulating myogenic autophagy. Inhibition of Pbk kinase activity using HI-TOPK-032 significantly reduced the protein levels of LC3, Atg5 and Becn1(Fig. 7A–C), while increasing p62 expression in differentiating C2C12 cells (Fig. 7D). These data suggest that Pbk regulates myogenic autophagy in differentiating C2C12 cells depending on its kinase activity.

Fig. 7.

Pbk promotes myogenic autophagy via enhancing AMPK mediated phosphorylation of ULK1 during myoblast differentiation. A–D C2C12 cells were treated with HI-TOPK-032 (2 μM) and induced to differentiation for 3 days. Cells were harvested and the protein levels of autophagy related genes (LC3, Atg5, Becn1 and p62) were assessed and quantified by western blot assay, n = 3 per group. E, F C2C12 cells were treated with HI-TOPK-032 (2 μM) and induced to differentiation for 3 days. Cells were harvested and the protein levels of AMPKα1/AMPKα2, phospho- AMPKα1(T183) + AMPKα2(T172), ULK1 and phospho-ULK1(S317) were assessed and quantified by western blot, n = 3 per group. G Immunofluorescent staining and quantification of the integrated density of phospho-ULK1(S317) (red) in differentiating C2C12 cells after treatment with HI-TOPK-032 (2 μM) for 3 days. Scale bar = 20 μm. n = 3 per group. H, I C2C12 cells treated with or without HI-TOPK-032 (2 μM) and LYN-1604 (2 μM) were induced to differentiation for 3 days. After that, cells were harvested and subjected to western blot to detect and quantify the protein levels of MyHC and autophagy related genes, n = 3 per group. J–L C2C12 cells treated with or without HI-TOPK-032 (2 μM) and LYN-1604 (2 μM) were induced to differentiation for 3 days. Then cells were fixed and stained for MyHC (green) and DAPI (blue) to detect and quantify the differentiation index and fusion index of differentiating C2C12 cells. n = 3 per group. Scale bar = 50 μm. All values are shown as mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001,versus the vehicle group. ^##p < 0.01, ^###p < 0.001, versus the HI-TOPK-032 treatment group

In the preceding section, we demonstrated that Pbk facilitates myoblast differentiation by augmenting myogenic autophagy signaling. Our subsequent objective was to elucidate the molecular mechanism through which Pbk modulates autophagy in differentiating C2C12 cells. It has been documented that under conditions of starvation or energy deficiency, AMPK is activated to promote autophagy via direct phosphorylation of ULK1 [29]. Consequently, we sought to determine whether Pbk enhances myogenic autophagy by activating AMPK-induced phosphorylation of ULK1. Initially, we assessed the activity of the AMPK/ULK1 axis during myoblast differentiation. The findings indicated a progressive upregulation of AMPK and ULK1 activity during myoblast differentiation, as evidenced by increased phosphorylation levels of AMPK and ULK1 (Supplementary Fig. S3C-D). Western blot analysis demonstrated that Pbk knockdown led to a significant decrease in the phosphorylation of AMPK and ULK1 compared to the siNC control group. Conversely, the overexpression of Pbk enhanced the phosphorylation of AMPK and ULK1 in differentiating C2C12 cells (Supplementary Fig. S4C-D). Consistently, the inhibition of Pbk kinase activity using HI-TOPK-032 also resulted in a marked reduction in the phosphorylation of AMPK and ULK1 in differentiating C2C12 cells (Fig. 7E, F). Furthermore, immunofluorescent staining demonstrated a notable decrease in ULK1 phosphorylation levels in HI-TOPK-032-treated differentiating C2C12 cells relative to the vehicle-treated group (Fig. 7G). These data indicate that Pbk modulates AMPK activation and subsequent phosphorylation of ULK1 in its kinase activity dependent manner. Importantly, the ULK1 activator LYN-1604 is capable of counteracting the inhibitory effects of HI-TOPK-032 on autophagy activation and myoblast differentiation in C2C12 cells. Treatment with LYN-1604 significantly elevated the expression levels of LC3, Atg5, and MyHC in differentiating C2C12 cells compared to those treated with HI-TOPK-032 (Fig. 7H, I). Furthermore, immunofluorescent assays further confirmed that the impaired differentiation and fusion of C2C12 cells, induced by HI-TOPK-032 or Pbk knockdown, were reversible upon treatment with LYN-1604 (Fig. 7J–L; Supplementary Fig. S5). These findings suggest that Pbk facilitates myogenic autophagy by promoting AMPK activation-induced phosphorylation of ULK1 during the differentiation of myoblasts.

Discussion

In this study, we elucidated the positive role of Pbk in the regulation of myoblast functions and muscle regeneration. In vitro analyses demonstrated that Pbk is essential for myoblast survival, proliferation, and differentiation. During myoblast differentiation, Pbk facilitates myogenic autophagy by enhancing AMPK-induced phosphorylation of ULK1. In vivo, Pbk expression is upregulated in newly formed myofibers in patients with DMD and IMNM, as well as in a mouse model of muscle injury. Notably, Pbk knockdown results in delayed muscle regeneration in mice, underscoring its critical role in skeletal muscle regeneration.

Through analysis of microarray expression profiles from datasets GSE45577 and GSE103684, derived from glycerol or cardiotoxin-induced models of muscle regeneration in mice, we discovered that Pbk expression is upregulated during skeletal muscle regeneration post-injury. Furthermore, clinical validation confirmed that Pbk is expressed in eMyHC-positive regenerative myofibers in muscle specimens from patients with DMD and IMNM. In the mouse model of muscle injury, Pbk expression is upregulated on days 3 and 7 post-injury, coinciding with the phases of myoblast proliferation and differentiation, thereby suggesting its involvement in myogenic progression. Our in vitro studies corroborated that Pbk is expressed in myoblasts and plays a crucial role in promoting cell proliferation while inhibiting apoptosis. Notably, Pbk also participates in the myoblast differentiation process. During myoblast proliferation, Pbk is predominantly localized in the nucleus; however, upon differentiation, its expression rapidly decreases and subsequently increases, with localization shifting primarily to the cytoplasm. Given Pbk's role in enhancing cell proliferation, its rapid decline in expression likely corresponds to cell cycle exit in response to differentiation signals.

To further elucidate Pbk's role in skeletal muscle regeneration, we established a muscle injury model through BaCl₂ injection into the tibialis anterior muscles of mice. Pbk knockdown via lentiviral-mediated delivery resulted in the formation of smaller myofibers with centrally located nuclei, indicating that Pbk is essential for effective muscle regeneration. While Pbk has been implicated in various cellular functions, its role in cell differentiation has been infrequently investigated, with the exception of a study demonstrating a significant decrease in Pbk expression during the terminal differentiation of leukemic cells [30]. To the best of our knowledge, this paper is the first to demonstrate that Pbk positively regulates myoblast functions and muscle regeneration. It is important to note that skeletal muscle is a complex tissue composed not only of muscle satellite cells but also of various other cell types involved in muscle regeneration, such as endothelial cells [31–33], fibroblasts [34, 35], and immune cells [36, 37]. Therefore, the potential influence of Pbk knockdown in other cell types, such as macrophages, on muscle regeneration cannot be dismissed. Previous studies have demonstrated that skeletal muscle regeneration is significantly reliant on immune responses and inflammatory signals [38–40]. Although inflammation is often viewed as harmful, it is essential for tissue recovery following injury. In cases of skeletal muscle injury, macrophages initiate an inflammatory response while simultaneously exerting a trophic effect on muscle stem cells. During the recovery phase, pro-inflammatory macrophages transition into anti-inflammatory cells, thereby suppressing inflammation and facilitating stem cell differentiation, angiogenesis, and matrix remodeling [41]. Notably, Pbk is also involved in the regulation of inflammation. It promotes macrophage polarization towards the anti-inflammatory M2 phenotype by inhibiting HDAC1/HDAC2 activity, thereby exerting neuroprotective effects against cerebral ischemia/reperfusion (I/R) [42]. Consequently, the potential role of Pbk in macrophages concerning skeletal muscle regeneration warrants further experimental investigation.

Regarding the underlying mechanisms, previous studies suggest that Pbk enhances cell proliferation and survival by promoting p38/MAPK activity, histone phosphorylation, and overcoming DNA damage [43, 44]. Utilizing a specific inhibitor of Pbk's kinase activity, HI-TOPK-032, we found that the promotion of myoblast differentiation by Pbk is dependent on its kinase activity. Additionally, we demonstrated that Pbk facilitates myoblast differentiation by augmenting the AMPK/ULK1 signaling pathway, which is crucial for myogenic autophagy. It is well-established that autophagy plays a crucial role in protecting myoblasts against apoptosis and in remodeling cellular organization for myofiber formation during myoblast differentiation and muscle regeneration [45]. The differentiation of myoblasts into myotubes triggers activation of AMPK, and the absence of AMPK impedes normal muscle regeneration following injury [16, 46]. Furthermore, AMPK can promote autophagy through the direct phosphorylation and activation of ULK1 [29]. In this study, Our findings indicate that inhibition of Pbk results in compromised autophagy, accompanied by a marked reduction in the phosphorylation of AMPK and ULK1 in differentiating C2C12 cells. Notably, the impaired autophagy and myoblast differentiation caused by the inhibition of Pbk can be reversed by the selective ULK1 activator LYN-1604. This observation confirms that Pbk enhances myoblast differentiation by modulating ULK1-mediated myogenic autophagy. In summary, we have identified Pbk as a novel upstream regulator of the AMPK/ULK1 signaling pathway, which governs myogenic autophagy during myoblast differentiation (Fig. 8). However, we have only identified the effect of Pbk regulated AMPK/ULK1 signaling on myoblast differentiation, the effects of AMPK/ULK1-mediated autophagy, particularly regulated by Pbk, on myoblast proliferation and apoptosis, need further explored.

Fig. 8.

Schematic diagram illustrating the regulation patterns of Pbk on myoblast functions during muscle regeneration. First, Pbk is required for myoblast survival and proliferation. Second, Pbk is increased and prevented from translocating from cytoplasm to nucleus during myoblast differentiation. Third, the upregulated cytoplasmic Pbk promotes AMPK signaling mediated phosphorylation of ULK1 and induces the myogenic autophagy, which ultimately enhances myoblast differentiation and fusion.

Conclusions

Together, this study presents novel evidence that Pbk is essential for the survival and proliferation of myoblasts. Additionally, Pbk has been shown to positively influence myoblast differentiation and skeletal muscle regeneration by enhancing AMPK/ULK1-mediated autophagy signaling pathways. A deeper understanding of the molecular mechanisms governing muscle recovery post-injury is crucial for developing treatments for myopathies such as IMNM, DMD, and sarcopenia. Our findings indicate that Pbk may serve as a promising gene target for myoblast-based therapeutic strategies aimed at addressing skeletal muscle injuries.

Limitations

This study is subject to several limitations. Firstly, the precise mechanism by which Pbk regulates AMPK/ULK1 phosphorylation remains undetermined. It is yet not clear whether Pbk influences AMPK phosphorylation through direct interaction or by modulating other molecular pathways. Secondly, the study employed lentiviral-mediated Pbk knockdown to assess its effect on muscle regeneration in vivo. Consequently, the potential impact of Pbk knockdown on other cell types, such as macrophages, cannot be excluded. Additionally, extrapolation of findings from mice to humans necessitates caution, as the potential side effects may differ between specie. Therefore, it is crucial to evaluate the suitability of Pbk for modulating myoblast function and its potential application in developing myoblast-based therapies for muscle injury and myopathy in humans.

Abbreviations

PDZ

PDZ binding kinase

TOPK

T-lymphokine-activated killer cell-originated protein kinase

siPbk

SiRNA targeting Pbk

Pbk-Flag

Pbk fusion with flag tag

rPbk-Flag

RNAi-resistant Pbk fusion with flag tag

DMD

Duchenne muscular dystrophy

IMNM

Immune-mediated necrotizing myopathy

MyHC

Myosin heavy chain

eMyHC

Embryonic myosin heavy chain

MyoG

Myogenin

GEO

Gene expression omnibus

GO

Gene ontology

DEGs

Differentially expressed genes

ATG

Autophagy-related protein

LC3

Microtubule-associated protein 1 light chain 3

iNOS

Inducible nitric oxide synthase

NO

Nitric oxide

TA

Tibialis anterior

CTX

Cardiotoxin

Author contributions

WDD and YCZ designed the study. WDD performed experiments, analyzed data and wrote the manuscript. SDD and ZDD performed experiments and analyzed data. DTJ, LFC, and YCZ guided the research and critically revised the manuscript.

Funding

This study received support from the Shandong Provincial Natural Science Foundation of China (No.ZR2023MH322, No.ZR2023LSW020) and the National Natural Science Foundation of China (82371410).

Data availability

The data supporting this study’s findings are included in the paper, the raw data can be obtained from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Human and Experimental Animal Ethics Board of Qilu Hospital Affiliated to Shandong University. All the individual participants have signed informed consent and have agreed with publishing the data and photographs from their specimens.

Consent for publication

This is to confirm that this submitted manuscript has never been published before. Also, it is not currently being considered for publication in any other journal. All the listed authors have carefully reviewed and approved this manuscript for publication.

Competing interests

The authors report no conflicts of interests.