NAMPT Activator P7C3-A20 Protects against Tunicamycin-Induced Cell Injury in C2C12 Murine Myoblast Cells

Abstract

Endoplasmic reticulum (ER) stress is linked to insulin resistance and several muscle diseases, including myopathies. ER defects are also implicated in skeletal muscle dysfunction associated with aging. Physical contact between the ER and mitochondria is essential for the local transport of materials and effective signal transduction between these organelles. Nicotinamide phosphoribosyltransferase (NAMPT) regulates mitochondrial biogenesis and maintains skeletal muscle function and integrity. Our study aims to investigate the effect of the NAMPT activator P7C3-A20 on tunicamycin-induced ER stress using an in-vitro myoblast cell model. We found that tunicamycin treatment decreased the expression of several mitochondrial proteins, such as NAMPT and the mitophagy regulator PINK1 in C2C12 murine myoblast cells. It also reduced the phosphorylation of Drp1, a master regulator of mitochondrial fission, in C2C12 cells treated with tunicamycin. JC-1 imaging revealed that treatment with P7C3-A20 for 6 h at concentrations of 1, 5, and 10 μM increased mitochondrial potential in C2C12 cells. Moreover, P7C3-A20 ameliorated tunicamycin-induced cell death in a concentration-dependent manner. In conclusion, the NAMPT activator P7C3-A20 can mitigate tunicamycin-induced cell damage in C2C12 murine myoblasts. Activation of NAMPT is a potential novel therapeutic approach for muscle diseases associated with ER stress, such as sarcopenia.

INTRODUCTION

Skeletal muscle has a rich endoplasmic reticulum (ER) network, which plays roles in protein homeostasis and calcium regulation. Environmental and genetic factors lead to the accumulation of misfolded and unfolded proteins in the ER in many cell types, activating the unfolded protein response (UPR) for processing these proteins.¹⁾ Skeletal muscle activates the UPR after exercise. Notably, UPR activation is reduced in older adults,²⁾ suggesting that decreased tolerance to ER stress with aging may lead to imbalances between protein synthesis and degradation, resulting in muscle cell dysfunction. ER stress has been implicated in nutritional insults and skeletal muscle diseases, including sarcopenia.³⁾ Sarcopenia is defined as a progressive and generalized loss of skeletal muscle mass and a decline in physical performance due to aging.⁴⁾ Various stresses, including hormonal balance,^5,6) oxidative stress,^7,8) nutritional status,^9–11) reduced physical activity,¹²⁾ and ER stress,³⁾ have been suggested to contribute to sarcopenia, although their individual contributions remain unclear. The defects of muscle regeneration relate to the functional decline in sarcopenia. Muscle satellite cells are activated to proliferate and differentiate into myoblasts, forming new skeletal muscles after injury. UPR also plays a pivotal role in these myogenic differentiation and regeneration,¹³⁾ implying that excessive ER stress may contribute to the deterioration of the myoblast and the pathological condition in sarcopenia.

Under prolonged and unresolved stress, UPR activation can lead to the induction of apoptosis. ER stress-induced apoptosis is mediated by Bak and Bax, two proteins that are localized at both ER and mitochondria and associated with ER calcium release with a concomitant increase of mitochondrial calcium, which triggers the depolarized inner membrane, cytochrome c release, and activation of apoptosis pathway.¹⁴⁾ ER stress can directly induce the activation of caspase 12.¹⁵⁾ The ER also regulates the fission of mitochondria via their interaction.¹⁶⁾Mitochondria are dynamic membrane organelles that undergo repeated cycles of division and fusion, forming a reticular network. Their quality is controlled by factors involved in fusion (e.g., mitofusin, Opa1) and fission (e.g., Fis1, dynamin-related protein 1 [Drp1]).^17–19) Mitochondrial homeostasis plays an essential role in skeletal muscle function. Under oxidative stress, Drp1 mediates mitochondrial fragmentation in myoblasts,²⁰⁾ and mitochondrial fragmentation in skeletal muscles promotes inflammation, muscle atrophy, and reduced physical performance.²¹⁾ Dysfunctional mitochondria become fragmented and are subsequently removed by mitophagy, which is impaired in aged skeletal muscles.²²⁾ Thus, modulation of mitochondrial metabolism and dynamics is gaining attention as a novel therapeutic approach for age-related diseases, including sarcopenia.

Nicotinamide adenine dinucleotide (NAD⁺) is essential for muscle integrity. Three pathways are involved in NAD⁺ synthesis: the de novo pathway starting from tryptophan, the Preiss-Handler pathway using nicotinic acid, and the salvage pathway using nicotinamide (NAM). The rate-limiting step in the salvage pathway is the conversion of NAM to nicotinamide mononucleotide (NMN) by nicotinamide phosphoribosyltransferase (NAMPT),^23,24) which regulates skeletal muscle function. Most cell types express NAMPT intracellularly in the cytoplasm, mitochondria, and nucleus.²⁵⁾ P7C3, an aminopropyl carbazole first identified in 2010, is a low molecular weight compound that enhances hippocampal neurogenesis in rats.²⁶⁾ Its mechanism is thought to increase NAD⁺ levels by directly activating NAMPT.²⁷⁾ P7C3 has been shown to improve skeletal muscle function in diabetic models.²⁸⁾ A derivative of P7C3, P7C3-A20, exhibits enhanced activity and has been reported to improve neurological function in several models.^29–31) These findings suggest that P7C3-A20 might improve myoblast function under stress conditions.

In this study, we investigated the expression levels of mitochondrial proteins under ER stress induced by tunicamycin in C2C12 cells. We then evaluated the effect of P7C3-A20 on mitochondrial function in C2C12 cells and examined whether P7C3-A20 protects mouse myoblasts C2C12 against tunicamycin-induced cell death.

MATERIALS AND METHODS

Reagents

The primary antibodies used in this study were as follows: phosphorylated-dynamin-related protein 1 (p-Drp1 [Ser616]; rabbit, Cell Signaling Technology, Beverly, MA, USA, 3455, 1:1,000 for immunoblotting [IB]), Drp1 (mouse, Santa Cruz, Dallas, TX, USA, sc-271583, 1:1,000 for IB), mitofusin-2 (Mfn2; mouse, Santa Cruz, sc-515647, 1:1,000), NAMPT (rabbit, Abcam, Cambridge, MA, USA, ab236874, 1:1,000 for IB), voltage-dependent anion-selective channel 1 (VDAC1; mouse, Santa Cruz, sc-390996, 1:1,000 for IB), PTEN induced putative kinase 1 (PINK1; mouse, Santa Cruz, sc-517353, 1:1,000 for IB), NIP3-like protein X (Nix; mouse, Invitrogen, Waltham, MA, USA, 39-3300, 1:1,000 for IB), and β-actin (mouse, Sigma-Aldrich, St. Louis, MO, USA, A2228, 1:20,000 for IB). Horseradish peroxidase (HRP)-conjugated secondary antibodies (goat anti-mouse, goat anti-rabbit, 1:2,000 for IB), Hoechst 33342, and propidium iodide (PI) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Tunicamycin was purchased from FUJIFILM-Wako Pure Chemical Corporation (Osaka, Japan). P7C3-A20 was purchased from Selleck Chemicals (Houston, TX, USA). The JC-1 MitoMP Detection Kit and Cell Counting Kit-8 (CCK-8) were purchased from Dojindo Laboratories (Kumamoto, Japan). Tauroursodeoxycholic acid (TUDCA) was purchased from Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan).

Cell Culture

C2C12 murine myoblast cells were purchased from the European Collection of Authenticated Cell Cultures (London, UK) and maintained as described previously.³²⁾ The cells were cultured in Dulbecco’s Modified Eagle’s Medium (Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum (Valeant, Costa Mesa, CA, USA), 100 U/mL penicillin (Meiji Seika, Tokyo, Japan), and 100 µg/mL streptomycin in a humidified atmosphere of 95% air and 5% CO₂ at 37°C. Cells were passaged by trypsinization every 2 days.

Immunoblotting

Immunoblotting was carried out as described previously.³²⁾ Briefly, cells were seeded in a 24-well plate (Corning, Corning, NY, USA) at a density of 25,000 cells per well and incubated for 24 h. After incubation with 61.2 nM (50 ng/mL) tunicamycin for 6, 12, or 24 h, cells were harvested in radioimmunoprecipitation assay buffer [50 mM Tris-HCl, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 0.1% NP-40] containing protease inhibitor cocktail, phosphatase inhibitor cocktail 2, and phosphatase inhibitor cocktail 3 (Sigma-Aldrich). Protein concentrations were determined using a BCA Protein Assay Kit (Thermo Fisher Scientific). Samples containing 2 µg of protein were separated by 5-20% gradient SDS-polyacrylamide gel electrophoresis (SuperSep Ace; FUJIFILM-Wako Pure Chemical Co.) and transferred to a polyvinylidene difluoride membrane (Immobilon-P; Merck Millipore, Billerica, MA, USA). The membranes were incubated with the primary antibodies described above, followed by further incubation with HRP-conjugated secondary antibodies. Immunoreactive bands were visualized using ImmunoStar LD (FUJIFILM-Wako Pure Chemical Co.) and detected with an Amersham Imager 680 (GE Healthcare, Chicago, IL, USA). Signal intensities were analyzed using Amersham Imager 680 software (GE Healthcare).

Mitochondrial Membrane Potential (MMP) Assay

MMP was evaluated using a JC-1 MitoMP Detection Kit (Dojindo) following the manufacturer’s protocol, with minor modifications as reported previously.³³⁾ Cells were seeded in a 96-well plate (Corning) at a density of 2,000 cells per well and incubated for 24 h at 37°C, followed by further incubation with P7C3-A20 for 6 h. Representative images were captured using a BZ-X700 all-in-one fluorescence microscope (Keyence, Osaka, Japan). JC-1 polymer (Ex: 535 nm/ Em: 585 nm) and JC-1 monomer (Ex: 485 nm/ Em: 525 nm) were measured using a Varioskan LUX microplate reader (Thermo Fisher Scientific).

Cell Death Rate and Cell Viability Assay

Cell death rate and cell viability assays were carried out as previously described.³²⁾ Briefly, cells were seeded in a 96-well plate (Corning) at a density of 5,000 cells per well and incubated for 24 h at 37°C. P7C3-A20 and TUDCA were dissolved in dimethyl sulfoxide (Nacalai Tesque) and diluted with phosphate-buffered saline to obtain the desired final concentrations of P7C3-A20 (1, 5, or 10 µM) and TUDCA (100 µM). One hour after cells were pretreated with or without P7C3-A20 and TUDCA, 61.2 nM (50 ng/mL) tunicamycin was added to the medium and followed by incubation for 24 h. For the cell death rate assay, cells were incubated with Hoechst 33342 and PI for 15 min at final concentrations of 8.1 and 1.5 µM, respectively. Images were acquired using Lionheart FX (BioTek, Winooski, Vermont, USA) and analyzed with Gen5 software (BioTek Instruments). For the cell viability assay, absorbance at 450 nm was measured at 0 and 2 h after adding 10 µL of CCK-8 reagent to each well using a Varioskan LUX microplate reader (Thermo Fisher Scientific).

Statistics

All data are presented as means ± standard error of the mean (SEM). Statistical analyses were performed using Tukey’s test, Student’s t-test, or Dunnett’s test, using SPSS Statistics software (IBM, Armonk, NY, USA).

RESULTS

Tunicamycin-Induced ER Stress Alters the Expression of Mitochondrial Proteins in C2C12 Cells

To investigate the effects of tunicamycin on myoblasts, we analyzed protein expressions in total cell lysates obtained from C2C12 cells at 6, 12, or 24 h after treatment with tunicamycin at a concentration of 61.2 nM (50 ng/mL). Interestingly, we observed decreased protein levels at 12 h after incubation with tunicamycin in several mitochondrial proteins (Fig. 1A). Drp1 regulates mitochondrial fission by forming oligomeric rings. At 6 h, Drp1 expression did not alter, whereas phosphorylation of Drp1 decreased significantly (Fig. 1B). From 12 h onwards, there was a decrease in both the total amount of Drp1 and phosphorylated Drp1 (Fig. 1B, C). However, protein levels of Mfn2 remained unchanged after treatment with tunicamycin (Fig. 1D). Regarding mitochondrial abundance, we examined the expression of VDAC1, a mitochondrial outer membrane protein, which did not change after treatment with tunicamycin (Fig. 1E). Intriguingly, NAMPT expression decreased at 12 and 24 h after tunicamycin treatment (Fig. 1F). For mitophagy-related proteins, PINK1 expression decreased after 12 h of tunicamycin treatment (Fig. 1G), whereas NIX expression showed no significant change (Fig. 1H). These results suggest that tunicamycin treatment affects the proteostasis of mitochondrial proteins in C2C12 cells.

Fig. 1

Tunicamycin-Induced ER Stress Reduces the Levels of Mitochondrial Proteins, Including NAMPT, in C2C12 Cells

(A) Representative immunoblot images of various mitochondrial proteins in C2C12 myoblast cell lysates treated with 61.2 nM (50 ng/mL) tunicamycin for 0 h (Control), 6 h, 12 h, and 24 h. (B-H) Graphs representing the phosphorylation of Drp1 (B) and the relative protein levels of Drp1 (C), Mfn2 (D), VDAC1 (E), NAMPT (F), PINK1 (G), and Nix (H), normalized to β-actin. Data are presented as means ± SEM (n = 5-6). ^#P < 0.05, ^##P < 0.01, ^###P < 0.005, Tukey’s test vs. Control.

P7C3-A20 Increases MMP

Next, we examined the effect of tunicamycin on mitochondrial function using JC-1, a membrane-permeable fluorescent dye that selectively enters mitochondria. JC-1 fluoresces green as a monomer, indicating low MMP and mitochondrial damage. Tunicamycin treatment increased the JC-1 monomer in C2C12 cells (Fig. 2A, green). The red/green ratio reduced significantly at concentrations of either 12.2 nM (10 ng/mL), or 61.2 nM (50 ng/mL; Fig. 2B). The rate-limiting step in the salvage pathway is the conversion of NAM to NMN catalyzed by NAMPT.^23,24) We investigated the effect of a NAMPT activator, P7C3-A20, on MMP in C2C12 myoblasts. JC-1 fluoresces red as a polymer, indicating high MMP. Treatment with P7C3-A20 for 6 h increased both the JC-1 polymer (red) and monomer (green; Fig. 2C). The fluorescence of the JC-1 polymer increased more than the one of the JC1 monomer, resulting in the elevation of the red/green ratio in C2C12 cells (Fig. 2D). This result suggests that P7C3-A20 enhances the mitochondrial function and concomitantly the production of mitochondrial ROS, which can lead the depolarization in part of the mitochondria.

Fig. 2

P7C3-A20 Increases MMP in C2C12 Cells

(A,C) Representative images of JC-1 staining, an indicator of MMP, in C2C12 cells after incubation either with tunicamycin at concentrations of 2.4 nM (2 ng/mL), 12.2 nM (10 ng/mL), or 61.2 nM (50 ng/mL; A), or with P7C3-A20 at concentrations of 1, 5, or 10 μM (C) for 6 h. The JC-1 aggregates in high membrane potential mitochondria and the JC-1 monomers in low membrane potential mitochondria are detected as red and green fluorescence, respectively. Scale bar, 100 µm. (B,D) The fluorescence intensity ratio of JC-1 aggregates to JC-1 monomers. Data are means ± SEM (n = 6). ^###P < 0.005, Tukey’s test vs. Control.

P7C3-A20 Inhibits Tunicamycin-Induced Cell Death in Murine Myoblasts Cell C2C12

Next, we investigated the effect of P7C3-A20 on cell death induced by tunicamycin in C2C12 mouse myoblasts. Cells were exposed to 61.2 nM (50 ng/mL) of tunicamycin along with varying concentrations of P7C3-A20 for 24 h. The cell death rate in the vehicle group increased to 63.5 ± 1.2% (Fig. 3A, B). In the presence of tunicamycin, the cell death ratio decreased to 56.9 ± 1.5% and 46.5 ± 0.3% when cells were treated with 5 and 10 µM of P7C3-A20, respectively (Fig. 3B). Evaluation of cell viability using the CCK-8 assay revealed a significant reduction in the tunicamycin-treated group (Fig. 3C, Vehicle). However, cell viability was restored with 5 and 10 µM P7C3-A20, as well as with 100 µM TUDCA, which is known to alleviate ER stress (Fig. 3C). These results suggest that P7C3-A20 has a protective effect on tunicamycin-induced ER stress injury in myoblast cells.

Fig. 3

P7C3-A20 Inhibits Tunicamycin-Induced Apoptosis in C2C12 Murine Myoblasts

(A, B) C2C12 cells were treated with 1 µM, 5 µM, or 10 µM of P7C3-A20 following exposure to 61.2 nM (50 ng/mL) tunicamycin and subjected to Hoechst 33342 and PI staining. (A) Representative images of Hoechst 33342 (blue) and PI (magenta) staining. Scale bar, 200 µm. (B) The number of PI-positive cells was counted and represented as a cell death ratio. (C) C2C12 cells were treated with 1 µM, 5 µM, or 10 µM of P7C3-A20, or 100 μM of TUDCA, followed by incubation with 61.2 nM (50 ng/mL) tunicamycin, and subjected to a cell viability assay measured by the CCK-8 assay. Bars represent a fold increase in cell viability compared to the control group. Data are presented as means ± SEM (n = 6). ***P < 0.001, Student’s t-test vs. Control, ^###P < 0.005, Dunnett’s test vs. Vehicle, ^†††P < 0.001, Student’s t-test vs. Vehicle.

DISCUSSION

In this study, we found that tunicamycin treatment decreased the protein levels of NAMPT and PINK1 in C2C12 murine myoblast cells (Fig. 1). Prior to these reductions in mitochondrial protein levels, decreased phosphorylation of Drp1 was observed 6 h after tunicamycin treatment (Fig. 1). P7C3-A20 increased the MMP at concentrations of 1, 5, and 10 μM in C2C12 cells (Fig. 2). P7C3-A20 also ameliorated the increase in cell death and the decrease in cell viability in C2C12 cells treated with tunicamycin (Fig. 3). Overall, we conclude that the NAMPT activator P7C3-A2 protects C2C12 myoblasts from tunicamycin-induced cell injury.

We investigated C2C12 cells as an in-vitro myoblast model. Skeletal muscles contain stem cells called satellite cells that exist in a quiescent state within muscle fibers. When myogenesis is required (e.g., in the case of muscle injury), satellite cells are activated and differentiate into myoblasts. Myoblasts proliferate and eventually fuse with other cells to form multinucleated myofibrils.^34,35) Skeletal muscles, including cardiomyocytes, contain a specialized smooth ER known as the sarcoplasmic reticulum. ER stress and activated UPR pathways are involved in degenerative muscle disorders and various types of myopathies.³⁶⁾ Conversely, mild ER stress is known to facilitate myofiber formation.³⁷⁾ We used tunicamycin to induce cell death (approximately 60%, Fig. 3B). Since ER stress is known to mediate mitochondria-independent apoptosis,¹⁴⁾ it requires an investigation of the involvement of this pathway to elucidate the mechanism underlying tunicamycin-induced C2C12 cell death. Our results suggest that ER stress also disrupts mitochondrial proteostasis in myoblasts (Fig. 1). We have not demonstrated whether this mitochondrial protein change occurs under ER stress in mature muscle fibers. Mature muscle fibers are rich in sarcoplasmic reticulum that contains fewer ribosomes than the rough ER in general cells, suggesting that ER stress more likely occurs in the undifferentiated muscle cells. It remains unclear and controversial whether the apoptosis of undifferentiated muscle cells, including myoblast, is associated with the cause of sarcopenia. Further study is necessary to elucidate the molecular mechanisms linking ER stress and mitochondrial dynamics to their roles in myogenesis.

Oxidative stress is an important contributor to sarcopenia. Sarcopenic muscles are characterized by increased production of reactive oxygen species, which are known to be responsible for mitochondrial biogenesis and quality control. Mitophagy plays an important role in maintaining mitochondrial homeostasis and functional capacity by managing the turnover of individual mitochondrial proteins, the regulation of which is disrupted in sarcopenic skeletal muscles.³⁸⁾ The reduction of MMP in intact mitochondria has been observed in single muscle fibers isolated from aged animals.³⁹⁾ In our study, the MMP increased in C2C12 cells treated with P7C3-A20 at a concentration of 1 μM (Fig. 2). Oral administration of P7C3-A20 at a 10 mg/kg dose provided sustained plasma exposure and showed a neuroprotective effect in adult male rhesus macaques.⁴⁰⁾ Further research is required to confirm whether this compound can modulate mitochondrial function in skeletal muscles.

Aging is associated with a decline in NAD⁺ biosynthesis and an increase in its consumption.⁴¹⁾ NAD⁺ levels are reduced in aged mice and Caenorhabditis elegans, and pharmacological restoration of NAD⁺ prevents age-associated metabolic decline and promotes longevity in worms.⁴²⁾ NAD levels are significantly reduced in skeletal muscle-specific NAMPT-KO mice.⁴³⁾ The activation of NAMPT and increased NAD⁺ levels, especially in the cytoplasm and mitochondria, may contribute to the increase of polarized mitochondria and the inhibition of tunicamycin-induced cell death. NAMPT activation in the nucleus is also involved in regulating gene expression.⁴⁴⁾ Further investigations, such as the evaluation of the intracellular NAD⁺ level in C2C12 cells treated with P7C3-A20, are required to conclude that the protective effect of P7C3-A20 relies on its enzyme activity and is mediated by NAD⁺. Moreover, NAMPT is also present in extracellular locations. Extracellular NAMPT is systemically circulated via NAMPT-containing vesicles, which are released from adipocytes and distributed throughout the body by blood circulation.⁴⁵⁾ A reduction in NAMPT-containing vesicles in the systemic circulatory system may contribute to the age-related decline in NAD⁺. Therefore, activation of NAMPT could be a novel therapeutic target for muscle diseases linked to ER stress, such as sarcopenia.

Acknowledgments

This work was supported by a Grant-in-Aid for Early-Career Scientists from the Japan Society for the Promotion of Science (No. 21K14999 awarded to W.O.).

Conflict of interest

The authors declare no conflict of interest.

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