Effects of α-dicarbonyl Compounds on the Differentiation of Chicken Embryonic Myoblasts

Abstract

Glyoxal (GO) and methylglyoxal (MGO) are highly reactive alpha-dicarbonyl compounds (α-DCs) generated through multiple pathways of glucose metabolism. Both GO and MGO are cytotoxic to mammalian cells, and are implicated in the pathogenesis of several diseases, including diabetes. In mammalian systems, MGO reduces myoblast viability, and inhibits their differentiation. Conversely, we previously demonstrated that GO and MGO do not induce cell death in chicken myoblasts; however, their effects on myoblasts differentiation remain unclear. Therefore, the aim of the present study was to investigate the effects of GO and MGO on cell differentiation in chicken embryonic myoblasts. First, we analyzed the temporal expression patterns of key myogenic regulatory factors, including paired box 7, myoblast determination protein 1, myogenin, and myomaker, during the differentiation of chicken embryonic myoblasts. Exposure to GO or MGO significantly decreased the mRNA levels of key myogenic regulatory factors. Furthermore, both α-DCs suppressed myoblast fusion, a process necessary for myotube formation. Collectively, these findings revealed that α-DCs suppress cell differentiation in chicken embryonic myoblasts.

Introduction

Alpha-dicarbonyl compounds (α-DCs) are highly reactive, and are characterized by the presence of two carbonyl groups. Glyoxal (GO) and methylglyoxal (MGO) are among the well-characterized α-DCs, which are generated through multiple pathways of glucose metabolism, endogenously[1,2,3,4]. Moreover, GO and MGO are also generated during the degradation of glycated proteins[5]. Protein glycation is a spontaneous, non-enzymatic chemical reaction in which proteins are modified by addition of reducing sugars such as glucose and fructose[6]. In mammalian cells, GO and MGO induce oxidative stress and reduce cell viability[7,8,9,10,11,12], and are associated with several diseases, including diabetes.

In mammals, MGO exerts cytotoxic effects on myoblasts by inducing oxidative stress and apoptosis in rat L6 myoblasts[13]. Similarly, in mouse C2C12 myoblasts, MGO promotes oxidative stress and reduces cell viability[14,15]. Consistent with these in vitro findings, in vivo studies have shown that MGO induces skeletal muscle atrophy in mice, accompanied by reduced physical performance, muscle mass, and muscle cross-sectional area[16]. Furthermore, skeletal muscle mass is reduced in patients with diabetes compared with healthy individuals, suggesting a potential contribution of MGO in loss of muscle mass in humans[17].

Additionally, MGO inhibits myoblast differentiation[14,15]. Myoblasts, derived from muscle satellite cells, exit the cell cycle, elongate, and then fuse to form multinucleated myotubes. The differentiation of myoblasts is tightly regulated by key myogenic factors, such as paired box 7 (PAX7), myoblast determination protein 1 (MYOD), myogenin (MYOG), and myomaker (MYMK)[18,19]. Differentiated myoblasts express myosin heavy chain (MYH), a contractile protein. MGO inhibits myoblast differentiation by reducing the mRNA and protein expression levels of MYOD and MYOG, and inhibiting myoblast fusion[14]. However, another study reported that MGO increases MYOG expression without affecting MYOD expression in C2C12 myoblasts[15].

We previously demonstrated that GO and MGO decrease intracellular NADH levels in chicken embryonic myoblasts and myotubes[20]. Notably, exposure to GO or MGO induced morphological changes in chicken embryonic myoblasts, which appeared thinner and shorter than control cells[20], suggesting impaired differentiation. Because myoblast differentiation is essential for skeletal muscle development, its inhibition by GO and MGO also inhibits meat production in chickens. However, the effects of GO and MGO on myoblast differentiation in chickens have not been investigated till date.

This study aimed to investigate the effects of GO and MGO on the differentiation of chicken embryonic myoblasts. Primary chicken embryonic myoblasts used in this study undergo spontaneous differentiation into myotubes under conventional culture conditions without the need for specialized differentiation media. We investigated time-course changes in mRNA expression levels of key myogenic regulatory factors during myoblast differentiation. Subsequently, the effects of GO and MGO exposure on mRNA expression of the myogenic regulatory factors were assessed. Finally, immunocytochemical staining of MYH was performed to assess the effects of GO and MGO on myoblast fusion.

Materials and Methods

Cell culture medium and drugs

Medium 199 (M199, pH 7.2, Thermo Fisher Scientific Inc., Waltham, MA, USA) containing 0.25 µg/mL of amphotericin B (Nacalai Tesque Inc., Kyoto, Japan), 100 unit/mL of penicillin (Nacalai Tesque), 10 µg/mL of streptomycin (Nacalai Tesque), 50 µg/mL of gentamicin sulfate (Biological Industries Ltd., Beit HaEmek, Israel), and 0.2 g/mL of NaHCO₃ was used. Fetal bovine serum (FBS, Biowest SAS, Maine-et-Loire, France) was added to the medium to yield M199 + 10% FBS, which was used for cell preparation, culture, and control treatment. Dulbecco’s phosphate-buffered saline (DPBS, pH 7.4) was used for myoblasts collection from chicken embryos. Red blood cell lysis buffer, consisting of 0.16 M NH₄Cl, 0.1 mM KHCO₃, and 0.1 mM EDTA-2Na (all purchased from Nacalai Tesque), was used to lyse red blood cells when chicken myoblasts were prepared. PBS without Ca²⁺ and Mg²⁺ (PBS (−), pH 7.1–7.3) and Hanks’ Balanced Salts Solution without Ca²⁺, Mg²⁺, and phenol red (HBSS (–), pH 7.1–7.5) (both purchased from FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) were used for washing cells.

GO and MGO (Nacalai Tesque) were dissolved in M199 + 10% FBS unless indicated otherwise.

Cell preparation and culture

Cell preparation and culture were performed as previously described[21], with slight modifications. Fertilized eggs of Single-Comb White Leghorn chickens (purchased from Japan Layer, Gifu, Japan) were incubated at 37.6 °C and 58%–73% relative humidity in an incubator (Showa Furanki Co., Ltd., Saitama, Japan). At embryonic day 17, embryos were removed from the eggs. Following decapitation, breast muscles were excised, and minced using scissors. M199 medium containing collagenase type II (100 U/mL, Worthington Biochemical Corporation, Worthington, USA) was added, and the tissue was incubated at 37 °C for 60 min in a CO₂ incubator (PHC Holdings Corporation, Tokyo, Japan) to facilitate gentle enzymatic digestion. The resulting suspension was filtered using a cell strainer (pore size: 100 µm, Greiner Bio-One Co., Ltd., Frickenhausen, Germany) to remove debris. The filtrate was centrifuged (700 × g, room temperature, 5 min), and the cell precipitate was treated with sterile red blood cell lysis buffer for 2 min. After a second centrifugation (700 × g, room temperature, 5 min), the cell precipitate was resuspended in M199 supplemented with 10% FBS. The cell suspension was plated in a polystyrene dish and incubated for 30 min in a CO₂ incubator (37 °C, 5% CO₂) to allow fibroblasts to adhere to the dish. The cells in the supernatant were collected, counted, and seeded onto type I collagen-coated 35 mm dishes (Iwaki, AGC TECHNO GLASS CO., Ltd., Shizuoka, Japan) at a density of 1.0 × 10⁵ cells/cm². The cells were precultured for 24 h in a CO₂ incubator, unless otherwise specified, and the culture medium was replaced every 2 days. In some experiments, cell aliquots were cryopreserved for later use.

Experiment 1: Time-course changes in the mRNA expression of cell differentiation markers during myoblast differentiation

Myoblasts were seeded in culture dishes and maintained for the indicated time duration prior to analysis. The mRNA expression levels of cell differentiation markers, including PAX7, MYOD1, MYOG, MYMK, and MYH1F, were analyzed in cells cultured for 6 h and up to 5 days.

Total RNA was extracted using the RNA Basic Kit 250 (NIPPON Genetics, Tokyo, Japan). Subsequently, cDNA was synthesized from total RNA using ReverTra Ace^TM qPCR RT Master Mix with gDNA Remover (TOYOBO CO, Ltd., Osaka, Japan). Real-time PCR was performed using gene-specific primers and THUNDERBIRD SYBR qPCR MIX (TOYOBO) on a StepOnePlus Real-Time PCR System (Thermo Fisher Scientific). Primer sequences are listed in Table 1. Amplified nucleotide sequences were determined (Eurofins Genomics, Ebersberg, Germany) to confirm amplification of target genes. Thermal cycling conditions were as follows: initial denaturation at 95 °C for 60 s, followed by 45 cycles of amplification, with each cycle consisting of denaturation at 95 °C for 10 s, annealing at 60 °C for 15 s, and extension at 72 °C for 30 s.

Table 1.  Primer sequences used for real-time PCR

Genes		Primer sequences	Product size (bp)
PAX7	Forward	5′-CCAGTAGAGACAGGCCAAGC-3′	104
	Reverse	5′-GCAGATCCTGGAAGCTGGTA-3′
MYOD1	Forward	5′-CGGAATCACCAAATGACCCAA-3′	173
	Reverse	5′-CAGGTTTGCTCCTTCCTGGG-3′
MYOG	Forward	5′-AAGGGGAGAAGTGTGATGGC-3′	177
	Reverse	5′-ACAGCGGTCAGTAGGAAAGC-3′
MYMK	Forward	5′-TGGGTGTCCCTGATGGC-3′	135
	Reverse	5′-CCCGATGGGTCCTGAGTAG-3′
MYH1F	Forward	5′-GAGCTGACCTACCAGTCTGAA-3′	295
	Reverse	5’-TGCCTCAGGTCACACTTTAGC-3′
RPS17	Forward	5′-CAAGATCGCGGGGTATGTCA-3′	161
	Reverse	5′-TGTCCGGGTCCACTTCAATG-3′

Abbreviations: PAX7, Paired box 7; MYOD1, Myoblast determination protein 1; MYOG, Myogenin; MYH1F, Myosin heavy chain 1F; MYMK, Myomaker; RPS17, Ribosomal protein S17.

Ribosomal protein S17 (RPS17) was used as an internal control. Relative mRNA expression levels were quantified using the ΔΔCt method.

Experiment 2: Effects of GO and MGO on the mRNA expression of cell differentiation markers in chicken embryonic myoblasts

Myoblasts pre-cultured for 24 h were treated with 100 µM GO or MGO for 12 h. Total RNA was extracted, cDNA was synthesized, and real-time PCR was performed as described in Experiment 1. The concentrations of GO and MGO were selected based on our previous study[20].

Experiment 3: Effects of GO and MGO on the fusion of chicken myoblasts

Myoblasts pre-cultured for 24 h were exposed to 100 µM GO or 100 µM MGO for 24 h. Cells were then fixed with 2% paraformaldehyde, permeabilized with 0.2% Triton X-100 (Nacalai Tesque), and blocked with PBS (−) containing 1% bovine serum albumin. Cells were incubated overnight at 4 °C with a primary antibody (2.0 µg/mL, mouse monoclonal anti-pan-MYH antibody, MF20, R&D Systems, McKinly Place NE, MN, USA), followed by incubation with a second antibody (15 µg/mL, Alexa Fluor 488-conjugated AffiniPure Donkey Anti-Mouse IgG antibody, Jackson ImmunoResearch, PA, USA) at room temperature for 1 h. Nuclei were stained with Hoechst (Hoechst 33342, Dojindo Laboratories) stain. Fluorescence images were acquired using a fluorescence microscope (Alexa Fluor 488, Ex/ Em: 470 nm/ 525 nm; Hoechst, Ex/ Em: 360 nm/ 460 nm, BZ-X810, KEYENCE CORPORATION, Osaka, Japan). MYH-immunoreactive cells containing two or more nuclei were defined as myotubes, and the number of nuclei within myotubes was counted. The average number of nuclei from three fields per dish was calculated as the representative value. The fusion index was calculated by dividing the number of nuclei within myotubes by the total number of nuclei stained with the Hoechst stain.

Statistical analyses

Multiple comparisons were performed using the Tukey–Kramer test. Comparisons between two groups were conducted using the t-test. Statistical significance was defined as p < 0.05. Data were represented as the mean ± S.E.M. The number of wells or dishes used in each experiment is indicated in the corresponding figure legends.

Results

Experiment 1: Time-course changes in the mRNA expression of cell differentiation markers during myoblast differentiation

Cells began to elongate from day 2 after the culture was initiated, with cell density gradually increasing over time. On the basis of the observed morphological characteristics, we established that myotube-like cells started to appear from day 2 (Figure 1A).

Fig. 1.

Time-course of changes in the mRNA expression of cell differentiation markers in chicken myoblasts. (A) Representative microscopic images of proliferating and differentiating cells. (B) mRNA expression of cell differentiation markers in chicken myoblasts from 6 h to 5 days after seeding. Results are shown as fold changes from the mRNA level at 6 h, and expressed as the means ± standard error of the mean (n = 3 for 6 h and n = 4 for 1 to 5 days). The values of treatments denoted by different lower-case letters above bars are significantly different (p < 0.05).

The mRNA expression of PAX7 significantly increased at day 1 d and then sharply decreased from day 2 onwards (Figure 1B). However, by day 5, the expression of PAX7 had returned to a level comparable to that observed at 6 h after the culture initiation. Similarly, we observed a significant increase in the expression of MYOD1 from day 1, which was maintained to the following day (Figure 1B), after which it gradually declined, with no significant differences being detected by day 4. In contrast, expression levels of MYOG, MYMK, and MYH1F started to increase from day 2, reaching peak levels on day 3 (Figure 1B), and subsequently declined over time, returning to initial levels by day 5.

Experiment 2: Effects of GO and MGO on the mRNA expression of cell differentiation markers in chicken embryonic myoblasts

The mRNA expression levels of all cell differentiation markers were significantly decreased after GO treatment (Figure 2). Among these markers, GO strongly suppressed the expression of MYOD1, MYOG, and MYMK, which play pivotal roles in the fusion of myocytes[19]. Exposure to GO similarly resulted in a reduction of the mRNA expression of MYH1F, which plays an important role in myotube formation. Comparatively, whereas MGO treatment had no measurable effect the mRNA expression of PAX7, we detected similar significant reductions in the expression of MYOD1, MYOG, MYMK, and MYH1F (Figure 2).

Fig. 2.

Effects of GO and MGO on the mRNA expression of cell differentiation makers in chicken myoblasts. Cells were exposed to 100 µM GO or MGO for 12 h. Results are shown as fold changes relative to the control group. Data are expressed as the means ± standard error of the mean. (n = 6). * denotes a significant difference from the control group (p < 0.05).

Experiment 3: Effects of GO and MGO on the fusion of chicken myoblasts

The intracellular levels of MYH protein, as visualized using immunocytochemical staining, were observed to be reduced following treatment with GO and MGO (Figure 3A), with corresponding significant reductions in the fusion index, an indicator of myoblast fusion and differentiation (Figure 3B).

Fig. 3.

Effects of GO and MGO on the expression of MYH protein and fusion index in chicken myoblasts. Cells were exposed to 100 µM GO or MGO for 24 h. (A) Representative fluorescent microphotograph of cell nuclei stained with Hoechst and intracellular MYH protein visualized via immunocytochemical staining. (B) Fusion index values for cells subjected to different treatments. Fusion index values were calculated based on the number of nuclei in myotubes divided by the entire number of nuclei stained using Hoechst. Cells containing two or more nuclei and stained for MYH were regarded as myotubes. Data are expressed as the means ± standard error of the mean (n = 3). The values of treatments denoted by different lower-case letters above bars are significantly different (p < 0.05).

Discussion

Under the culture conditions established in this study, we observed clear morphological changes in the cultured myoblasts from day 2 of the onset of culture, which were closely synchronized with changes in the mRNA expression of selected myogenic regulatory factors (Figure 1). Among these, we detected increases in the expression of PAX7 and MYOD1 mRNAs on day 1 following the onset of culture, with subsequent sharp declines from day 2 onwards. Given that PAX7 has been established to be associated with the proliferation of muscle satellite cells[18,19], these findings would tend to indicate that chicken embryonic myoblasts actively proliferate for up to 2 days after seeding. Likewise, we detected an increase in the expression of MYOD1 mRNA from day 1, although in this case, levels remained elevated until day 3. Given that MYOD1 is involved in both the proliferation and differentiation of myoblasts[18,19], it is reasonable to assume that peak levels of expression might persist for longer than those observed for PAX7. For the other assessed markers MYOG, MYMK, and MYH1F, following an initial latent period, we observed significant increases in mRNA expression of from day 2 after seeding. Among these factors, MYOG and MYMK have been established to promote myoblast fusion[19], whereas MYH1F encodes a contractile protein expressed in myotubes. Collectively, these findings provide evidence to indicate that under the evaluated culture conditions, chicken myoblasts undergo differentiation to myotubes from approximately day 2 d after seeding. On the basis of these findings, we subsequently examined the effects of α-DCs on myoblast differentiation using cells cultured for 2 days.

Exposure of mouse C2C12 myoblast cells to MGO for 4 days has been reported to inhibit cell differentiation by reducing the expression of MYOD and MYOG[14], although it has also been reported that C2C12 myoblasts treated with MGO for 24 h were characterized by an increase MYOG expression in the absence of any significant changes in the expression of MYOD[15]. In the present study, we found that exposure of chicken embryonic myoblasts to MGO caused reductions in the mRNA expression of both MYOD1 and MYOG (Figure 2). We speculate that these discrepant findings regarding the effects of MGO could be ascribed to differences in experimental conditions, such as MGO concentration or cell type (cell line vs. primary cells). Furthermore, we found that treatment with MGO also reduced the mRNA expression of MYMK and MYH1F in chicken embryonic myoblasts (Figure 2). Collectively, these findings indicate that in chickens, MGO negatively influences the differentiation of myoblasts.

In contrast to MGO, to the best of our knowledge, there have to date been no studies that have sought to determine the effects of GO on the differentiation of myoblasts in any animal species. In this study, we found that treatment with GO reduced the mRNA expression of PAX7, MYOD1, MYOG, MYMK, and MYH1F (Figure 2), thereby providing evidence that similar to MGO, GO may also inhibit myoblast differentiation. However, unlike in the response to MGO, we detected a reduction on the expression of PAX7 mRNA (Figure 2), indicating that GO may also suppress the proliferation of muscle satellite cells.

A further finding was that MYH-positive cells appeared less abundant following exposure to either GO or MGO, and there were corresponding reductions in the fusion index (Figure 3). This reduction in the expression of MYH protein was consistent with the detected reduction in the expression of MYH1F mRNA (Figure 2), whereas the lower fusion index values indicate that these α-DCs inhibit the fusion of myoblasts and thus their differentiation to myotubes. Given that MYMK promotes myoblast fusion[19], we speculate that the GO- and MGO-induced reductions in MYMK mRNA expression may have contributed to the observed reduction in fusion index values. Furthermore, as it has been established that MYOD1 and MYOG contribute to the regulation of MYMK expression[19], it would appear that the α-DC-induced inhibition of chicken myoblast differentiation is mediated via the downregulated expression of MYOD1 and MYOG.

In mammals, MGO has been shown to induce oxidative stress in myoblasts, thereby inhibiting the expression of MYOD1 and MYOG and subsequently suppressing myotube formation[14]. However, whereas in mammalian systems, myoblast differentiation is also impaired by a reduction in mitochondrial activity[22], we have demonstrated that GO and MGO do not induce oxidative stress or mitochondrial dysfunction in the embryonic myoblasts of chickens[20]. Consequently, it is plausible that the mechanisms underlying the inhibitory effects of α-DCs on myoblast differentiation in chickens differ from those in mammals.

Among the putative mechanisms proposed for the inhibitory effects of α-DCs on myoblast differentiation is the suppression of cell proliferation. Efficient myoblast differentiation is dependent on a high density of cells[23], and given that GO and MGO have been shown to cause reductions in the proliferation of chicken embryonic myoblasts[20], it is conceivable that differentiation may have been impaired under the conditions assessed in the present study. A further potential mechanism involves heme oxygenase-1 (HMOX1), a well-established antioxidant enzyme[24] that has also been reported to suppress the expression of MYOD1 and its downstream targets, including MYOG and MYH[25]. In chicken embryonic myoblasts, we have previously detected increases in the expression of HMOX1 mRNA in response to treatment with GO and MGO[20]. Consequently, it is feasible that an α-DC-induced increase in HMOX1 expression may contribute to the suppression of myoblast differentiation in chickens.

Whereas we found that both GO and MGO influenced the mRNA expression of myogenic regulatory factors, the effects were notably somewhat stronger in cells treated with GO (Figure 2). In addition, unlike MGO, GO reduced the expression of PAX7 mRNA (Figure 2), which is consistent with our previous findings indicating different effects on the mRNA expressions of antioxidant genes[20]. Consequently, it is plausible that although the overall effects of GO and MGO are similar, the respective mechanisms of action might differ. GO and MGO non-enzymatically bind to proteins and form advanced glycation end products (AGEs)[26], and it is thus conceivable that such differences in the mechanisms of action could be attributable to the production of different types of AGEs. Notably in this regard, metformin, a biguanide antidiabetic medication, has been reported to inhibit the production of AGEs by reacting with α-DCs[27], and given that chicks fed a metformin-containing diet have been found to develop heavier breast muscles[28], it is proposed that inhibition of the production of GO- and MGO-derived AGEs might contribute to enhancing the production of chicken meat.

In this study, we established that GO and MGO inhibit the differentiation of embryonic myoblasts in chickens, thereby indicating that α-DCs may negatively influence myogenesis, muscle development, and the production of meat in chickens. Although naturally hyperglycemic animals[29], chickens rarely develop diabetic complications[30], and given that GO and MGO have similar inhibitory effects on the differentiation myoblasts in both chickens and mammals, it is conceivable that rather than having an inherent tolerance to α-DCs, the relative resistance of chickens to diabetic complications is attributable to a more efficient detoxification system for these compounds. Consistent with this interpretation, in chickens, the levels of circulating MGO are lower than those in rodents[31]. However, further studies will be necessary to clarify the precise effects of α-DCs on myogenesis and meat production in chickens.

Ethical approval

All animal experiments were approved by the Committee of Animal Care and Use of Ehime University, Japan (No. 08-o15-10) and were conducted in accordance with the Ehime University Animal Experiment Regulations.

Funding

This study was supported by the Japan Society for the Promotion of Science KAKENHI (Grant Number JP23K05508).

Acknowledgements

We are grateful to Mr. H. Kayooka for his valued technical assistance.

Author contributions

Meiko Okino conceived the study, contributed to the experimental design, conducted experiments, analyzed the data, and wrote the manuscript; Ryosuke Makino contributed to the experimental design, discussed the results, and reviewed the manuscript; Tetsuya Tachibana contributed to the experimental design, analyzed the data, and wrote the manuscript.

Conflicts of interest

The authors declare that they have no conflicts of interest associated with this manuscript.

Declaration of AI and AI-assisted technologies

The authors declare that no AI and AI-assisted technologies were used for any aspects of this study.

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