Identification of Agents That Ameliorate Hyperphosphatemia-Suppressed Myogenin Expression Involved in the Nrf2/p62 Pathway in C2C12 Skeletal Muscle Cells

Hyperphosphatemia can occur as a result of reduced phosphate (Pi) excretion in cases of kidney dysfunction, which can induce muscle wasting and suppress myogenic differentiation. Higher Pi suppresses myogenic differentiation and promotes muscle atrophy through canonical (oxidative stress-mediated) and noncanonical (p62-mediated) activation of nuclear factor erythroid 2-related factor 2 (Nrf2) signaling. However, the crosstalk between myogenin and Nrf2/p62 and potential drug(s) for the regulation of myogenin expression needed to be addressed. In this study, we further identified that myogenin may negatively regulate Nrf2 and p62 protein levels in the mouse C2C12 muscle cell line. In the drug screening analysis, we identified N-acetylcysteine, metformin, phenformin, berberine, 4-chloro-3-ethylphenol, cilostazol, and cilomilast as ameliorating the induction of Nrf2 and p62 expression and reduction in myogenin expression that occur due to high Pi. We further elucidated that doxorubicin and hydrogen peroxide reduced the amount of myogenin protein mediated through the Kelch-like ECH-associated protein 1/Nrf2 pathway, differently from the mechanism of high Pi. The dual functional roles of L-ascorbic acid (L-AA) were found to be dependent on the working concentration, where concentrations below 1 mM L-AA reversed the effect of high Pi on myogenin and those above 1 mM L-AA had a similar effect of high Pi on myogenin when used alone. L-AA exacerbated the effect of hydrogen peroxide on myogenin protein and had no further effect of doxorubicin on myogenin protein. In summary, our results further our understanding of the crosstalk between myogenin and Nrf2, with the identification and verification of several potential drugs that can be applied in rescuing the decline of myogenin due to high Pi in muscle cells.


Introduction
Serum phosphate (P i ) levels are primarily regulated by the kidney, and hyperphosphatemia may occur because of reduced Pi excretion in kidney dysfunction [1,2]. Many studies have demonstrated that P i is a pro-aging factor linked with muscle wasting [3][4][5]. Sarcopenia is a geriatric syndrome that involves an imbalance between anabolic and catabolic pathways for muscle mass [6,7]. Hyperphosphatemia could induce muscle wasting and suppress myogenic differentiation through oxidative stress-mediated activation of nuclear factor erythroid 2-related factor 2 (Nrf2) signaling [5]. However, the detailed molecular mechanisms underlying muscle atrophy induced by higher concentrations of P i are not fully understood.
Excess reactive oxygen species (ROS) production could lead to muscle atrophy mediated through the inhibition of muscle protein synthesis and the promotion of protein 2. Results 2.1. Crosstalk of Myogenin with the Nrf2/p62 Complex and Other Proteins in High P i -Treated C2C12 Cells C2C12 cells, a subgroup of C2 myoblast, differentiate and fuse into a myotube when exposed to low-serum differentiation media [18]. These properties of C2C12 myotubes expressing contractile proteins and contracting spontaneously make C2C12 cells a tool for understanding the molecular mechanisms of muscle development. The limitation of C2C12 cells is the detachment of myotubes which subsequently leads to cell death. However, in our previous work in C2C12 cells, we demonstrated that high P i could decrease myogenin gene and protein expression via the Nrf2/p62 pathway [5]. We confirmed that the induction of Nrf2 by high P i was not primarily mediated through the Keap1/Nrf2 pathway as a response to ROS since proteasome inhibitor MG132 treatment was ineffective. It is a puzzle whether protein stability was involved in the regulation of myogenin. Hence, to clarify this possibility, we applied cycloheximide (CHX), an inhibitor of de novo protein synthesis ( Figure 1A). The C2C12 cells treated with higher concentrations of P i resulted in the suppression of myogenin protein stability. We observed the stability of myogenin was decreased with increasing concentrations of P i , whereas Nrf2 and p62 proteins were stabilized by higher concentrations of P i . Overexpression of myogenin proteins via a transient transfection strategy consistently suppressed the amounts of endogenous Nrf2 and p62 proteins in C2C12 cells, suggesting that myogenin might be involved in the negative effects on Nrf2 and p62 proteins ( Figure 1B). In combination with high P i , transient overexpression of HA-myogenin in C2C12 cells reversed the effects of high P i in the reduction of myogenin protein and the induction of Nrf2 and p62 proteins ( Figure 1C). suppressed the amounts of endogenous Nrf2 and p62 proteins in C2C12 cells, sugge that myogenin might be involved in the negative effects on Nrf2 and p62 proteins (F 1B). In combination with high Pi, transient overexpression of HA-myogenin in C2C12 reversed the effects of high Pi in the reduction of myogenin protein and the induct Nrf2 and p62 proteins ( Figure 1C). Myogenin is an important transcription factor for muscle terminal different [12,15]. In addition to the suppression of myogenin protein expression by high Pi re to the Nrf2/p62 pathway, we further examined other proteins involved in the le myogenin proteins. Here, we checked signaling pathways, such as ERK1/2 and antioxidative enzymes (superoxide dismutase 1, SOD1, and glutathione peroxid GPX-1/2), enzymes for calcitriol inactivation (cytochrome P450 24A1, cyp24A1), pro involved in RNA binding (HuR), cellular proliferation (proliferating cell nuclear an PCNA), survival (survivin), and inflammation (interleukin-6, IL-6). Our data showe high Pi suppressed the ratio of p-ERK/ERK and p-p38/p38 and SOD1 in C2C12 ( Figure 2A). We observed that high Pi increased GPX-4, HuR, PCNA, survivin, vitam receptor (VDR), cyp24A1, and IL-6 in C2C12 cells. No apparent effect on the suppr of cytokine signaling 3 (SOCS3) protein was observed under the current conditions. C2C12 is a pluripotent mesenchymal cell line for which osteoblast differentiatio be induced by bone morphogenetic protein-2 (BMP-2) [19,20]. Mature muscle can un a reversal of differentiation in the presence of high Pi, which causes a decr Myogenin is an important transcription factor for muscle terminal differentiation [12,15]. In addition to the suppression of myogenin protein expression by high P i related to the Nrf2/p62 pathway, we further examined other proteins involved in the level of myogenin proteins. Here, we checked signaling pathways, such as ERK1/2 and p38, antioxidative enzymes (superoxide dismutase 1, SOD1, and glutathione peroxide-1/2, GPX-1/2), enzymes for calcitriol inactivation (cytochrome P450 24A1, cyp24A1), proteins involved in RNA binding (HuR), cellular proliferation (proliferating cell nuclear antigen, PCNA), survival (survivin), and inflammation (interleukin-6, IL-6). Our data showed that high P i suppressed the ratio of p-ERK/ERK and p-p38/p38 and SOD1 in C2C12 cells (Figure 2A). We observed that high P i increased GPX-4, HuR, PCNA, survivin, vitamin D receptor (VDR), cyp24A1, and IL-6 in C2C12 cells. No apparent effect on the suppressor of cytokine signaling 3 (SOCS3) protein was observed under the current conditions. of Pi using the dye Alizarin Red S, which is a stain commonly used to identify calciumcontaining osteocytes [21]. Our staining data showed that higher concentrations of Pi enhanced the Alizarin Red S staining in C2C12 cells ( Figure 2B). Our data suggested that myogenin downregulation might have the ability to reverse the differentiation of horse serum-induced C2C12 muscle cell maturation into pluripotent mesenchymal cells, which then potentially trans-differentiate into osteocytes in response to higher concentrations of Pi.

The Identification of Compounds Involved in the Regulation of Myogenin Expression via a Drug Screening Strategy with the Myogenin/Nrf2/p62 Platform
Our previous work provided evidence that phosphonoformic acid, N-acetylcysteine (NAC), metformin, and phenformin significantly diminish the stimulatory effect of high Pi on Nrf2 and p62 expression in addition to its inhibitory effect on myogenin expression [5]. We further examined the effects of 36 drugs on high Pi-mediated Nrf2, p62, and myogenin expression ( Figure 3). In addition to NAC, metformin, and phenformin, our screening data showed that berberine, 4-chloro-3-ethylphenol (4-CEP), cilostazol, and cilomilast ameliorate the induction of Nrf2 and p62 expression and reduction of myogenin expression caused by high Pi. Thapsigargin and oltipraz reported effects similar to the above in the absence of high Pi. Doxorubicin, hydrogen peroxide, MitoQ, and CoCl2 had direct negative effects on myogenin expression in C2C12 cells. C2C12 is a pluripotent mesenchymal cell line for which osteoblast differentiation can be induced by bone morphogenetic protein-2 (BMP-2) [19,20]. Mature muscle can undergo a reversal of differentiation in the presence of high P i , which causes a decreased abundance of the muscle terminal differentiation transcription factor myogenin. Hence, we examined the differentiation status of C2C12 cells treated with different concentrations of P i using the dye Alizarin Red S, which is a stain commonly used to identify calciumcontaining osteocytes [21]. Our staining data showed that higher concentrations of P i enhanced the Alizarin Red S staining in C2C12 cells ( Figure 2B). Our data suggested that myogenin downregulation might have the ability to reverse the differentiation of horse serum-induced C2C12 muscle cell maturation into pluripotent mesenchymal cells, which then potentially trans-differentiate into osteocytes in response to higher concentrations of P i .

The Identification of Compounds Involved in the Regulation of Myogenin Expression via a Drug Screening Strategy with the Myogenin/Nrf2/p62 Platform
Our previous work provided evidence that phosphonoformic acid, N-acetylcysteine (NAC), metformin, and phenformin significantly diminish the stimulatory effect of high P i on Nrf2 and p62 expression in addition to its inhibitory effect on myogenin expression [5]. We further examined the effects of 36 drugs on high P i -mediated Nrf2, p62, and myogenin expression ( Figure 3). In addition to NAC, metformin, and phenformin, our screening data showed that berberine, 4-chloro-3-ethylphenol (4-CEP), cilostazol, and cilomilast ameliorate the induction of Nrf2 and p62 expression and reduction of myogenin expression caused by high P i . Thapsigargin and oltipraz reported effects similar to the above in the absence of high P i . Doxorubicin, hydrogen peroxide, MitoQ, and CoCl 2 had direct negative effects on myogenin expression in C2C12 cells.

Comparison of the Effects of Doxorubicin, Hydrogen Peroxide, and L-Ascorbic Acid on the Regulation of Myogenin Expression in C2C12 Cells with High P i
We further examined the detailed mechanisms of doxorubicin and hydrogen peroxide (H 2 O 2 ) regarding their effects on myogenin regulation related to the Nrf2/p62 pathway. We treated C2C12 cells with 0, 0.01, 0.1, 0.25, 0.5, and 1 µM doxorubicin for 20 h and then analyzed related proteins and mRNAs. Our Western blotting and RT-PCR analyses demonstrate that proteins and mRNA expression of myogenic factors, including myogenin, myosin light chain-2v (MLC-2v), and myosin heavy chain 3 (MYHC 3), were suppressed by doxorubicin in a dose-dependent manner ( Figure 4A,B). However, p62 and Nrf2 proteins were initially suppressed and levels finally increased at the highest dosage, whereas their mRNA levels were unchanged. Examination of the well-known Keap1/Nrf2 pathway showed that Keap1 expression was suppressed by doxorubicin in a dose-dependent manner. Hence, we observed Keap1 downregulation at doxorubicin concentrations above 0.5 µM, whereby Nrf2 protein levels were accordingly increased.

Comparison of the Effects of Doxorubicin, Hydrogen Peroxide, and L-Ascorbic Acid on the Regulation of Myogenin Expression in C2C12 Cells with High Pi
We further examined the detailed mechanisms of doxorubicin and hydrogen peroxide (H2O2) regarding their effects on myogenin regulation related to the Nrf2/p62 pathway. We treated C2C12 cells with 0, 0.01, 0.1, 0.25, 0.5, and 1 μM doxorubicin for 20 h and then analyzed related proteins and mRNAs. Our Western blotting and RT-PCR analyses demonstrate that proteins and mRNA expression of myogenic factors, including myogenin, myosin light chain-2v (MLC-2v), and myosin heavy chain 3 (MYHC 3), were suppressed by doxorubicin in a dose-dependent manner ( Figure 4A,B). However, p62 and Nrf2 proteins were initially suppressed and levels finally increased at the highest dosage, whereas their mRNA levels were unchanged. Examination of the well-known Keap1/Nrf2 pathway showed that Keap1 expression was suppressed by doxorubicin in a dosedependent manner. Hence, we observed Keap1 downregulation at doxorubicin concentrations above 0.5 μM, whereby Nrf2 protein levels were accordingly increased. C2C12 cells were treated with 0, 0.5, 1, 2, 5, and 10 mM H2O2 for 24 h, and the related proteins and mRNAs were then analyzed. Our Western blotting and RT-PCR analyses demonstrated that protein and mRNA expression of myogenic factors, including myogenin, MLC-2v, and MYHC3, were suppressed by H2O2 in a dose-dependent manner ( Figure 5A,B). Similar to the doxorubicin response, p62 and Nrf2 proteins were initially suppressed and levels finally increased at the highest dosage, whereas mRNA levels were unchanged. Keap1 protein was suppressed by H2O2 in a dose-dependent manner, whereas Keap1 mRNA was partially induced. We further combined H2O2 with 4 mM Pi, 100 μM sodium phosphonoformate tribasic hexahydrate (NaPTH), and 10 mM NAC to examine myogenin, Keap1, and Cox-2 levels using Western blotting analysis ( Figure 5C). In C2C12 cells, H2O2 induced the expression of Cox-2 protein, a well-known H2O2 stress biomarker [22]. H2O2 suppressed the expression of Keap1 in a dose-dependent manner, suggesting that Nrf2 levels increase. Hence, the results in C2C12 cells confirmed that the suppression of myogenin expression was mediated through the Keap1/Nrf2 pathway. High Pi alone had positive effects on the expression of Cox-2, Keap1, and myogenin, and it potentiated C2C12 cells were treated with 0, 0.5, 1, 2, 5, and 10 mM H 2 O 2 for 24 h, and the related proteins and mRNAs were then analyzed. Our Western blotting and RT-PCR analyses demonstrated that protein and mRNA expression of myogenic factors, including myogenin, MLC-2v, and MYHC3, were suppressed by H 2 O 2 in a dose-dependent manner ( Figure 5A,B). Similar to the doxorubicin response, p62 and Nrf2 proteins were initially suppressed and levels finally increased at the highest dosage, whereas mRNA levels were unchanged. Keap1 protein was suppressed by H 2 O 2 in a dose-dependent manner, whereas Keap1 mRNA was partially induced. We further combined H 2 O 2 with 4 mM Pi, 100 µM sodium phosphonoformate tribasic hexahydrate (NaPTH), and 10 mM NAC to examine myogenin, Keap1, and Cox-2 levels using Western blotting analysis ( Figure 5C). In C2C12 cells, H 2 O 2 induced the expression of Cox-2 protein, a well-known H 2 O 2 stress biomarker [22]. H 2 O 2 suppressed the expression of Keap1 in a dose-dependent manner, suggesting that Nrf2 levels increase. Hence, the results in C2C12 cells confirmed that the suppression of myogenin expression was mediated through the Keap1/Nrf2 pathway. High P i alone had positive effects on the expression of Cox-2, Keap1, and myogenin, and it potentiated the effect of H 2 O 2 on the Keap1/Nrf2/myogenin pathway. A P i transporter inhibitor, NaPTH, had negative effects on the expression of Keap1 and myogenin but not on the expression of Cox-2 protein. The application of a ROS scavenger, NAC, in isolation suppressed the expression of Keap1 but not of Cox-2 or myogenin. NAC was observed to totally suppress H 2 O 2 -induced Cox-2 expression and partially rescue H 2 O 2 -repressed myogenin expression in C2C12 cells. Except for in the presence of 4 mM P i , no changes in Alizarin Red S staining were observed regardless of whether cells were in H 2 O 2 -repressed myogenin expression conditions or otherwise ( Figure 5D).  Figure 5D). L-ascorbic acid (L-AA) has been reported as either an antioxidant or prooxidant depending on the applied cellular concentration [23,24]. Our data showed that antioxidant NAC has the ability to rescue the repression of myogenin expression by high Pi in C2C12 cells [5]. Hence, we applied various concentrations of L-AA to examine whether it had the ability to rescue the repression of myogenin expression by high Pi in C2C12 cells. We first L-ascorbic acid (L-AA) has been reported as either an antioxidant or prooxidant depending on the applied cellular concentration [23,24]. Our data showed that antioxidant NAC has the ability to rescue the repression of myogenin expression by high P i in C2C12 cells [5]. Hence, we applied various concentrations of L-AA to examine whether it had the ability to rescue the repression of myogenin expression by high P i in C2C12 cells. We first measured the status of cytosolic ROS in the presence of high P i , NAC, L-AA, and NAC combined with L-AA using the dye DCFH-DA ( Figure 6A-C). Our data showed that 1000 µM L-AA reduced cytosolic ROS, suppressed high P i -induced ROS, and synergistically worked with NAC to reduce cytosolic ROS and high P i -induced ROS in C2C12 cells. According to Western blotting analysis, L-AA had no apparent effect on the expression of p62, Nrf2, and myogenin at the tested concentrations ( Figure 6D, compared with lanes 1-3). Moreover, L-AA did not appear to suppress the effects of high P i regarding the expression of p62, Nrf2, and myogenin ( Figure 6D, compared with lanes 4-6). Thus, L-AA appeared to synergistically function with NAC to suppress the effects of high P i on the expression of p62, Nrf2, and myogenin proteins ( Figure 6D, compared with lanes 7-12). measured the status of cytosolic ROS in the presence of high Pi, NAC, L-AA, and NAC combined with L-AA using the dye DCFH-DA ( Figure 6A-C). Our data showed that 1000 μM L-AA reduced cytosolic ROS, suppressed high Pi-induced ROS, and synergistically worked with NAC to reduce cytosolic ROS and high Pi-induced ROS in C2C12 cells. According to Western blotting analysis, L-AA had no apparent effect on the expression of p62, Nrf2, and myogenin at the tested concentrations ( Figure 6D, compared with lanes 1-3). Moreover, L-AA did not appear to suppress the effects of high Pi regarding the expression of p62, Nrf2, and myogenin ( Figure 6D, compared with lanes 4-6). Thus, L-AA appeared to synergistically function with NAC to suppress the effects of high Pi on the expression of p62, Nrf2, and myogenin proteins ( Figure 6D, compared with lanes 7-12). We further elevated the concentration of L-AA up to 5 mM to examine its effect on C2C12 cells using cell cycle profile analysis ( Figure 7A). We observed a higher population We further elevated the concentration of L-AA up to 5 mM to examine its effect on C2C12 cells using cell cycle profile analysis ( Figure 7A). We observed a higher population of subG1 phase cells at concentrations higher than 3 mM, accompanied by the downregulation of G1 populations and the upregulation of S and G2/M populations. The cell cycle profiling results suggested that 5 mM L-AA is harmful to C2C12 cells and affects their survival. Hence, we selected 3 mM L-AA as the highest concentration for Western blotting analysis. Over 2 mM L-AA alone increased the expression of p62 and Nrf2 proteins and decreased the expression of myogenin proteins to the same degree as high P i ( Figure 7B). It was difficult to examine the functional interaction between high P i and L-AA on p62, Nrf2, and myogenin proteins under this condition. The comparison between L-AA and high P i treatments suggested that higher L-AA concentrations as well as high P i increased the expression of Nrf2 and p62 via ROS induction. Higher concentrations of L-AA partially rescued high P i -repressed myogenin proteins ( Figure 7C, compared with lanes 4-6) and further enhanced the suppressive effects of H 2 O 2 in repressing myogenin proteins in C2C12 cells ( Figure 7C, compared with lanes 7-9). of subG1 phase cells at concentrations higher than 3 mM, accompanied by the downregulation of G1 populations and the upregulation of S and G2/M populations. The cell cycle profiling results suggested that 5 mM L-AA is harmful to C2C12 cells and affects their survival. Hence, we selected 3 mM L-AA as the highest concentration for Western blotting analysis. Over 2 mM L-AA alone increased the expression of p62 and Nrf2 proteins and decreased the expression of myogenin proteins to the same degree as high Pi ( Figure 7B). It was difficult to examine the functional interaction between high Pi and L-AA on p62, Nrf2, and myogenin proteins under this condition. The comparison between L-AA and high Pi treatments suggested that higher L-AA concentrations as well as high Pi increased the expression of Nrf2 and p62 via ROS induction. Higher concentrations of L-AA partially rescued high Pi-repressed myogenin proteins ( Figure 7C, compared with lanes 4-6) and further enhanced the suppressive effects of H2O2 in repressing myogenin proteins in C2C12 cells ( Figure 7C, compared with lanes 7-9).

Discussion
Hyperphosphatemia can induce muscle wasting and suppress myogenic differentiation [1,2,4]. Our recent study demonstrated that the treatment of differentiating C2C12 cells with Pi induced protein degradation and reduced protein synthesis in a dosedependent manner [5]. Treatment with either Pi influx, cytosolic ROS, or Nrf2 inhibitors abrogated the inhibitory and stimulatory effect of high Pi on myogenin and Nrf2 and p62

Discussion
Hyperphosphatemia can induce muscle wasting and suppress myogenic differentiation [1,2,4]. Our recent study demonstrated that the treatment of differentiating C2C12 cells with P i induced protein degradation and reduced protein synthesis in a dose-dependent manner [5]. Treatment with either P i influx, cytosolic ROS, or Nrf2 inhibitors abrogated the inhibitory and stimulatory effect of high P i on myogenin and Nrf2 and p62 expression, respectively. Nrf2 could serve as a repressor of myogenin and activator of p62 transcription. High P i concentrations disrupt the mitochondrial membrane potential and oxidative phosphorylation in differentiating C2C12 cells. These data demonstrate that hyperphosphatemia suppresses skeletal muscle cell differentiation through induction of oxidative stress, activation of the Nrf2/p62 signaling pathway, and disruption of mitochondrial function. In this study, we further identified that myogenin might negatively regulate Nrf2 and p62 at the protein level. In the drug screening analysis, we identified NAC, metformin, phenformin, berberine, 4-CEP, cilostazol, and cilomilast as significantly diminishing the stimulatory effect of high P i on Nrf2 and p62 expression as well as its inhibitory effect on myogenin expression. In our work, we further elucidated that doxorubicin and hydrogen peroxide reduce the amount of myogenin protein, which is mediated through the Keap1/Nrf2 pathway and different from the mechanism of high P i . The dual functional roles of L-AA were dependent on the working concentration, where concentrations below 1 mM L-AA reversed the effect of high P i on myogenin and those above 1 mM L-AA, applied in isolation, had an effect similar to that of high P i on myogenin. L-AA exacerbated the effects of hydrogen peroxide on myogenin protein and did not influence the effect of doxorubicin on myogenin protein.
There is growing evidence for the association of drugs with muscle, where they may act as a trigger in the development of sarcopenia and frailty. Sarcopenia is a geriatric syndrome involving an imbalance between the anabolic and catabolic pathways that regulate muscle mass [4,6,7]. Innovative anticancer strategies have contributed to improving the survival of cancer patients, transforming cancer into a chronic rather than terminal disease, and include the use of doxorubicin, with its well-known cardiotoxicity, which results in myocardial fibrosis and necrosis via ROS production [25,26]. Myopathy refers to diseases that affect skeletal muscles. Myopathies are usually degenerative, but they are sometimes caused by drug side effects, electrolyte imbalance, or chronic immune system disorders [27,28]. For chronic kidney disease patients, hyperphosphatemia is one of the side effects of long-term hemodialysis [1,2]. Our current study focuses on high P i and highlights the protein-protein interaction between myogenin and Nrf2 via the modulation of ROS, which can serve as potential therapeutic targets for developing drugs reducing the risks associated with hemodialysis [5].
These myopathies are often related to a drug's ability to alter the balance of metabolism and protein, induce necrosis, or impair autophagy [28]. In our drug-screening results, we identified many potential drugs, including metformin and phenformin. The effects of metformin on muscle are still uncertain and, therefore, a matter of debate [29,30], because metformin use does not alter mTOR expression, thus appearing to negate this theory [31]. The use of metformin in preventing the development of sarcopenia in older people with prediabetes is supported by the results of a clinical trial [32]. Vitamin D is related to muscle strength and frailty, which can be either dependent or independent of binding to its receptor, VDR [33][34][35]. Vitamin D supplementation is demonstrated to have beneficial effects in increasing muscle strength and performance [35]. Here, our screening data failed to support the role of vitamin D in rescuing the regulation of myogenin via the Nrf2/p62 pathway. However, the induction of VDR expression by high P i provides further issue in addressing the functional role of vitamin D/VDR and muscle differentiation. The nuclear localization of VDR as a transcription factor is more important than the degree of expression. Hence, we will address the transcriptional function of vitamin D/VDR in the presence of high P i in the future.
In addition to myogenin, retinoblastoma protein was identified as prompting the reversal of differentiation in terminally differentiated muscle cells [36]. The Alizarin Red S dye is a commonly used stain to identify calcium-containing osteocytes [21]. P i acts as an intracellular signaling molecule to regulate the expression of numerous osteogenic genes, including osteopontin and BMP-2 [37]. C2C12 is a pluripotent mesenchymal cell line for which osteoblast differentiation can be induced by BMP-2 [19,20]. In this study, we applied this dye to determine whether the decline of myogenin could reverse the differentiation status of C2C12 cells. In such a scenario, terminally differentiated C2C12 cells might be dedifferentiated into immature muscle cells or pluripotent mesenchymal progenitor, which might then differentiate into osteocytes in response to high P i . Our current data imply two possibilities, one in which high P i can cause differentiation of myogenin-deficient C2C12 cells into osteocytes, and the other where high P i causes misleading responses of Alizarin Red S. Without the solid evidence of osteocytic protein expression, we could not rule out the crosstalk between P i and calcium in the presence of a higher concentration of exogenous P i . We failed to observe a positive response of Alizarin Red S staining similar to the effect of hydrogen peroxide on myogenin expression. One study demonstrated that activation of ERK and p38 are both essential components in BMP-2 signaling that lead to the induction of the osteoblast phenotype in C2C12 cells [38]. The activation of the ERK signaling pathway by P i is biphasic, whereby the two phases are separated by a time interval of several hours. The second phase of ERK activation is required for gene regulation in cases where the first alone is insufficient. In our current data, high P i decreased the activation of the ERK signaling pathway in C2C12 cells, suggesting that exogenous high P i disfavors the osteoblastic commitment process in our study condition.
L-AA induces hydrogen peroxide formation at pharmacologic concentrations (>100 µM) achieved by intravenous administration [24,[39][40][41], in contrast to physiological concentrations that neutralize the damage of ROS. In our study, physiologic concentration L-AA failed to serve as a ROS scavenger as NAC for the relief of P i effects on C2C12 cells, whereas pharmacological concentration L-AA exacerbated the effects of P i in C2C12 cells. Our Figure 5C data showed that NAC had the ability to rescue the effect of hydrogen peroxide on myogenin, but pharmacological concentration L-AA exacerbated this effect ( Figure 7C). In normal cells, hydrogen peroxide can initiate Fenton's reactions and cause oxidative damage to cellular macromolecules [42][43][44]. The recycling of endogenous ascorbate enables continuous production of hydrogen peroxide which supports pharmacological concentration L-AA exacerbated the effects of P i on C2C12 cells in this study. Many studies demonstrated that the steady-state levels of ROS stress in cancer cells are higher than in normal cells and L-AA kills some tumor cells via hydrogen peroxide-related mechanisms but not normal cells [45,46]. In addition to the involvement of redox homeostasis, L-AA can also serve as a cofactor for the Fe 2+ -2-oxoglutarate-dependent dioxygenase family for the stability of hypoxia-inducible factor 1 alpha (HIF-1α) and ten-eleven translocation 2 (Tet2) DNA hydroxylases, which catalyze the demethylation of modified DNA [40,41,[47][48][49]. Hence, the applications of L-AA as a repurposing drug for the modulation of the redox homeostasis involved in ROS and hydrogen peroxide productions and the enzymatic activity of dioxygenase involved in HIF-1α and Tet2 protein stabilities remain highlight potential.

C2C12 Myotube Culture and P i Treatment
Mouse C2C12 myoblasts seeded into 6-well plates at a density of 2 × 10 5 cells/well were first cultured for 3 days in DMEM/HG (Dulbecco's Modified Eagle's Medium/high glucose (4500 mg/L)) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Waltham, MA, USA) at 37 • C under a 5% CO 2 . They were then cultured for an additional 4 days in differentiation medium consisting of DMEM/HG supplemented with 2% adult horse serum (HS) (Thermo Fisher Scientific). Sodium phosphate buffer (1 M Na 2 HPO 4 /NaH 2 PO 4 (pH 7.4)) was then added to achieve the final Pi concentrations, and the C2C12 myotubes were incubated for 24 h.

Immunoblotting
C2C12 cells were lysed in RIPA lysis buffer (50 mM HEPES (pH 7.6), 150 mM sodium chloride, 0.5% Triton X-100, 10% glycerol, and 0.1% dodecyl sulfate) for 10 min at 4 • C. The resultant protein extracts (30 µg) were separated by 12% SDS-PAGE at 80 V and electro-transferred onto PVDF membrane (Immobilon-P; Millipore, Bedford, MA, USA) using a semi-dry Trans-Blot Turbo System (Bio-Rad, Hercules, CA, USA) at 25 V, 1.0 A for 30 min. To check transfer efficiency, Ponceau S staining was performed on PVDF membranes. The membrane was blocked with 5% nonfat milk in PBS with 0.01% Tween-20 (TBS-T) for 1 h and then incubated with diluted primary antibodies (Table 1) at 4 • C overnight with shaking. The membrane was washed in TBS-T for 15 min and repeated three times and incubated with HRP-conjugated secondary antibodies at room temperature for 1 h. The membrane was then washed in TBS-T for 15 min and repeated three times. The immunoreactive proteins were detected using ECL TM Western Blotting Detection Reagent and Amersham Hyperfilm TM ECL (GE Healthcare, Waukesha, WI, USA).

Alizarin Red S Staining
Mouse C2C12 myoblast cells seeded into 12-well culture plates at a density of 1 × 10 5 cells/well were first cultured for 3 days in DMEM/HG supplemented with 10% FBS at 37 • C under 5% CO 2 and then cultured for an additional 4 days in differentiation medium consisting of DMEM/HG supplemented with 2% HS. The spindle-shaped C2C12 myoblast cells then differentiated and fused, gradually forming multinucleate myotubes. Sodium phosphate buffer (1M Na 2 HPO 4 /NaH 2 PO 4 , pH 7.4) was then added to the indicated Pi concentrations of 0, 0.5, 1, 2, 3, or 4 mM or treated with H 2 O 2 dose of 1 and 2 mM combined with P i (4 mM), NaPTH (100 µM), or NAC (10 mM), and C2C12 myotubes were incubated for 24 h. After treatment, the medium was carefully aspirated, and the cells were washed with Dulbecco's PBS w/o Ca 2+ /Mg 2+ twice. After carefully aspirating the PBS, the cells were fixed by incubation in 10% formalin for at least 30 min. After fixation, the formalin was aspirated, and the cells were washed with distilled water twice. The distilled water was then carefully aspirated, and the cells were stained with Alizarin Red S staining solution (Alizarin, 94%, A14404, Alfa Aesar (Ward Hill, MA, USA); the Alizarin Red S staining solution was prepared by dissolving 2 g powder in 100 mL distilled water, mixed, and pH adjusted to 4.1~4.3 with HCl or NH 4 OH) with incubation at room temperature in the dark for 45 min. After staining, the cells were washed with distilled water four times and PBS added to analyze the cells (calcium deposits are specifically stained bright orange-red by Alizarin Red S).

Flow Cytometric Analyses of Cell Cycle Profile and ROS Generation
The cell cycle profile was determined by measuring the DNA content of cells using fluorescence-activated cell sorting (FACS). Differentiated C2C12 cells were fixed in 70% ice-cold ethanol and kept at 20 • C overnight. Before analysis, the harvested C2C12 cells were washed twice with ice-cold PBS and stained with propidium iodide (PI) (5 µg/mL PI in PBS, 0.5% Triton X-100, and 0.5 µg/mL RNase A) for 30 min at room temperature in the dark. All the samples were analyzed using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA). The data were analyzed using the Cell Quest Pro software (BD Biosciences).
Cytosolic ROS levels were measured using the fluorescent indicator H2DCFDA (2 ,7 -dichlorodihydrofluorescein diacetate). Myotubes treated for 24 h with the indicated concentrations of P i were stained with 10 µM H2DCFDA at 37 • C. After incubation for 30 min, ROS levels were assessed using a FACSCalibur flow cytometer (BD Biosciences). The excitation and emission wavelengths were 488 and 525 nm, respectively.

Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
Total RNAs were isolated from differentiated C2C12 cells using TRIzol reagent (Invitrogen, Waltham, MA, USA). Reverse transcription for first-strand cDNA synthesis was carried out using MMLV reverse transcriptase (Epicentre Biotechnologies, Madison, WI, USA) with 1 µg of total RNA at 37 • C for 60 min. Specific primers ( Table 2) were designed and evaluated by the program "Pick Primers" on the National Center for Biotechnology Information website (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) (accessed on 1 December 2021). Primers, dNTP, and Taq DNA polymerase were added for subsequent PCR reactions, which were processed using a Veriti™ Simpli Thermal Cycler (Applied Biosystems, Foster City, CA, USA).

Statistical Analyses
The values are expressed as the mean ± SD of at least three independent experiments. All the comparisons between groups were conducted using unpaired two-tailed t-tests. The statistical significance was set at p < 0.05.

Conclusions
Our data demonstrate that myogenin might negatively regulate Nrf2 and p62 at the protein level, and Nrf2 is a repressor of myogenin expression. In addition to NAC, metformin, and phenformin, berberine, 4-CEP, cilostazol, and cilomilast were identified to diminish the induction of Nrf2 and p62 expression and the reduction of myogenin expression by high P i in the drug screening analysis. Doxorubicin and hydrogen peroxide reduced the amount of myogenin protein mediated through the Keap1/Nrf2 pathway, different from the mechanism of high P i . The dual functional roles of L-ascorbic acid were dependent on the working concentration, where concentrations below 1 mM L-AA reversed the effects of high P i on myogenin and those above 1 mM L-AA, applied in isolation, had effects similar to those of high P i on myogenin. L-AA exacerbated the effect of hydrogen peroxide on myogenin protein and did no influence the effect of doxorubicin on myogenin protein.