ATF4 is considered to be a key regulator of the ISR, and it is also involved in the regulation of myogenic differentiation. During myogenic differentiation, the transcription factor c-Myc is thought to promote myoblast proliferation and hinder myoblast differentiation by regulating the expression of its target genes, miRNAs and lincRNAs (Luo et al., 2019). Recently Memme JM et al. (Memme et al., 2023) found that ATF4 can affect myotube formation via c-Myc and MyoD and also control mitochondrial biogenesis via the PPAR-gamma coactivator-1alpha (PGC-1α). In addition, Sestrin2 (SESN2) is thought to be a downstream gene of ATF4 and is involved in mitochondrial stress-induced ISR (Garaeva et al., 2016). Piochi LF et al. (Piochi et al., 2021) proposed a theoretically possible role for SESN2 during myogenic differentiation: after the onset of differentiation, an increase in SESN2 expression, which induces the activation of AMP-activated protein kinase (AMPK) and the upregulation of nuclear factor erythroid 2-related factor 2 (NRF2) transcriptional levels, could enhance the efficiency of differentiation through three pathways:a) promotion of mitophagy b) generation of antioxidant responses c) increase in mitochondrial biogenesis. Meanwhile, another article (Brearley et al., 2019) noted that mRNA transcript levels of ISR-related genes, including SESN2 and ATF5, showed a similar trend with MyoG, reaching a maximum after about 48 h of induced differentiation and then starting to decline. It is suggested that SESN2 and ATF5 may coordinate MyoG to play some roles in the differentiation of the mouse myoblast cell line C2C12, but experiments are still needed to further verify this.

CHOP is an important transcription factor regulating the ISR, and Alter J et al. (Alter and Bengal, 2011) demonstrated that CHOP can inhibit MyoD transcription to delay myoblast differentiation. Normally, CHOP expression is downregulated early in the differentiation of C2C12 cells. Upon knockdown of CHOP, the fusion index and differentiation rate of C2C12 cells were significantly increased. In contrast, overexpression of CHOP significantly delayed C2C12 cell differentiation, and the resulting result was caused by CHOP inhibition of MyoD. This suggests that downregulation of CHOP expression is required for the activation of the myogenic differentiation programme in C2C12 myoblasts.

Although no direct evidence for the involvement of ATF5 in myogenic differentiation was found, the differentiation effects of ATF5 for tissues such as bone, liver, brain and adipose were well characterised (Sears and Angelastro, 2017). This suggests that ATF5 has more potential roles in developmental and normal physiological processes, and may provide new research directions for exploring the regulatory mechanisms of myoblast proliferation and differentiation.

The effect of the OMA1-mediated DELE1-HRI axis on myogenic differentiation has not been reported in detail. Whether the OMA1-mediated DELE1-HRI axis is then a necessary pathway for the activation of the ISR-associated transcription factors ATF4, CHOP, and ATF5 involved in myogenic differentiation is worth exploring subsequently. Although, a direct effect of this signaling axis on myogenic differentiation was not seen, it was described in a study of mitochondrial myopathy. CHCHD10, a nuclear gene-encoded mitochondrial protein, is mainly situated in the mitochondrial intermembrane space (IMS) and, in conjunction with its homologue CHCHD2, has a vital function in preserving mitochondrial inner membrane integrity and electron transport chain (ETC) function (Jiang et al., 2022). Mutations in the CHCHD10 G58R gene can lead to mitochondrial myopathy. According to this study (Shammas et al., 2022), OMA1 is activated in response to misfolding of the CHCHD10 G58R protein. This activation triggers the ISR through the OMA1-DELE1-HRI signaling pathway, ultimately providing a protective effect that prolongs the survival of newborn infants with CHCHD10 G58R gene mutations. Given the correlation between mitochondrial myopathy and myogenic differentiation, this suggests that the OMA1-mediated DELE1-HRI axis may have an effect on myogenic differentiation.

ClpP is an important protease within the mitochondrial matrix involved in the initiation of UPRmt. ClpP expression is higher in skeletal muscle compared to other tissues (Bross et al., 1995). In C2C12 cells after ClpP knockdown (KD), Drp1 was upregulated, leading to altered mitochondrial morphology, while the ETC subunit protein expression level was reduced, attenuating the activity of the ETC complex and thus affecting mitochondrial respiration, suggesting that ClpP is essential for maintaining mitochondrial function in myoblasts (Deepa et al., 2016). In addition, treatment of ClpP KD C2C12 cells using doxycycline (UPRmt inducer) showed no upregulation of the UPRmt-associated protein Hsp60 in both the dosed and undosed groups, suggesting that ClpP is one of the major pathways for mitochondrial stress-induced activation of UPRmt. Moreover, in the experimental group of ClpP KD C2C12 cells, enhanced expression levels of PKR and phosphorylation of eIf2α (p-eIf2α) were also found, whereas the expression of myogenic differentiation-associated proteins, such as MyHC and MyoG, was not upregulated, suggesting that myogenic differentiation is significantly impaired when protein translation is inhibited (Deepa et al., 2016). The aforementioned findings indicate that ClpP has an impact on myogenic differentiation via UPRmt and also plays a role in regulating myogenic differentiation through ISR. This highlights the strong correlation between UPRmt and ISR.

HtrA2 is a protease located in the mitochondrial IMS that is involved in UPRmt to maintain mitochondrial homeostasis (Riar et al., 2017). Previously published results from our team (Sun et al., 2022) showed that inhibition of HtrA2 enzyme activity using UCF101 resulted in blocked differentiation of C2C12 myoblasts. Considering that the loss of HtrA2 enzyme activity-induced proteostasis imbalance and mitochondrial IMS stress are the main triggers of UPRmt, we hypothesised that UPRmt could be associated with impaired differentiation due to HtrA2 enzyme activity inhibition. To this end, C2C12 cells in the differentiated state were treated with UCF101, and Western blotting results showed upregulation of the expression of the UPRmt-related protein CHOP, the HSP family of molecular chaperones, and the LonP1 protein. This suggests that loss of HtrA2 enzyme activity activates UPRmt to inhibit the translation of myogenic differentiation-related proteins, resulting in blocked myogenic differentiation.

The effect of the mtROS/c-mtProt-driven DNAJA1-HSF1 axis on myoblast differentiation has not been reported in detail. However, relevant signaling molecules targeting this axis have been described. A study (Kim et al., 2018) indicated that mtROS rapidly increases during differentiation and that mtROS can activate the phosphatidylinositol three kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway by inducing tensin homolog deleted on chromosome 10 (PTEN) oxidation. After activation, mTORC1 promotes phosphorylation at uncoordinated-51 like kinase 1 (ULK1) S317 and upregulates the expression of Atg protein, leading to autophagy and participating in the myogenic differentiation process. In addition, several studies have shown that heat shock proteins (HSPs) are involved in myogenic differentiation (Table 1). While muscle repair is significantly diminished in aged mice undergoing severe injury, aged mice overexpressing HSP70 reversed this outcome, which may be related to the promotion of myoblast fusion by HSP70 (McArdle et al., 2006; Thakur et al., 2020). HSPs are transcriptionally regulated by two members of the heat shock transcription factor family, HSF1 and heat shock factor 2 (HSF2), with HSF1 functioning primarily by responding to a variety of stressful environments, while HSF2 functions under non-stressful conditions such as differentiation and development (Santopolo et al., 2021). It has been shown (McArdle et al., 2006) that during myogenic differentiation, HSF1 levels increase significantly in the late stages, whereas HSF2 levels increase rapidly in the early stages and then return to normal levels. It is suggested that the early development and recombination of skeletal muscle cells is regulated by HSF2, and the ability of cells to respond to acute stress depends mainly on HSF1 and the UPRmt regulated by it.

In conclusion, both ISR and UPRmt are mitochondria-nuclear interactive regulatory responses, which can activate the same signaling molecules to exercise different signaling pathways (Roca-Portoles and Tait, 2021). Some literature indicates that in mammals, ISR is a prerequisite for the induction of UPRmt, as the expression of the three basic leucine zip (bZIP) transcription factors ATF4, CHOP, and ATF5 involved in UPRmt requires the activation of ISR (Rainbolt et al., 2013; Melber and Haynes, 2018; Anderson and Haynes, 2020). A typical example is arsenite-induced MSRs (Rainbolt et al., 2013), in which arsenite first activates the ISR and the transient phosphorylation of eIF2α leads to a reduction in Tim17A synthesis, which reduces the import efficiency of mitochondrial proteins and induces the expression of UPRmt-related genes. It has also been noted in the literature (Khan et al., 2017) that UPRmt is part of ISRmt. This interactive regulation at the level of front and back, whole and part, reflects the close connection between the two. Even though both are “saviours” of the cell against stress, myogenic differentiation is affected during differentiation, whether they are over-activated or inhibited (Figure 1). This is because the general translational decline caused by overactivation affects the expression of differentiation-related proteins, and overrepression weakens the ability of cells to withstand stress during differentiation. It is thus clear that the interactive effects between such signaling pathways induced by the stress response are extremely complex, and the mechanisms of their regulation on myogenic differentiation still need to be further explored.

The process of myogenic differentiation is accompanied by increased levels of mitochondrial biogenesis, resulting in an increase in the number and activity of mitochondria, enabling myoblasts to adapt to stressful conditions and increase cell viability (Fortini et al., 2016; Piochi et al., 2021). As a transcriptional co-activator, the role of PGC-1α in myogenic differentiation has been elucidated. PGC-1α serves a dual role in both triggering mitochondrial biogenesis to increase mitochondrial mass and buffering the expression of genes involved in mitophagy mediated by ROS/FOXO1 (Baldelli et al., 2014). This not only indicates that PGC-1α is an important integrating molecule for mitochondrial biogenesis and mitophagy, but also suggests that there is a close collaboration between mitochondrial biogenesis and mitophagy during myogenic differentiation. A study (Sin et al., 2016) showed that downregulating PGC-1α in C2C12 myoblasts increases ROS generation, causes mitochondrial damage, and leads to poor differentiation. In contrast, in vivo studies in mice, upregulation of PGC-1α expression alters MRFs and exhibits a positive protective effect on skeletal muscle regeneration by promoting myogenic differentiation (Washington et al., 2022). In addition, some important transcription factors, such as NRF1/2 and TFAM, have been found to show a trend of transcriptional upregulation during differentiation (Kraft et al., 2006; Remels et al., 2010), and NRF1 has been shown to mediate PGC-1β-induced mitochondrial biogenesis and activation of cellular respiration in conjunction with ERR alpha (Shao et al., 2010). So far, a number of studies have shown that activation of mitochondrial biogenesis-related signaling pathways can promote myogenic differentiation (Li et al., 2007; Barbieri et al., 2016; Lee and Choi, 2018; Niu et al., 2021; You et al., 2023). Thus, mitochondrial biogenesis plays an important role in myogenic differentiation.

There is a link between mitochondrial dynamics and myogenic differentiation (Figure 2). In the early stages of muscle differentiation, mitochondrial fission is activated while fusion is inhibited, resulting in a loose mitochondrial network; in the later stages, the opposite occurs, resulting in a tight mitochondrial network (Huang et al., 2020). This fully reflects the adaptive response of mitochondrial fusion and fission activity to environmental changes during myogenic differentiation. It was recently shown (Luo et al., 2021) that the expression of MFN2 proteins involved in mitochondrial fusion is upregulated during myogenic differentiation, and that myoblasts isolated from MFN2-null mice exhibit abnormal mitochondrial respiration with elevated ROS. However, interestingly, these alterations do not negatively affect the proliferation and differentiation of myoblasts, which may be due to a potential compensatory function of MFN1 that prevents these alterations from affecting muscle development and regeneration (Luo et al., 2021). In conjunction with some existing work a) inhibition of dihydroorotate dehydrogenase (DHODH) activity increases MFN2 expression and promotes mitochondrial fusion via the pyrimidine de novo synthesis pathway (Miret-Casals et al., 2018) b) knockdown of MFN2 limits the synthesis and consumption of aspartate, a raw material for the pyrimidines de novo synthesis (Yao et al., 2019) c) DHODH is localised in the inner mitochondrial membrane and is linked to the respiratory chain via the coenzyme Q pool and is involved in oxidative phosphorylation (Boukalova et al., 2020) d) DHODH in skeletal muscle mitochondria can produce H₂O₂ directly or indirectly (Hey-Mogensen et al., 2014). This suggests that there may be a feedback mechanism for MFN2 knockout to affect DHODH activity through the pyrimidine de novo synthesis pathway. If so, can it be argued that the abnormal mitochondrial respiration and elevated ROS exhibited in MFN2-null cells may be caused by DHODH affecting the mitochondrial respiratory chain? Further work is needed to verify this. This further suggests the potential involvement of DHODH in myogenic differentiation through the regulation of MFN2-mediated mitochondrial fusion. In addition to MFN2, another key molecule of mitochondrial fusion, OPA1, has also been reported to have an effect on myogenic differentiation, and cells under oxidative stress induced by the application of tert-butylhydroperoxide (t-BHP) or FCCP treatments to C2C12 cells can inhibit myogenic differentiation by cleaving OPA1 to reduce MyHC expression (Wang et al., 2014).

The effect of mitochondrial fission activity on myogenic differentiation has been described. One study (Kim et al., 2013) noted that inhibition of Drp1 activity using Mdivi-1 impairs myogenic differentiation. Interestingly, another study (De Palma et al., 2010) pointed out that nitric oxide inhibition of Drp1-mediated mitochondrial fission is an essential step in the process of early myogenic differentiation, which seems to contradict the previous findings. Further studies are needed to fully explain this discrepancy. In addition, a study on muscle regeneration after frostbite (Wagatsuma et al., 2011) found that Fis1 expression was upregulated early in muscle regeneration, along with its concomitant upregulation of the myogenic differentiation-associated proteins MyoD and MyoG. This suggests that mitochondrial fission may not only regulate myogenic differentiation by affecting mitochondrial dynamics and morphological alterations, but also that mitochondrial fission-associated proteins may be involved as signaling molecules in regulating the expression of factors associated with myogenic differentiation. These findings re-emphasise that tight regulation of the activity of mitochondrial fusion and fission proteins and, more importantly, the maintenance of a dynamic balance between mitochondrial morphology and function are essential for the initiation and progression of myogenic differentiation.

McMillan EM et al. (McMillan and Quadrilatero, 2014) demonstrated for the first time that myogenic differentiation requires the involvement of autophagy, which prevents apoptosis generated during myogenic differentiation. Afterwards, some workers directed their research specifically to mitophagy to explore the link between mitophagy and myogenic differentiation. Sin J et al. (Sin et al., 2016) proposed that the shift in energy metabolism from glycolysis to oxidative phosphorylation during the differentiation of myoblasts to myotubes requires the involvement of mitophagy. Notably, Brown HMG et al. found that mitophagy is a transient phenomenon that is specifically upregulated only in the early stages of differentiation (Figure 2), whereas the overall cellular autophagic flux persists throughout the differentiation phase (Brown et al., 2021). Mitochondrial network remodeling is an important feature of myogenesis (Rahman and Quadrilatero, 2021). One study (Li et al., 2022) claimed that tetrandrine can inhibit myogenic differentiation by blocking autophagic flux leading to impaired mitochondrial network remodeling, which shows that autophagy-mediated mitochondrial network remodeling can also have an effect on myogenic differentiation. The impact of mitophagy on the entire myogenic differentiation event is profound, specifically regulating multiple aspects of mitochondrial network remodeling, oxidative stress, ER-associated stress, mitochondria-mediated apoptotic signaling that occur during differentiation (Baechler et al., 2019; Jiang et al., 2021).

(1) Impact of SLR-dependent mitophagy pathways on myogenic differentiation

PINK1, an important participant in mitophagy (Lazarou et al., 2015), has been shown to affect myoblast differentiation, and interfering with PINK1 using siRNAs in C2C12 cells leads to a decrease in the transcriptional level of MyoG (Baldelli et al., 2014). Similarly, PRKN, another important player in mitophagy, has now been shown to be recruited into mitochondria during early differentiation of C2C12 (Esteca et al., 2020), which supports the conclusion that mitophagy is involved in early myogenic differentiation as suggested by Sin J et al. (Sin et al., 2016). Importantly, PRKN deficiency results in myoblasts remaining in a proliferative state, delayed differentiation and even a reduction in myofiber area (Peker et al., 2018; Esteca et al., 2020); in contrast, overexpression of PRKN has been shown to increase muscle mass and strength in aged mice (Leduc-Gaudet et al., 2019). This suggests that PRKN is involved in the regulation of skeletal muscle growth and development. Moreover, Huang S et al. (Huang et al., 2020) found that LonP1 regulates mitochondrial network remodeling through the PINK1/PRKN-mediated mitophagy pathway to promote myogenic differentiation. This suggests that the regulatory role of Lonp1 on myogenic differentiation is not only limited to its role in UPRmt, but its role in mitophagy is also involved. It indicates that Lonp1 may be an integrated molecule of different MSRs during myogenic differentiation.

(2) Impact of SLR-independent mitophagy pathways on myogenic differentiation

Baechler BL et al. (Baechler et al., 2019) used CRISPR-Cas9 technology to construct a Bcl-2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3)^−/− C2C12 cell model, and the experimental results showed that knockout of BNIP3 impaired myogenic differentiation, which was associated with mitophagy defects. And supplementation of Insulin-like growth factor 1 could attenuate the impaired myogenic differentiation caused by defective mitophagy (Guan et al., 2022). Meanwhile, knockout of BNIP3 also caused increased caspase-3 and caspase-9 activities and DNA fragmentation, which could lead to apoptosis of myoblasts. Another mitophagy receptor, FUN14 domain containing 1 (FUNDC1), has not been found to have a direct correlation with myogenic differentiation but has been shown to be closely related to skeletal muscle mitochondrial function, and FUNDC1 deficiency leads to defects in mitophagy mediated by microtubule-associated protein light chain 3 (LC3), causing impaired skeletal muscle mitochondrial function (Fu et al., 2018). Of interest, FUNDC1 not only mediates mitophagy in skeletal muscle cells involved in the differentiation process, but also plays a role in the differentiation process of human cardiac progenitor cells (Lampert et al., 2019).

Apoptosis-related events (e.g., cytoskeletal reorganization (Qu et al., 1997), phosphatidylserine receptor expression (Hochreiter-Hufford et al., 2013)) are necessary for myogenic differentiation. However, either excessive or absent apoptosis affects myotube formation (Fernando et al., 2002), suggesting that the level of apoptosis must be tightly regulated during myogenic differentiation.

(1) Impact of Bcl-2 family-related members on myogenic differentiation

In summary, myogenic differentiation is a highly dynamic and tightly coordinated process, with mitochondrial dynamic processes such as mitochondrial biogenesis, mitochondrial fission, fusion, mitophagy, and apoptosis running through the entire process of myogenic differentiation (Figure 2), which regulates the mass, structure, function and distribution of mitochondria. Myogenic differentiation coupled with metabolic reprogramming ultimately leads to an increase in mitochondrial mass and OXPHOS to support newly formed myotubes, which requires replacement of the original mitochondria of myoblasts (Rahman and Quadrilatero, 2021). Thus, mitochondrial fission and subsequent activation of mitophagy are critical processes in the early differentiation of myoblasts. Interestingly apoptosis activates mitophagy, which in turn inhibits apoptosis, thus maintaining the stability of myogenic differentiation (McMillan and Quadrilatero, 2014; Baechler et al., 2019; Jiang et al., 2021). After pre-existing mitochondria are removed, mitochondrial biogenesis and mitochondrial fusion drive the formation of a dense network of mitochondria to complement and sustain cellular energy metabolism (Sin et al., 2016; Rahman and Quadrilatero, 2021). All these results suggest that the individual stress responses during myogenic differentiation are not separate entities but an intricate network. In addition, it is worth noting that the mitochondrial dynamics processes associated with the above are regulated by Ca²⁺ signaling. For example, Ca²⁺ stimulates mitochondrial biogenesis (Yeh et al., 2005). Ca²⁺ can regulate mitochondrial fusion and fission homeostasis by mediating Drp1 phosphorylation or dephosphorylation (Cribbs and Strack, 2007; Cereghetti et al., 2008). Mitochondrial permeability transition pore (mPTP) opening is a crucial event in the induction of mitophagy and apoptosis, and Ca²⁺ overload is one of the key factors in inducing mPTP opening (Rimessi et al., 2013). Interestingly, the current study also found that Ca²⁺ and its associated signaling molecules are strongly associated with myogenic differentiation. Earlier studies have shown that myogenic differentiation is accompanied by transmembrane inward flow of extracellular Ca²⁺ and that lowering extracellular Ca²⁺ concentrations inhibits myoblast fusion (Adamo et al., 1976; Naro et al., 2003). Sun W et al. (Sun et al., 2017) proposed that intracellular Ca²⁺ may promote myogenic differentiation via the MLCK-MLC-myosin-actin pathway, a signaling pathway that mediates cytoskeletal dynamics. Meanwhile, some important molecules involved in the regulation of Ca²⁺ signaling, such as calcineurin, may be involved in the early process of myogenic differentiation by regulating nuclear factor of activated T cells 3(NFATc3) to promote MyHC expression (Delling et al., 2000); MyoG expression requires the activation of calmodulin-dependent protein kinase II (CaMII) (Terruzzi et al., 2017); S100 calcium binding protein B (S100B) is a chiral EF Ca²⁺-binding protein that is downregulated in the early stages of myogenic differentiation as an inhibitor of myogenic differentiation, whereas in the late stages S100B expression is upregulated and reduces apoptosis through activation of NF-κB (Tubaro et al., 2011). It remains to be elucidated whether Ca²⁺ can be involved in the regulation of myogenic differentiation by influencing mitochondrial dynamics.