Role of PAX7 in Muscular Dystrophies: Comparison
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Myogenesis is a series of progressive development of skeletal muscle tissue over a lifetime where myoblasts, the early mononucleated committed precursor cells of skeletal muscle fuse together and differentiate into myotubes, the multinucleated muscle cells that later undergo further differentiation and fusion to form myofibers. Myoblast heterogeneity stems from three types of myoblasts: embryonic, fetal, and adult myoblasts with distinct genetic backgrounds that are traditionally distinguished by desmin, myogenin (MyoG), and myosin heavy chain isoform (MyHC) expression. These myoblast transitions are thought to overlap at multiple points during myogenesis, due to the activation of several factors. Four myogenic regulatory factors (MRF), Myogenic factor 5 (Myf5), Mrf4, Myogenic Differentiation 1 (MyoD), and MyoG play critical roles in the precise differentiation of progenitor myoblasts into myofibers during embryonic-to-adult myogenesis. Pax7 is closely associated with myogenesis, which is governed by various signaling pathways throughout a lifetime and is frequently used as an indicator in muscle research. 

  • signaling pathways
  • muscular dystrophy
  • myogenesis
  • Pax7

1. Pax7 as a Key Indicator for Myogenesis

Pax7, also known as Paired-Box Gene-7, is a transcription factor that belongs to the PAX family and is strongly linked to adult satellite cells, which are precursors of skeletal muscle cells [1][2]. Satellite cells are derived from embryonic progenitor cells during development, which divide only a limited number of times, whereas adult satellite cell activation induces differentiation, proliferation, and fusion in muscle regeneration, transforming it into myofibers [3][4]. Quantifying the Pax7 gene and/or protein expression as a marker for determining the function of a novel molecular factor at different phases of myogenesis is unavoidable in muscular dystrophy research. Pax7-expressing satellite cells are well-established models for determining myoblast activation and myogenic status [5][6]. MyoD belongs to the basic helix-loop-helix transcription factor family, and is frequently used as a marker for myogenic and activated satellite cell identification in conjunction with Pax7 [6][7].

Pax7 and MyoD interact with each other during myogenesis, making them ideal markers for determining the cells’ quiescent, activation, and myogenic status. The Pax7 and MyoD proportions of satellite cell subpopulations have well-established symmetric expression patterns; Pax7+/MyoD−, Pax7+/MyoD+, and Pax7−/MyoD+ [6][8][9]. The Pax7+/MyoD− cell population pattern, known as self-renewal satellite cells, highly expresses Pax7 to keep the cells quiescent, and is necessary for the maintenance of stem cell homeostasis [5][10]. Pax7+/MyoD+ cells are activated satellite cells that express myogenic factors and permit the cells to differentiate and proliferate, whereas Pax7−/MyoD+ cells are differentiating cells that are also controlled by other MRFs [11]. The symmetric expression pattern quantification is also often quantified using Pax7 and Myf5 instead of MyoD.

To investigate muscle regenerative capacity, Fujimaki et al. and Pisconti et al. measured Pax7 gene and/or protein expression levels in satellite cells during all phases of myogenesis before and after injury. Differential expression of Pax7 as a proliferation marker was observed at different degrees of injury to reveal the state of the satellite cell niche, where Fujimaki et al. discovered that Pax7+ cell number per myofiber in satellite cell-specific conditional knockout mice for Notch1/Notch2 (scDKO) is almost entirely ablated compared to control mice after 5 and 19 days of final tamoxifen (TMX) treatment, suggesting impaired muscle regeneration. Pax7+/MyoD− cell population expansion in scDKO mice was suppressed, which agreed with the reduction of proliferative cells and induced MyoG expression, compared to control mice. Fiore et al. investigated the muscle fiber regenerative capacity even after acute injury by observing the exogenous and endogenous expression of Pax7, which reflects the self-renewable and myogenic ability of satellite cells at different ages of the mice model. The team quantified the number of Pax7+ cells in tibialis anterior (TA) sections of the mice at different ages, of 6 weeks, 12 weeks, 6 months, and 12 months old, and compared calcium-dependent and phospholipid-dependant protein kinase (PKCθ) null mice with the controls. They found that PKCθ null mice showed over 50% more Pax7+ cells than the controls. The presence of Pax7+/MyoD− and Pax7+/MyoD+ cell populations implied an increase in both quiescent and proliferative cells in PKCθ null mice. Furthermore, Pisconti et al. discovered that the cell population, Pax7−/Myf5+, was approximately 25% higher in transmembrane heparan sulphate proteoglycan syndecan-3 (Sdc3) null dystrophic mice (Sdc3−/−) than in wild-type (WT), indicating that reduced Pax7 expression in the absence of Sdc3 resulted in a reduction of self-renewable cells. In contrast, the Pax7+/Myf5+ population proportion in Sdc3−/− mice was nearly twofold that of WT, indicating an increase in proliferating myogenic progenitors, and histopathology analysis revealed improved muscle fibrosis followed by decreased sarcolemmal permeability.
Servián-Morilla et al. discovered the D233E mutation on POGLUT1, a gene asssociated with enzymatic activity related to glycosylation pathway(s). α-dystroglycan is an essential component of the dystrophin-glycoprotein complex at the sarcolemmal junction which connects the extracellular matrix to the cytoskeleton through Laminin-2 [12]. Any mutation in POGLUT1 may induce disruption in α-dystroglycan, which therefore compromises the integrity of the dystrophin-glycoprotein complex, leading to degenerative muscle diseases including Duchenne Muscular Dystrophy (DMD) [13][14][15] The mutation resulted in a decrease in Pax7 mRNA level and Pax7+ cells, indicating a loss of the self-renewal ability of satellite cells in the patients’ muscle compared to controls, presumably due to the flawed O-glucosylation. They thus propose that impaired myogenesis mediated by inactivation of Notch signaling is a causal mechanism of muscular dystrophy in siblings in relation to the post-translational modification and maintenance of muscle stem cells, leading to muscular degeneration and α-dystroglycan hypoglycosylation. The essential role of measuring Pax7 in muscular dystrophy research has been demonstrated in all phases of muscle development, in the effort to characterize some novel molecular factor functions, as it is dependent on the ability of satellite cells to regenerate.

2. Pax7 in the Context of Embryonic, Fetal, and Adult Myogenesis

Muscle development occurs as a sequential event, starting with embryonic, to fetal, and ending with adult myogenesis. The majority of embryonic myoblasts and satellite cells contain Paired-Box Gene-3 (Pax3), a member of the PAX family of transcription factors, which is a pivotal gene involved in organ development [16][17]. Satellite cells which are capable of self-renewal and myogenic differentiation facilitate postnatal muscle formation and regeneration. The remnants of embryonic-origin Pax7-expressing cells from development give rise to post-embryonic myogenesis, and are required for fetal myogenesis as well as adult skeletal muscle regeneration, which is critical for skeletal muscle repair. Using a dyW mice model, which is derived from homologous recombinant in mouse embryonic stem (ES) cells with laminin alpha 2 (Lama2)-knockout [18], Nunes et al. explored the merosin-deficient congenital muscular dystrophy type 1A (MDC1A) to trace the advent of the disease during muscle development in utero. They observed the homozygous dyW mice embryo to have significantly smaller muscle than the wild type at the fetal age of E15.5 to E18.5, although expressing the same number of myofibers. They discovered that the increase in the Janus kinase/signal transducers and activators of the transcription (JAK-STAT) signaling pathway, together with the decreased expression of myostatin and Pax7, caused the satellite cells to lose their normal level of self-renewal capacity. Nunes et al. also discovered an increase in MyoD expression at E18.5, as the cells exited the cell cycle faster, and reduced the differentiation and fusion rate. dyW mice had an aging phenotype that could not recover during the development event, due to low Pax7 levels, reflecting a low level of self-renewal capacity. Fetal myogenesis is closely associated with the formation of myofibers (hyperplasia) and myofiber fusion (hypertrophy). Improper execution of this sequential event may result in fewer differentiated and competent cells in utero.
Farini et al. investigated the muscle development stage in mice model fetuses and muscular biopsies from 12-week-old DMD and healthy human fetuses, using histological evaluation. They revealed a decrease in fiber density, where dystrophic fetal muscles expressed significantly higher levels of fast-type genes such as sarcoplasmic reticulum calcium ATPase-1 (ATP2A1), troponin T type 3 (TNNT3) and troponin fast C Type 2 (TNNC2). Dystrophic muscle is known to have dystrophin protein loss, which causes membrane tears and sarcolemmal destabilization, resulting in abnormal Ca2+ reflux [19][20]. Farini et al. examined myogenic marker expression in 12-week-old healthy and DMD human fetal muscles, and discovered a significantly higher number of Pax7+ cells per myofiber in DMD samples compared with healthy. PKCα is a serine/threonine kinase belonging to the PKC family, and it is involved in numerous cellular processes. In human fetal dystrophic muscle, PKCα is upregulated, and its activity is regulated by Ca2+ levels, contributing significantly to muscle development and plasticity. They hypothesized that inositol 1,4,5-trisphosphate (IP3)-IP3R binding-mediated Ca2+ signaling determines calcium cell accumulation in DMD muscle. Farini et al. discovered that enhanced PKCα activation causes altered myogenesis in DMD muscles, by overexpressing Pax7 while suppressing MyoD, along with a delay in fiber maturation and a modification in fiber type composition. Therefore, by attuning the IP3/IP3R pathway, satellite cells could exit the stemness state and express myogenic markers accordingly, during myogenesis, for the next regeneration cycle, and limit DMD muscle damage by Ca2+ deposition.
Tierney et al. in their research in 2016 demonstrated the extracellular matrix (ECM) function in muscle stem cell regulation at different stages of muscle development. Myogenesis regulation in prenatal muscle stem cells focuses primarily on cellular contribution to myofibers, laying the foundation for subsequent muscle growth. Conversely, in postnatal or adult muscle stem cell myogenesis, the regulation is more focused on the balance between muscle growth and repair, as well as the maintenance of the stem cell pool population for the next regeneration. Fetal muscle stem cells adapt to the microenvironment by autonomously secreting ECM. This leads to stem cell expansion support and enhances stem cell function intrinsically by actively dividing and maintaining the Pax7 expression level in growing conditions. They revealed that in differentiation conditions, Pax7 expression increased while Myf5 and MyoG expression decreased, showing that fetal muscle stem cells resist myogenic lineage progression. This implies that fusion efficiency increases during development. Fetal muscle stem cells expressed higher level of Notch targets and a reduced level of canonical Wnt targets, compared to activated adult muscle stem cells, suggesting innate and robust expansion. Adult muscle stem cells profited from co-transplantation of fetal and adult muscle stem cells by boosting Pax7 expression. ECM molecule remodeling was influenced by fetal stem cells in a paracrine manner, improving adult stem cells’ regenerative potential for long-term self-renewal, implying that fetal muscle stem cells are responsible for local microenvironmental remodeling and promoting neighboring adult muscle stem cells’ regenerative potential. They concluded that fetal muscle stem cells have enhanced regenerative potential during tissue repair and the ability to repopulate the stem cell pool during development. Fetal muscle stem cells possess remarkable regenerative potential through more efficient expansion and selective expression of ECM proteins to remodel their local microenvironment.
In a context of adult myogenesis, Jiang et al. investigated how satellite cell number and activity decline with age in the mdx mice model. The mdx model is a universal mice model used as DMD murine strain with a point mutation in the dystrophin gene, and exhibits DMD phenotypes [21][22][23]. They found that mdx mice expressed higher Pax7 than in the wild-type, at different age groups. Their findings were suggested severe age-dependent deficiencies in satellite cell activation and/or proliferation, causing the mice to have reduced satellite cell self-renewal capacity, due to ongoing muscle injuries. Mdx mice had a decrease in Pax7+/MyoD− cells but an increase in MyoG+/MyoD+ cells, indicating terminal differentiation. They demonstrated that aberrant Notch signaling, which was supposed to be activated and improve the self-renewal capacity, was actually responsible for defective satellite cell renewal in dystrophic muscles. Dystrophin and the Notch signaling cascade may be perturbed in mdx mice satellite cells in dystrophic muscles, as they play an important role in embryonic muscle formation and postnatal myogenesis. The Notch signaling pathway is inhibited in dystrophic muscle satellite cells, leading to a defective self-renewal capacity and potential side effects. The research demonstrated the significance of quantifying Pax7 in the sequential event of myogenesis from embryonic and fetal, to adult myogenesis, to understand the relevance of certain signaling pathways and the role of a novel molecular factor in the rescue of dystrophic muscle.

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