Introduction
Skeletal muscle is the most abundant tissue in the human body, accounting for about 40–45% of total body weight. It plays an important role in controlling physical activity, including voluntary locomotion, postural behavior, and breathing. Moreover, it has an extraordinary ability to adapt to physiological demands, such as growth, and to regenerate new muscle fibers after damage by injury or intense physical activity [
1].
The regeneration and remodeling of skeletal muscles are extremely complex biological processes, in which skeletal muscle stem cells (also known as satellite cells, SCs) are involved. The SCs are located under the basal lamina of the myofiber; this position, between the myofiber and the surrounding extracellular matrix (ECM), is the reason Alexander Mauro gave them this name in 1961 [
2]. In healthy adult mammalian muscle, SCs are predominantly quiescent (phase G
0) and represent 2.5–6% of all nuclei of a given fiber; however, after injury or degeneration, SCs become activated and can generate large numbers of new myofibers within just few days [
3]. Like stem cells, satellite cells also self-renew to maintain their own population, re-establishing their numbers and quiescent state by homing back to highly specialized niches, thus allowing future rounds of regeneration [
4]. Specific temporal factors, called Myogenic Regulatory Factors (MRFs), members of the basic helix-loop-helix (bHLH) family of transcription factors including Myf-5, MyoD1, Myogenin and MRF4, are an essential group of four muscle-specific proteins responsible for acting at multiple points in the muscle lineage to cooperatively establish the skeletal muscle phenotype [
4,
5].
Besides its well-known structural and biomechanical functions for the purpose of movement, the skeletal muscle is considered a secretory organ, capable of producing several substances called myokines, which can act on the muscle itself, on nearby tissues, and on distant organs, in an autocrine, paracrine and endocrine fashion, respectively [
6‐
8]. These functions can be physiologically modulated by physical stimuli and by cytokines, mineral ions and hormones, or can be modified by sarcopenia, morphological modification of muscle fibers and/or endocrinopathies.
The evaluation of the skeletal muscle as a secretory organ is not fully understood, neither it is the endocrine control of muscle function and differentiation [
9]. A future chapter in the endocrine discipline will certainly be musclecrinology. The aim of our study was to evaluate the endocrine machinery in an in vitro cellular model of myogenesis obtained from human skeletal muscle biopsies. The understanding of hormonal production and regulation in the skeletal muscle remodeling may contribute to the identification of new possible therapeutic targets in pathologies in which the myogenesis and/or the function of mature myocytes is affected.
Discussion
The last decade has been an exciting period for the study of the biology of skeletal muscle stem cells and tissue regeneration and the development of novel human in vitro cell models can contribute to the identification of new mechanisms that control myogenesis [
14‐
20]. These human cell cultures appear more suitable for predictive screening strategies when compared to rodent cell lines, such as C2C12 or rat L6 myoblasts [
21,
22].
In this study, we have isolated and characterized skeletal muscle-derived cells from human biopsies to be used for the in vitro study of myogenesis. The developed cellular model was enriched in satellite cells, as confirmed by analyzing the presence of PAX-7 in more than 99% of the hSkMCs and the disappearance of the PAX-7 gene with each passage in culture [
23]. For this reason, we decided to use cells not over the fourth passage in vitro.
Although CD34 antigen is a marker of satellite cells in murine models, our results indicate that, in human SCs, CD34 expression is not the hallmark of SCs, as it is present in only 0.5% of hSkMCs [
24]. The same is true for CD56 antigen, which is present in only 9% of the isolated hSkMCs, a result that may be controversial considering other data in the literature, since it also marks natural killer lymphocytes [
25,
26]. Flow cytometry analysis in hSkMCs has permitted the verification of the presence of the principal markers to be expressed on the surface of mesenchymal stem cells, specifically CD44, CD105 and CD90, and the negativity of the hematopoietic CD45 antigen, confirming the mesenchymal stemness of our cells, as largely recognized in the literature. The multipotentiality of the isolated hSkMCs was confirmed by demonstrating their own capacity to differentiate into the adipogenic, osteogenic and myogenic phenotypes, as assessed by cytochemical staining performed on cells, properly induced with specific differentiating media, as reported in the literature [
27].
Using appropriate differentiation medium with confluent cells at 70–80% density in the plate, we were able to induce the alignment of the activated cells (myoblasts), the subsequent fusion with each other and, finally, the differentiation into multinucleated myofibers. Gene expression analyses have shown significant increases of MRFs, MyoD-1, MRF-4, and Myogenin, 9 days after myogenic induction which, together with Desmin and MHC gene expression augmentation during the entire study period, have confirmed myoblast determination and muscle differentiation, confirming the suitability of our in vitro myogenesis model. The only non-significance increase in gene expression was detected for the Myf-5 gene, in agreement with data reported in the literature. The reason may the fact that this transcription factor is the main controller of the activation of SCs toward myogenic differentiation and is expressed in a majority of quiescent SCs in adult muscle [
3]. It is also reported that Myf-5, in the absence of MyoD-1, MRF-4 and Myogenin during development, is unable to drive myogenic differentiation, so Myf-5 may contribute to controlling "stemness" in the niche [
4] [
28].
Once validated the in vitro model, we embarked in the project of characterization of the endocrine machinery in the different stages of myocytic differentiation. This analysis was never performed before in in vitro models. The analysis was focused on selected receptor genes known to mediate a specific hormonal action on skeletal muscle tissue or suggested to mediate an endocrine action on skeletal muscles: VDR, TRα, TRβ, IGF-1R, PTH-1R, GCR, LRP-5 and LRP-6.
In the literature, it has been reported that vitamin D deficiency is a condition associated with skeletal muscle weakness and small muscle fiber size [
29]. In animal models, the skeletal muscle dysfunction observed in vitamin D deficiency is reversed by vitamin D repletion, whereas vitamin D supplementation in humans has been found to increase skeletal muscle strength [
30]. Many reports suggest that the VDR is expressed in skeletal muscle [
31]. VDR deletion in mice results in alterations in muscle function and strength [
32], and its association with interleukin-6 may play a role in intramuscular inflammation [
33].
The thyroid hormone plays an essential role in myogenesis; it acts as a pleiotropic factor during development and regulates genes involved in growth and differentiation [
34,
35]. In particular, data on C2C12 cells and primary myoblasts from mice have suggested the essential role of TRα in the optimal fusion and regeneration of myofibers after muscle injury and to maintain the SC niche during aging [
36,
37]. Moreover, it has been reported that TRα is the dominant isoform thyroid receptor in C2C12 and murine primary myoblasts. This is in agreement with our results in which mRNA expression is higher for TRα with respect to TRβ.
Insulin-like Growth Factor-1 (IGF-1), the mediator of growth hormone function, strongly promotes the proliferation and differentiation of skeletal myoblasts. The anabolic effects of IGF-1 are mediated through specific binding with IGF-1R to promote the activation of the PI3K/art/mTOR signaling pathway, which is associated with protein synthesis and muscle hypertrophy [
38,
39]. Moreover, IGF-1R is required for normal muscle growth, and its loss on mouse muscle leads to increased basal glucose uptake due to increases in levels of Glu1 and Glu4 transporters, chronic activation of Akt and AMPK signaling, and a loss of TBC1D1, data confirmed also in L6 myotubes [
40].
Regarding parathyroid hormone (PTH) and its receptor (PTH-1R), very few data in the literature are reported about their effects on skeletal muscle cells. PTH has been shown to enhance the differentiation of mesoderm to various cell types, including osteoblasts and smooth muscle cells [
41,
42]. Since skeletal muscle cells are derived from the mesoderm, it is conceivable that PTH may also influence the differentiation of these cells. It has been reported that PTH and the expression of PTH-1R accelerate the differentiation of SCs to myotubes in a mouse model [
43].
Skeletal muscle is a notable target for glucocorticoids (GCs) in health and disease. GCs convey their signals mainly through an intracellular glucocorticoid receptor (GCR). Chronically increased levels of endogenous or exogenous GCs can lead to proteolysis, muscle wasting, myopathy, and induce insulin resistance with severe perturbation in systemic energy metabolism, while short exposures to high GC concentrations have been involved in the development of crucial illness myopathy [
44,
45]. Despite its importance, data on GC signaling during human skeletal muscle regeneration and how GCR primary target genes confer metabolic function of GCs remain incomplete [
46]. In the literature, it has been reported that GCR is involved in a positive regulation of muscle regulatory gene Myf-5 in the mouse myogenic cell line C2 [
47].
LRP-5 and LRP-6 are highly homologous proteins with key functions in canonical Wnt signaling. Alteration in genes encoding these receptors or their interacting proteins is linked to human diseases and, for that reason, they have been a major focus of drug development efforts to treat several human conditions, including osteoporosis, cancer and metabolic disease [
48]. Sclerostin is a circulating osteocyte-derived glycoprotein produced by the osteocytes that in a paracrine fashion negatively regulates Wnt signaling after binding the LRP5/LRP6 co-receptors in osteoblastic cells and its pharmacologic inhibition produces bone anabolic effects [
49]. Conversely, endocrine effects of sclerostin on muscle morphology remain unknown, and very little data are reported in the literature [
50].
In the present study, the genes encoding the receptors for the above outlined hormones were detected in a limited number of collected hSkMCs. Moreover, all the assayed receptor genes significantly increase during in vitro myogenesis of hSkMCs, supporting their role in the maturation of the human skeletal muscle. The fact that the two sclerostin receptors LRP-5 and LRP-6 are expressed in hSkMCs is opens for this important osteocytic protein a function as a hormone in the reciprocal interaction between bone and skeletal muscle. Interestingly, also the expression of the gene encoding Irisin, an important hormone produces by mature skeletal muscle tissue that affects cortical bone [
51] increases during the process of in vitro differentiation of the hSkMCs.
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