In this study, we have shown for the first time that FGF23 in hSMCs influences myogenic differentiation but not proliferation. FGF23 belongs to the large family of FGFs, of which 22 members are known to exert paracrine or endocrine effects in bone, kidney, liver, and brain physiology [9, 26]. High FGF23 levels are responsible for the main clinical manifestations of XLH, such as hypophosphatemia, rickets, and bone mineralization defects [12]. XLH also leads to muscular disorders, such as muscle weakness, pain, reduced muscle density, peak strength, and power [17,18,19, 27].

The presence of muscular symptoms in XLH is not surprising as the importance of biomechanical and biochemical crosstalk by which muscle and bone, through the secretion of myokines and osteokines, respectively, communicate and influence each other has become increasingly apparent in recent years [28,29,30].

As very few reports take into consideration the role of FGF23 on skeletal muscle processes, our study focuses on the effects of FGF23 treatments in an in vitro model of skeletal muscle cells derived from human biopsies.

Based on previous studies, we selected three hSMC cell lines, isolated and characterized by their stem phenotype [31], in which we did not find FGF23 mRNA expression (data not shown). This finding, on the one hand, confirms that FGF23 is mainly synthesized by osteocytes and osteoblasts and, on the other, is in line with data reported in the literature that mRNAs of only 4 FGFs were found in satellite cells [32].

In this study, we used three different FGF23 concentrations (1, 10, and 100 ng/mL), which were not only in line with those used by other groups but also reflected FGF23 levels measured in the serum of patients with hypophosphatemic rickets or other diseases characterized by high FGF23 levels [33,34,35,36,37,38,39,40,41]. This enabled us to attempt to reproduce in vitro experimental conditions that were as similar as possible to those demonstrated in vivo.

Under our experimental conditions, FGF23 is not able to influence the proliferation of hSMCs. These data agree with the literature, where it is reported that different concentrations of FGF23 do not affect the proliferation of human or murine muscle cells [33, 35, 41]. Conditions of hypophosphataemia and moderate to severe hyperphosphataemia do not alter the proliferation rates of C2C12 murine myoblasts [42, 43]. To our knowledge, this is the first in vitro study to examine the effect of different concentrations of FGF23 on cell proliferation in the presence of low phosphate levels. Therefore, we hypothesize that there is no activation of pathways affecting cell proliferation under these experimental conditions.

However, FGF23 affects the process of myogenic differentiation, a complicated and finely regulated biological process involving Myf-5, MyoD-1, myogenin, and MRF4, which make up the family of MRFs [44, 45]. MRFs are muscle-specific proteins that guide progenitor cells to establish the skeletal muscle phenotype [44, 45]. Besides these, other important factors involved in the myogenic differentiation process are desmin, irisin, and myostatin, which are involved in skeletal muscle development, and MHC, a protein essential for skeletal muscle contraction and, therefore, of primary importance along with actin for muscle movement [46,47,48,49]. FGF23 under our experimental conditions resulted in a significant decrease in the expression of these genes in hSMCs.

Although some works report no effect of FGF23 on murine C2C12 muscle lines, some data support our results [29, 31]. In fact, other members of the FGFs family (such as FGF2, 9, 16, 20) cause an inhibition of the differentiation of C2C12 cells, human skeletal muscle cells, and myoblasts [35, 50, 51]. Furthermore, FGF23 has been shown to induce the senescence of mesenchymal stem cells derived from skeletal muscle, which, although having different properties from satellite cells, support muscle differentiation and regeneration [41].

FGF23 exerts its actions through its FGFRs receptors with the presence of the α-KLOTHO coreceptor [5]. We confirmed the presence of FGFRs and α-KLOTHO in GM-cultured hSMCs, as reported in the literature [33, 52, 53]. In our experimental conditions, FGF23 resulted in a significant reduction in the levels of the 4 FGFRs and the α-KLOTHO coreceptor compared to untreated hSMCs. We hypothesize that this is due to the establishment of a negative feedback mechanism that aims to reduce the effects induced by high FGF23 levels. Latic et al. also showed that in the kidney of the Hyp mice, the animal model of hypophosphatemia, the abundance of the α-Klotho protein decreases by half compared to wild-type controls [54].

All these collected data suggest that FGF23 is indeed involved in the muscle disorders that occur in XLH. Indeed, in 2018 in Europe and the USA a fully human immunoglobulin IgG1 monoclonal antibody directed against FGF23 (Burosumab), which prevents FGF23 binding to FGFRs, was approved for the treatment of XLH [55]. In preclinical studies performed on Hyp mice, Burosumab resulted in improved hypophosphatemia, 1,25(OH)2D levels, and it was also observed that it was able to improve muscle strength. Aono et al. demonstrated that administration of anti-FGF23 antibodies in adult Hyp mice increased not only muscle strength but also the frequency of spontaneous movements [56]. In patients with XLH, the administration of Burosumab resulted in a better perception of their physical performance [57].

Although further studies are needed, we can speculate that the improvement in muscle function observed in patients with XLH after treatment with Burosumab is due to increased serum phosphate levels and reduced FGF23 action in skeletal muscle. In particular, patients with hypophosphatemic rickets exhibit qualitative and quantitative muscle deficits, including reduced muscle density and volume, as well as altered contractions and elongation-shortening cycles [17]. Our results could explain how Burosumab positively affects on skeletal muscle of XLH patients reducing FGF23 action, since we have reported that FGF23 is able to impair myogenic differentiation reducing the expression of MRFs. The regulation of MRFs expression is fundamental, as MRFs are involved in the activation of satellite cells and myogenic differentiation, as well as being closely connected with signaling pathways involved in adaptation, muscle development, and regeneration [58]. Therefore, a reduction in MRFs expression levels may be associated with structural and functional deficits in skeletal muscle [59]. Therefore, if it is caused by the presence of FGF23, a reduction of FGF23 action, as reported under treatment with Burosumab, could be one of the positive effects of this treatment, since it is able to block FGF23 activity.

Despite, this first study on the effects of FGF23 on muscle physiology presents some limitations such as the small number of tested hSMCs and the fact that we do not know the FGF23 levels of the three donors, an aspect that could be interesting to evaluate the effects of FGF23 among patients which present different circulating levels of FGF23 [60].

However, the reported results have shown the in vitro effects of FGF23 on the proliferation and myogenic differentiation of hSMCs. Although such treatments do not affect cell proliferation at the experimental tested condition, it induces a decrease in MRFs, desmin, irisin, myostatin, MHC, FGFRs, and α-KLOTHO gene expression during differentiation, indicating the possible involvement of FGF23 in muscular dysfunction characterizing hypophosphatemic rickets and the possible presence of negative feedback providing protection against high FGF23 levels.

Further studies, using a human in vitro model for clarifying these effects at the molecular and cellular levels, are needed for a better comprehension of XLH muscular impairment and, consequently, of its management. A deeper knowledge of the precise mechanism in human cellular models may lead to the development of new therapeutic strategies to prevent and treat this disease. These results will make possible to evaluate whether the muscular symptoms in XLH are related to FGF23 excess in skeletal muscle.