The present study explored the potential role of FGF23/FGFR4 signaling pathway in the pathogenesis of sarcopenia. The results demonstrated that the expressions of FGF23 and FGFR4 in muscle tissue of mice with sarcopenia were significantly decreased compared with control mice. In vitro studies indicated that overexpression of FGF23 promoted the myogenic differentiation of muscle satellite cells and inhibited the myogenic differentiation of muscle satellite cells after interference. FGFR4 overexpression reversed the inhibition of FGF23 interference on myogenic differentiation of muscle satellite cells. The results suggested that the FGF23/FGFR4 signaling pathway played an important role in the myogenic differentiation of muscle satellite cells.
FGF23 is an endocrine hormone released primarily by osteocytes that regulates phosphate and vitamin D metabolism through the mediation of the FGF receptor 1 (FGFR1) and the co-receptor Klotho[18]. Liu S et al. found that the rank order of FGF23 mRNA expression in normal mouse tissues was bone > thymus > brain > heart > skeletal muscle > spleen > skin > lung > testes[19]. In our study, qRT-PCR and WB analysis showed that FGF23 mRNA and protein were indeed expressed in skeletal muscle. The mRNA and protein expression levels of FGF23 in muscle tissue of mice with sarcopenia were significantly lower than those of mice without sarcopenia. The results support the important role of FGF23 in skeletal muscle biology. It should be noted that FGFR4, which is another receptor of FGF23, has been shown to be a necessary step in limb muscle differentiation.The inhibition of FGFR4, but not FGFR1 signaling, leads to a dramatic loss of limb muscles[12]. In our study, we constructed an animal model of sarcopenia and found that FGFR4 expression level was significantly down-regulated in the skeletal muscle tissue of mice with sarcopenia, which also demonstrated the role of FGFR4 in skeletal muscle myogenesis at the histological level.
Studies on the potential effect of FGF23 in skeletal muscle are rare. Chisato et al. found that FGF23 treatment significantly reduced the number of human skeletal muscle mesenchymal stem cells, and FGF23 induced cellular senescence of human skeletal muscle mesenchymal stem cells through the p53/p21/oxidative-stress pathway[10]. The interaction between skeletal muscle mesenchymal stem cells and muscle satellite cells played a key role in skeletal muscle regeneration and homeostasis maintenance. Joe et al. showed that skeletal muscle mesenchymal stem cells promote myogenic differentiation of co-cultured muscle satellite cells[20]. Chisato et al. showed that FGF23 treatment reduced the number of skeletal muscle mesenchymal stem cells, which may affect the differentiation of muscle satellite cells. However, Chisato found that the number of muscle satellite cells remained unchanged after FGF23 treatment, indicating that FGF23 may not affect the proliferation of muscle satellite cells, but whether FGF23 affects the differentiation of muscle satellite cells is unclear. Our study explored the effect of FGF23 on the differentiation function of muscle satellite cells, which was a supplement to previous studies on the effect of FGF23 on muscle satellite cells.
Another study found that acute exercise, exhaustive exercise and chronic exercise all increased serum FGF23 levels. Chronic exercise up-regulated the expression of FGF23 mRNA and protein in skeletal muscle. Exercise-stimulated FGF23 promoted exercise performance via controlling the excess reactive oxygen species production and enhancing mitochondrial function in skeletal muscle[11]. Oxidative stress and mitochondrial damage were the pathogenesis of sarcopenia, and the above study demonstrated the positive effects of FGF23 on muscle function[21, 22]. Muscle satellite cells are the only cells that can proliferate and differentiate after being activated in adult muscle. The regenerative capacity of adult skeletal muscle after injury is mainly attributed to the proliferation and differentiation of muscle satellite cells[4, 5]. Our study showed that the marker protein Pax7 of muscle satellite cells was significantly reduced in muscle tissue of mice with sarcopenia, suggesting that the number of muscle satellite cells decreased in sarcopenia. Our study also found that protein expression of FGF23 increased during induction of muscle satellite cell differentiation. Overexpression of FGF23 promoted the expression of MyoD and MHC in muscle satellite cells. Muscle development is regulated by members of the myogenic regulatory factors (MRFs), of which MyoD is a key regulator of myogenic early differentiation and muscle fiber formation[23, 24]. The MRFs gene family encodes actin, myosin and troponin. Myosin consists of two heavy chains and multiple light chains[25]. Myosin heavy chain (MHC) is a specific contractile protein expressed during muscle development[26]. In our study, while promoting FGFR4 expression, FGF23 overexpression also promoted MyoD and MHC expression, indicating that FGFR4 signaling not only regulates the early differentiation of muscle satellite cells, but also controls the top of the molecular cascade of muscle differentiation in skeletal muscle.
Previous studies showed that FGF23 stimulated the expression and secretion of inflammatory cytokines such as C-reactive protein in hepatocytes by activating FGFR4[27]. FGF23 enhanced myocardial contractility and induced left ventricular hypertrophy by activating FGFR4. These processes did not depend on the co-receptor Klotho[16, 28]. Graves JM et al. found that FGF23 induced ventricular arrhythmias and prolonged QTc interval in mice in an FGFR4-dependent manner[15]. The role of FGF23/FGFR4 signaling in muscle development has not been studied. Our study found that overexpression of FGF23 promoted FGFR4, MyoD, and MHC expression in muscle satellite cells. The interference of FGF23 inhibited the expression of FGFR4, MyoD and MHC in muscle satellite cells. FGFR4 overexpression reversed the inhibitory effect of FGF23 interference on the differentiation of muscle satellite cells, suggesting that the FGF23/FGFR4 signaling pathway played an important role in the myogenic differentiation of muscle satellite cells.
Our research has several limitations. First, this study mainly reflected the role of FGF23 in skeletal muscle atrophy similar to natural aging. The causes of skeletal muscle atrophy are malnutrition, infection, endocrine and metabolic abnormalities such as diabetes and so on. Therefore, the role of FGF23 in different muscle atrophy conditions remains to be explored. Second, we only explored the role of FGF23/FGFR4 signaling pathway in muscle satellite cell differentiation at the cellular level, and the role of FGF23/FGFR4 signaling pathway in skeletal muscle atrophy needs to be further explored at the animal level in the future. Third, the study did not explore the interaction between FGF23/FGFR4 signaling pathway and Notch signaling pathway, Wnt signaling pathway, and downstream signaling pathway of muscle differentiation, etc. We will further explore in subsequent studies. Finally, we did not detect the expression level of FGF23 in the blood of people with skeletal muscle atrophy, and we will add relevant content in further studies.