Study of the Expression and Function of Calcium-Sensing Receptor in Human Skeletal Muscle

Skeletal muscle has an outstanding capacity for regeneration in response to injuries, but there are disorders in which this process is seriously impaired, such as sarcopenia. Pharmacological treatments to restore muscle trophism are not available, therefore, the identification of suitable therapeutic targets that could be useful for the treatment of skeletal reduced myogenesis is highly desirable. In this in vitro study, we explored the expression and function of the calcium-sensing receptor (CaSR) in human skeletal muscle tissues and their derived satellite cells. The results obtained from analyses with various techniques of gene and protein CaSR expression and of its secondary messengers in response to calcium (Ca2+) and CaSR drugs have demonstrated that this receptor is not present in human skeletal muscle tissues, neither in the established satellite cells, nor during in vitro myogenic differentiation. Taken together, our data suggest that, although CaSR is a very important drug target in physiology and pathology, this receptor probably does not have any physiological role in skeletal muscle in normal conditions.


Introduction
Skeletal muscle tissue has an inherent capacity for regeneration in response to minor injuries. However, there are circumstances that can cause irreversible loss of muscle mass and function, for example, severe trauma, tumor ablation, and different types of congenital or acquired muscle diseases, such as muscular dystrophies and sarcopenia [1][2][3][4][5][6].
Pathological situations, in which skeletal muscle regeneration is seriously impaired, represent conditions that affect motor unit function with significant disabilities and reduced quality of life (QoL) in people who are affected. Pharmacological treatments are not available for these muscle disorders. Hence, there is an unmet need for pharmacological strategies for the treatment of skeletal muscle degenerative disorders.
Postnatal skeletal muscle stem cells, called satellite cells (SCs), are responsible for skeletal muscle regeneration during the lifespan. Several studies report that some myopathies, such as sarcopenia and muscular dystrophies, are related to a failure of SCs to enter into the myogenic program [7,8]. As SCs are committed myogenic precursors, and possess the capability of self-renewal, a unique feature of stem cells, they play a central role in the search for therapies for skeletal muscle disorders [9]. The identification of new therapies,

Established hSC Lines
Satellite cells (hSCs) were isolated from human skeletal muscle biopsies, as described recently [14], and subcultured in Matrigel-coated plates ( Figure 1A,B).
Gene expression of Pax7, a nuclear transcriptional factor and the main marker of SCs [30], was confirmed by RT-PCR ( Figure 1C). By flow cytometry, Pax7 protein expression was demonstrated in 98.29% of all cells ( Figure 1D) and confirmed by Western blot analysis ( Figure 1E).  Gene expression of Pax7, a nuclear transcriptional factor and the main marker of SCs [30], was confirmed by RT-PCR ( Figure 1C). By flow cytometry, Pax7 protein expression was demonstrated in 98.29% of all cells ( Figure 1D) and confirmed by Western blot analysis ( Figure 1E).

In Vitro Myogenic Differentiation of hSCs
Confluent hSCs, at 70-80% density in the plate, were differentiated toward the myogenic phenotype using an appropriate myogenic differentiation medium for 9 days. During this period, the activated cells align (myoblasts), then subsequently fuse with each other and, finally, differentiate into multinucleated myofibers. Phase contrast microscopy revealed the presence of multinucleated elongated cells, containing from three to more than eight nuclei, related to myotubes (Figure 2A).

In Vitro Myogenic Differentiation of hSCs
Confluent hSCs, at 70-80% density in the plate, were differentiated toward the myogenic phenotype using an appropriate myogenic differentiation medium for 9 days. During this period, the activated cells align (myoblasts), then subsequently fuse with each other and, finally, differentiate into multinucleated myofibers. Phase contrast microscopy revealed the presence of multinucleated elongated cells, containing from three to more than eight nuclei, related to myotubes (Figure 2A).
Myogenic differentiation was confirmed by expression of the myosin heavy chain (MHC), which is the most important terminal myogenic differentiation marker protein [31]. After 9 days of myogenic induction, the multinucleated cells exhibited high MHC expression, as confirmed by immunofluorescence microscopy ( Figure 2B,C). The myogenic phenotype was also confirmed by RT-PCR analysis to evaluate the expression of the myogenic regulatory factor genes Myf5, MyoD1, Myogenin, and the terminal differentiation marker MHC. We observed that these genes were expressed at the same time during the last period (9 days) of myogenic differentiation when myotubes were formed, while during the first phase (T0) of the myogenic differentiation, only Myf5 and MyoD1 were expressed, in accordance with the literature, while Myogenin and MHC were completely absent ( Table 1). The earlier detection of MyoD1 that we found in the primary cultured hSCs is due to the fact that after collecting biopsies, the surgical procedure per se is a form of injury that initiates muscle regeneration, which leads to the activation of satellite cells and to their commitment to myogenic differentiation.
MHC protein expression was also evaluated by Western blot analysis at different time points (T0-3-6-9 days) of myogenic differentiation and revealed a gradual increase Myogenic differentiation was confirmed by expression of the myosin heavy chain (MHC), which is the most important terminal myogenic differentiation marker protein [31]. After 9 days of myogenic induction, the multinucleated cells exhibited high MHC expression, as confirmed by immunofluorescence microscopy ( Figure 2B,C).
The myogenic phenotype was also confirmed by RT-PCR analysis to evaluate the expression of the myogenic regulatory factor genes Myf5, MyoD1, Myogenin, and the terminal differentiation marker MHC. We observed that these genes were expressed at the same time during the last period (9 days) of myogenic differentiation when myotubes were formed, while during the first phase (T 0 ) of the myogenic differentiation, only Myf5 and MyoD1 were expressed, in accordance with the literature, while Myogenin and MHC were completely absent ( Table 1). The earlier detection of MyoD1 that we found in the primary cultured hSCs is due to the fact that after collecting biopsies, the surgical procedure per se is a form of injury that initiates muscle regeneration, which leads to the activation of satellite cells and to their commitment to myogenic differentiation.
MHC protein expression was also evaluated by Western blot analysis at different time points (T 0 -3-6-9 days) of myogenic differentiation and revealed a gradual increase in MHC over time during myogenic differentiation progression ( Figure 2D), validating the suitability of the in vitro myogenesis model. We performed qualitative RT-PCR using different pairs of primers that target exons 2/3, 4/5, and 6/7 of the human CaSR gene ( Table 2). CaSR mRNA expression was neither detected in human skeletal muscle tissues (hSMt1, hSMt2, hSMt3), nor in the respective established human satellite cell lines (hSCs1, hSCs2, hSCs3), using any of the primer settings ( Figure 3A-C). A primary cell culture of human adenoma parathyroid cells (PTcs) was used as a positive control. On the other hand, human embryonic HEK293, used as a negative control, did not show CaSR expression, as expected [32]. Housekeeping gene β-actin was used to verify the appropriate quality of the assayed samples ( Figure 3D). Table 2. List of the human gene-specific primers used in the analysis.
Real-time qPCR detected very low CaSR expression levels in hSMts and in the established hSC lines, but since the quantity was extremely small in all of the assayed samples, it was not possible to sequence the amplicons to confirm the detected mRNA products ( Figure 3E).   Subsequently, since during in vitro myogenesis satellite cells mature and gradually express receptors [14], we analyzed CaSR gene expression by RT-PCR at T 0 and after 9 days of myogenic differentiation, using all three different pairs of primers previously utilized. The analyses showed no CaSR gene expression at these experimental time points, demonstrating the absence of CaSR gene during the in vitro myogenesis of the three primary hSC lines. CaSR gene expression was demonstrated in PTcs, while it was absent from HEK293 cells. Housekeeping gene β-actin was used in order to verify the appropriate quality of the assayed samples ( Figure 4A-D). Additionally, real-time qPCR was also performed to evaluate the possible expression of CaSR during myogenesis. We evaluated CaSR gene expression at T 0 , 3, 6, and 9 days of myogenic differentiation of the three primary hSC lines. Data verify the absence of CaSR during myogenic induction, confirming the data reported in Figure 3, and assuming that this gene is not present during the differentiation process of the hSCs ( Figure 4E,F).

CaSR Protein Expression Analysis in Human Skeletal Muscle Tissues (hSMts), in hSCs, and during In Vitro Myogenesis
After analyzing the CaSR gene expression, our study was focused on the evaluation of a possible expression of CaSR protein in hSMt sections, in the established hSC lines, and during myogenic differentiation. We performed: (a) Western blot analysis, (b) immunofluorescence staining, and (c) flow cytometry, in order to assess the presence of the protein CaSR.
CaSR protein expression in primary hSC lines was investigated by Western blot analysis, by which it is possible to evaluate the size of the CaSR protein either in monomers of 140 and 160 kDa (in reducing condition with β-mercaptoethanol or 50 mM DTT, dithiothreitol, disulfide bond breaker) or in dimers of 240 kDa (non-reducing condition) forms.
The analysis, in standard reducing conditions, was performed in primary hSC lines at different time points: T 0 , 3, 6, and 9 days of myogenic differentiation. The specificity of the antibody was demonstrated using HEK293 cells, stably transfected with human CaSR (HEK CaSR ), and used as a positive control. The obtained results showed very faint bands of about 140 kDa and 160 kDa (size of CaSR monomers) in the basal hSC lines and during myogenic differentiation ( Figure 5A).
Since we had not observed the CaSR gene expression in the established hSC lines and during myogenic differentiation, but did observe faint bands in Western blot analyses with similar molecular weight of CaSR monomer bands in the same samples, we analyzed the samples under reducing and non-reducing conditions by Western blot.
The obtained results showed that the monomer CaSR bands, observed in the positive control HEK CaSR in a standard reducing condition, shifted to the CaSR dimer band at 240 kDa in non-reducing conditions. On the other hand, the monomer bands in the hSC lines, obtained in standard reducing conditions, did not shift to the dimer band at 240 kDa in the non-reducing condition ( Figure 5B).
Then, immunofluorescence analysis of CaSR protein was performed. The hSMt sections ( Figure 6A-C) show how human skeletal muscle appears in phase contrast microscopy, with the longitudinal view of the skeletal muscle fibers.
The results of immunofluorescence analysis revealed no staining of CaSR in hSMt sections ( Figure 6D,F). In comparison to that, positive detection of CaSR expression in human parathyroid tissue sections validated our method of analysis ( Figure 6G,I). The specificity of the anti-CaSR antibody was verified by one extra internal negative control in the assay, where the antibody binding was blocked by pre-absorption with the blocking peptide [33,34]. The results demonstrate the absence of CaSR protein expression in hSMts ( Figure 6E,H).
The subsequent immunostaining performed in hSC lines also showed no detection of CaSR protein in satellite cells ( Figure 7A

CaSR Protein Expression Analysis in Human Skeletal Muscle Tissues (hSMts), in hSCs, and during In Vitro Myogenesis
After analyzing the CaSR gene expression, our study was focused on the evaluation of a possible expression of CaSR protein in hSMt sections, in the established hSC lines, and during myogenic differentiation. We performed: (a) Western blot analysis, (b) immunofluorescence staining, and (c) flow cytometry, in order to assess the presence of the protein CaSR. In addition to immunostaining, we performed flow cytometry using two different dilutions (1:500 and 1:100) of the primary anti-CaSR antibody (5C10, ADD). The obtained data showed the absence of CaSR protein in hSC lines even at the higher concentration of the anti-CaSR antibody; indeed, with both antibody dilutions used, 1:500 and 1:100, the rate of stained cells is close to zero (0.27% and 0.41%, respectively), confirming the results obtained with immunofluorescence.
during myogenic differentiation, but did observe faint bands in Western blot an with similar molecular weight of CaSR monomer bands in the same samples, we an the samples under reducing and non-reducing conditions by Western blot.
The obtained results showed that the monomer CaSR bands, observed in the p control HEKCaSR in a standard reducing condition, shifted to the CaSR dimer band kDa in non-reducing conditions. On the other hand, the monomer bands in the hSC obtained in standard reducing conditions, did not shift to the dimer band at 240 the non-reducing condition ( Figure 5B).  non-reducing conditions. The analysis for CaSR protein in three hSC lines showed that the observed bands are not CaSR as they did not shift to dimer form at 240 kDa in non-reducing conditions. HEK CaSR were used as positive control. The red and yellow boxes surround the analyzed bands.

CaSR Secondary Messenger Analysis in hSC Lines
CaSR signals to downstream pathways via three main groups of the heterodimeric G protein: Gα q/11 , Gα i/0 , and Gα 12/13 [35]. We verified the absence of CaSR protein expression and function in hSCs by analyzing some secondary messengers upon treating the hSC lines with Ca 2+ and CaSR drugs: NPS2143 (calcilytic) and NPS R-568 (calcimimetic).

Intracellular Calcium Mobilization Imaging
We performed the analysis of the intracellular calcium mobilization triggered by extracellular calcium in three live hSC lines. The obtained results showed that hSCs do not respond to CaSR stimulation by 0.5 mM, 3 mM, and 5 mM Ca 2+ and by 1 µM CaSR drugs NPS2143 or 1 µM NPS R-568 along with Ca 2+ , in an intracellular calcium mobilization assay evaluated by Fura 2-AM ( Figure 8B), confirming the absence of CaSR protein in the cellular model. HEK CaSR , used as a positive control in the assay, responded well to all of the CaSR stimulations ( Figure 8A). The results of immunofluorescence analysis revealed no staining of CaSR in hSMt sections ( Figure 6D,F). In comparison to that, positive detection of CaSR expression in human parathyroid tissue sections validated our method of analysis ( Figure 6G,I). The specificity of the anti-CaSR antibody was verified by one extra internal negative control in the assay, where the antibody binding was blocked by pre-absorption with the blocking peptide [33,34]. The results demonstrate the absence of CaSR protein expression in hSMts ( Figure 6E,H).
The subsequent immunostaining performed in hSC lines also showed no detection of CaSR protein in satellite cells ( Figure 7A,B). Human PTcs were used as a positive control, to validate the method and anti-CaSR antibody ( Figure 7C,D). To verify the absence of CaSR protein in hSC lines, we performed the analysis of IP 3 secondary messenger using an IP 1 ELISA assay upon inducing hSC lines, independently, in technical quadruplicates with different concentrations of Ca 2+ (1.2 mM and 3.5 mM) for 1 h. The results are shown in Figure 8C and confirm absence of any Ca 2+ dose-dependent increase in IP 1 response in hSCs.

CaSR Secondary Messenger Analysis in hSC Lines: Gα i/0 and Cyclic Adenosine Monophosphate (cAMP)
We analyzed the second messenger cAMP upon stimulation with 3 mM Ca 2+ alone and with CaSR drugs (1 µM NPS R-568 or 1 µM NPS2143), by ELISA. The results show significant decreases in cAMP production in hSC lines with slight effects with 3.0 mM Ca 2+ treatment (−24%) and with co-treatments with 1 µM NPS R-568 (−32.7%) or 1 µM NPS2143 (−12.7%), with respect to the control with FSK, used to induce the maximum cAMP response in cells ( Figure 8D). In addition to immunostaining, we performed flow cytometry using two different dilutions (1:500 and 1:100) of the primary anti-CaSR antibody (5C10, ADD). The obtained data showed the absence of CaSR protein in hSC lines even at the higher concentration of the anti-CaSR antibody; indeed, with both antibody dilutions used, 1:500 and 1:100, the rate of stained cells is close to zero (0.27% and 0.41%, respectively), confirming the results obtained with immunofluorescence.

CaSR Secondary Messenger Analysis in hSC Lines
CaSR signals to downstream pathways via three main groups of the heterodimeric G

Analysis of a CaSR Homologous Protein in Human Skeletal Muscle: GPRC6A
While we confirmed the absence of the CaSR gene and protein expression in hSMts and hSCs, we got a response, albeit low, in the cAMP secondary messenger production upon stimulation of the hSCs with 3.0 mM Ca 2+ and in co-treatment of CaSR drugs. This evidence led us to consider the presence of a recently discovered CaSR homologous protein, the G protein-coupled receptor class C group 6 member A (GPRC6A). It is reported to respond to calcium with low affinity, and also to the drugs NPS2143 and NPS R-568, and was found to produce cAMP secondary messenger in response to its activation, although these studies used a murine model of GPRC6A [36][37][38]. Considering all the information reported about GPRC6A, we hypothesized the presence of a GPRC6A receptor in human skeletal muscle as a potential drug target for skeletal muscle disorder. To analyze the expression of the GPRC6A gene, we performed RT-PCR in the hSMts and in the established hSC lines, using a commercial human kidney cDNA (hKcDNA) as a positive control. However, the results verified the absence of GPRC6A gene in hSMts and in the established hSC lines (Figure 9).

Discussion
The study of regenerative mechanisms that regulate skeletal muscle physiology to find new therapeutic drug targets represents a challenging field of research. Satellite cells are the postnatal stem cells of skeletal muscles which, in response to injury or daily wear and tear, undergo myogenesis, therefore being responsible for skeletal muscle regeneration [39]. The failure of satellite cells to undergo a myogenic differentiation program is one of the main causes of skeletal muscle disorders, such as sarcopenia and muscular dystrophies [5,6,40,41]. However, the pathogenetic mechanisms in these myopathies are not well understood.

Discussion
The study of regenerative mechanisms that regulate skeletal muscle physiology to find new therapeutic drug targets represents a challenging field of research. Satellite cells are the postnatal stem cells of skeletal muscles which, in response to injury or daily wear and tear, undergo myogenesis, therefore being responsible for skeletal muscle regeneration [39]. The failure of satellite cells to undergo a myogenic differentiation program is one of the main causes of skeletal muscle disorders, such as sarcopenia and muscular dystrophies [5,6,40,41]. However, the pathogenetic mechanisms in these myopathies are not well understood.
Thanks to the research advances in the last decade, it has become possible to isolate and culture populations of human satellite cells that have unambiguously shown the capacity to differentiate into myotubes in vitro [11][12][13], providing invaluable tools with which muscle physiology can be studied.
Since Ca 2+ plays an important role in skeletal muscle development [19], we have hypothesized that CaSR, along with Ca 2+ channels, can have a role in myogenic differentiation, becoming a possible molecular target for skeletal muscle disorders.
Expression of CaSR in cells with functions unrelated to systemic calcium homeostasis has been demonstrated in many cases, such as in neurons, skin, and placenta [17]. Unlike the case with other muscle tissues like cardiac and vascular smooth muscle cells, there has been little attempt at analyzing GPCRs in skeletal muscle [42]. In particular, CaSR is actually expressed in cardiomyocytes [43] and in airway smooth muscle cells [29]. Therefore, the assumption that this receptor might be present in skeletal muscle is plausible.
The aim of our work was to analyze the CaSR expression in hSMts, in their respective established hSC lines, and during the in vitro myogenic differentiation. In our study, we have analyzed both gene and protein expression, using different techniques and appropriate positive/negative internal controls, in order to strengthen the obtained results. As far as we know, this is the first report that takes into consideration the question of CaSR expression in skeletal muscle.
The presented results did not reveal any evidence of the expression of CaSR in skeletal muscle cells. This conclusion is based on followed lines of proof: (i) transcripts of CaSR were not detected in hSMts, in their derived SCs, or during myogenesis using three different pairs of primers which fall in different exons. Moreover, the analysis with a highly sensitive real-time qPCR technique with a TaqMan probe showed very low detectable limit values, in which it was not possible to sequence the amplicons to confirm the presence of mRNA of CaSR; (ii) protein expression of CaSR by immunofluorescence staining did not evidence the protein in skeletal muscle tissue or satellite cells, neither by flow cytometry analysis nor by Western blot analysis.
As CaSR is a GPCR, it can activate many G proteins, providing biologically important amplification steps in the signaling pathway. This underlines the importance of even a very low level of expression in physiological and pathological conditions [44]. Thus, to evaluate the possibility that the presence of CaSR mRNA and protein might be very low in skeletal muscle tissues and in the established cellular model of satellite cells, and therefore the inability to detect by CaSR probe and anti-CaSR antibodies used in our analysis, we performed analyses of CaSR functionality by studying CaSR secondary messengers (intracellular calcium mobilization triggered by extracellular calcium, IP 3 , and cAMP), using the main ligand of CaSR Ca 2+ , calcimimetic NPS R-568 (positive CaSR allosteric modulator), and calcilytic NPS2143 (negative CaSR allosteric modulator) [24].
The analysis of the intracellular calcium mobilization triggered by stimulation with increasing calcium concentrations, alone and together with calcilytic NPS2143 or calcimimetic NPS R-568, showed no detection of signal variations in intracellular calcium, supporting the absence of CaSR in hSCs, and as we expected and found in HEK CaSR , used as a positive control for our experiments. If our hypothesis was true, and if skeletal muscle had the CaSR, our data would be in contrast with results in the literature which report that an increase in extracellular calcium induced an increment in intracellular calcium concentration in cells, such as cardiac myocytes, where the receptor is functionally present [35,43].
The analysis of CaSR secondary messenger IP 3 in the Gα q /11 CaSR signaling pathway in human satellite cells stimulated with increased calcium concentrations showed no significant results and the absence of a dose response in IP 3 production. The data are in contrast with previous reports in cardiomyocytes and in pulmonary arterial smooth muscle cells where CaSR is functionally expressed, and calcium and calcimimetic induce IP 3 accumulation [35,43,45]. However, a study reported that in aortic vascular smooth muscle cells, stimulation with calcium did not causes an IP 3 increase, although the authors detected the expression of CaSR mRNA and protein [46].
In order to have an overall analysis of CaSR functionality in hSCs, cAMP secondary messenger in the Gα i/0 CaSR signaling pathway has been investigated, because CaSR signaling pathways can vary from cell to cell, depending on their specific physiology [17]. In our cellular model, we detected significant variations in cAMP response in hSCs stimulated with 3 mM Ca 2+ alone and together with calcilytic or calcimimetic, in agreement with a study on juxtaglomerular cells expressing CaSR, that if treated with the CaSR agonist cinacalcet, showed a significant decrease in cAMP [47].
Since data on cAMP were the only data supporting a role for CaSR in skeletal muscle cells, a possible explanation is that a homologous receptor related to CaSR is present, as suggested for osteoblasts and osteoclasts [48][49][50].
A CaSR homologous protein is the GPRC6A receptor, a recently discovered physiologically important GPCR [36,51], which responds to Ca 2+ and to calcilytic (NPS 2143) and calcimimetic (NPS R-568) through a cAMP secondary messenger, although studies have been performed using mouse models [36][37][38]. Considering this information, we hypothesized the presence of GPRC6A, instead of CaSR, in our model, which could be a possible drug target for skeletal muscle disorder. However, although a study in the literature reports the expression of the GPRC6A gene from multiple tissue cDNA panels that contain normalized adult human cDNA preparations, including skeletal muscle [51], the analysis of GPRC6A gene expression, performed in hSMts and in cultured hSC lines, gave negative data. The mechanisms responsible for the observed changes in cAMP levels remain unknown and need to be analyzed in the future.

Primary Culture and Development of Lines of Human Satellite Cells (hSCs)
Human skeletal muscle biopsies were obtained from the discarded tissues of patients undergoing plastic surgery, after signing an informed consent in accordance with a protocol approved by the Local Ethics Committee of the University Hospital Careggi (AOUC), Florence (Italy), for human studies (Rif. N. 16.022), as well as the ethical standards stated in the Declaration of Helsinki (1964) and its later amendments or comparable ethical standards. The biopsies were put in Dulbecco's modified Eagle medium (DMEM) and, within 24 h of collection, they were processed in the laboratory to obtain primary cultures, called hSCs [14]. Cells at the early passages (from 1 to 4) were used for all experiments.

In Vitro Myogenic Differentiation
In order to differentiate hSCs, cells were cultured to 60-70% confluency in Matrigel (BD Company, Franklin Lakes, NJ, USA) -coated culture dishes and subsequently induced with myogenic differentiation medium (MDM) for 9 days, as previously reported [14].

Flow Cytometry Analysis
Flow cytometry was performed to characterize the cultured hSC lines for the presence of Pax7 protein, using anti-Pax7 antibody (Sigma Aldrich, St. Louis, MO, USA, SAB1412356) dilution 1:10, and the presence of CaSR protein using anti-CaSR (5C10, ADD) antibody (Thermo Fisher Scientific, Waltham, MA, USA, MA1-934) at two different dilutions of 1:500 and 1:100, both followed by goat anti-mouse IgG (H + L) Superclonal™ Secondary Antibody, Alexa Fluor ® 488 conjugate (Thermo Fisher Scientific, Waltham, MA, USA, A28175, dilution 1:200). The FIX & PERM ® Cell Permeabilization Kit (Invitrogen, Walthan, MA, USA, GAS004) was used for fixing and permeabilizing cells in suspension. Then, 1 × 10 5 cells were labeled with primary antibodies for 20 min at room temperature (RT) in PBS with 1% bovine serum albumin (BSA), then removed and washed two times and centrifuged for 5 min at 1500 rpm; secondary antibody was incubated for 30 min at RT in the dark, then washed once and promptly analyzed. The stained cells were analyzed in a CyFlow ® Space cytometer, equipped with FlowMax ® software (Sysmex Partec, Norderstedt, Germany).

Gene Expression Analysis by Qualitative Reverse Transcriptase Polymerase Chain Reaction
Qualitative RT-PCR was performed to characterize the hSC lines and the in vitro myogenic differentiation by analyzing the presence of specific marker genes (Pax-7, Myf5, MyoD1, Myogenin, and MHC). The RT-PCR was also used to evaluate the expression of the CaSR gene in human skeletal muscle tissue, in the established hSC lines and during in vitro myogenesis, using different pairs of primers targeting various exons, 2/3, 4/5, and 6/7, of the human CaSR [44]. Finally, the RT-PCR was performed to analyze the expression of the GPRC6A gene in human skeletal muscle tissue and in established hSC lines using human GPRC6A-specific primers. The information about primers used in the analyses is given in Table 2.
RNA was isolated from the cellular pellets using QIAzol Lysis Reagent (Qiagen, Hilden, Germany, 79306), according to the manufacturer's protocol, and 500 ng of the isolated RNA was reverse transcribed using the QuantiTect Reverse Transcription kit (Qiagen, Hilden, Germany, 205310), according to the manufacturer's protocol. In CaSR gene expres-sion analysis, the cDNA isolated from primary cultured human parathyroid cells (PTcs) and HEK293 cells was used as the positive and negative controls, respectively. Human PTcs were obtained from fresh parathyroid adenoma tissue, as previously reported [52]. HEK 293 (Sigma-Aldrich, St.Louis, MO, USA, 85120602) cells have been reported not to express the CaSR gene and protein, thus they can be used as a good negative control for CaSR expression analysis [32]. The β-actin house-keeping gene was used as an internal control. All the obtained amplicons were verified by sequencing analysis using a BigDye™ Terminator v1.1 Cycle Sequencing Kit based on Sanger sequencing reactions (Thermo Fisher Scientific, Waltham, MA, USA, 4337451), according to the manufacturer's protocol.

Gene Expression Analysis by Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction
Real-time quantitative reverse transcription polymerase chain reaction (real-time qPCR) was performed in human skeletal muscle tissues, in proliferating hSCs, and during myogenic differentiation. The gene included in the analysis was CaSR. Target gene expression was normalized to ribosomal protein S18 (RPS18). The information of the probe and primers used in the analysis is given in Table 3. Table 3. List of the human gene-specific TaqMan probes and primers used in the analysis. TaqMan probes with F as reporter fluorochrome (6-carboxyfuorescein ) and ZEN as quencher. Fluorochrome (Iowa Black FQ).

Name of Gene Probe and Primer Sequences Tm ( • C) Amplicon Size (bp)
RPS18
HEK CaSR were used as a positive control in the CaSR protein expression analysis [53].

Secondary Messenger Analysis
The secondary messengers Gq-coupled inositol monophosphate (IP 1 ), intracellular calcium mobilization, and Gi-coupled cyclic adenosine monophosphate (cAMP) were analyzed in hSC lines to investigate the effects of Ca 2+  For this analysis, the hSCs were cultured in Matrigel (BD Company, Franklin Lakes, NJ, USA) -coated 24 multiwell plates in growth medium. Upon reaching 60-70% confluency of the cells in wells of 24 multiwell plates, the cells were washed once with DPBS and then stimulated independently in quadruplicates of each stimulation with 250 µL of 0.5 mM Ca 2+ , 250 µL of 1.2 mM Ca 2+ , and 250 µL of 3.5 mM Ca 2+ prepared in experimental buffer (0.5 mM CaCl 2 , 125 mM NaCl, 4 mM KCl, 0.5 mM MgCl 2 , 20 mM HEPES, 0.1% (w/v) glucose) for 1 h at 37 • C in modified air with 5% CO 2 . Each buffer was freshly prepared and supplemented with 50 mM LiCl; then, pH was adjusted to 7.4 using NaOH and HCl. The IP 3 lifetime within the cell is very short (less than 30 s) before it is transformed into IP 2 and IP 1 . When LiCl is added to the stimulation buffers, the degradation of IP 1 into myo-inositol is inhibited. Therefore, IP 1 can be accumulated in the cells and be measured. Following incubation, 250 µL of absolute EtOH (frozen at −80 • C) was added to each well and subsequently incubated overnight at −20 • C. The cold temperature of the EtOH stops the reaction and solubilizes the cell membrane in order to remove IP 1 in buffer solution. The next day, the cell lysate from each well was transferred to 1.5 mL centrifuge tubes. The pipetting was carried out slowly, in order to obtain all the volume and avoid the aspiration of precipitated cell debris and proteins on the surface of the wells. The solution in each vial was lyophilized and dissolved in 50 µL of diluent provided with the IP 1 ELISA assay kit (Cisbio, Waltham, MA, USA, 72IP1PEA/D rev03). The lyophilization step was performed to concentrate the low IP 1 response which could be present in the cells. Then, the IP 1 ELISA assay was performed according to the kit instructions. A one-way ANOVA with Bonferroni's post hoc test was performed on data obtained to analyze the significant differences in IP 1 response in the presence of the different concentrations of Ca 2+ .

Intracellular Calcium Mobilization Imaging
The intracellular calcium mobilization imaging analysis was performed in 3 live primary hSC lines, upon stimulation with various Ca 2+ concentrations and CaSR drugs: calcilytic (NPS 2143) and calcimimetic (NPS R-568), along with Ca 2+ .
For this analysis, the primary hSCs were cultured in growth medium on 13 mm Matrigel-coated glass coverslips for 48 h. For use as a positive control in the analysis, HEK CaSR (HEK239 transfected with human CaSR) cells were cultured on 13 mm glass coverslips in DMEM with 10% FBS for 48 h. The experimental buffer (0.5 mM CaCl 2 , 125 mM NaCl, 4 mM KCl, 0.5 mM MgCl 2 , 20 mM HEPES, 0.1% (w/v) glucose) was freshly prepared, and pH was adjusted to 7.4 using NaOH and HCl. The cells were loaded with 1 µM FURA2-AM in 1.2 mM Ca 2+ experimental buffer supplemented with 0.1% (w/v) BSA for 1-2 h at RT in the dark. Then, cells on a coverslip were mounted in a perfusion chamber and observed through a 40X oil immersion lens on a Nikon Diaphot inverted microscope (Miato, Tokio, Japan) equipped with MetaFluor ® Fluorescence Ratio Imaging Software (Molecular Devices, San Jose, CA, USA); dual-excitation-wavelength microfluorometry was then performed. The baseline free ionized Ca 2+ in cells was measured in 0.5 mM Ca 2+ experimental buffer. Subsequently, the cells were stimulated serially for 3 min each with 3 mM Ca 2+ , 3 mM Ca 2+ + 1 µM R568 (calcimimetic), 0.5 mM Ca 2+ , 5 mM Ca 2+ , 5 mM Ca 2+ + 1 µM NPS2143 (calcilytic), 0.5 mM Ca 2+ prepared in experimental buffer, and free ionized Ca 2+ in cells was measured. Each stimulation was performed in technical triplicates.

Cyclic Adenosine Monophosphate (cAMP) ELISA Assay
The secondary messenger cAMP was analyzed using the cAMP complete ELISA kit (Abcam, Cambridge, UK, ab133051) to evaluate the functionality of Ca 2+ /CaSR in primary hSC lines upon stimulation with Ca 2+ and calcilytic (NPS R-568) and calcimimetic (NPS2143) drugs. The experiment was performed in 3 primary hSC lines.
For the assay, the hSCs were cultured in growth medium in Matrigel (BD Company, Franklin Lakes, NJ, USA) -coated 24-well plates. Upon reaching 60-70% confluency, the cells were washed once in DPBS and then stimulated. Since Ca 2+ /CaSR inhibit the cAMP response in healthy cells [54], the cells for this assay were stimulated with experimental buffers supplemented with forskolin (FSK). FSK is a potent activator of stimulatory G protein (Gs), and stimulates the maximum level of cAMP in the cells. Therefore, FSK was used as an internal positive control for the assay. For the Ca 2+ /CaSR cAMP assay, the primary hSCs were stimulated independently in triplicate for each stimulation in wells of a 24-well tissue dish, with 250 µL of experimental buffers containing 0.5 mM Ca 2+ (control), 0.5 mM Ca 2+ + 10 µM FSK (positive control), 3 mM Ca 2+ + 10 µM FSK (Ca 2+ effect), 3 mM Ca 2+ + 10 µM FSK + 1 µM R568 (calcimimetic effect), and 3 mM Ca 2+ + 10 µM FSK + 1 µM NPS2143 (calcilytic effect), for 15 min at 37 • C in modified air with 5% CO 2 .
Following incubation, 150 µL of HCl 0.1 N + 0.5% Tryton X100 was added to each well and after 15 min at room temperature, the lysates were collected and centrifuged at 1000× g to pellet the cellular debris. Then, the cAMP ELISA assay was performed according to the kit instructions (Abcam, Cambridge, UK, ab133051). A one-way ANOVA with Bonferroni's post hoc test for Ca 2+ /CaSR cAMP was performed on results obtained to analyze the statistical differences.

Conclusions
In this work, we have presented for the first time the lack of CaSR expression and functionality in human skeletal muscle cells. Although CaSR is a very important receptor in the physiology of several organs and tissues, and also in several pathologies, it is absent from human skeletal muscle tissue, derived SCs, and cells differentiating into myoblasts, proving that CaSR is not involved in myogenesis. As Ca 2+ is very important in skeletal muscle physiology, it is through voltage-gated Ca 2+ channels that Ca 2+ ions enter the cells, functioning as second messengers in the control of cell functions.
Since this is a first study exploring CaSR in healthy human skeletal muscle, future studies should address the same question in the evaluation of CaSR expression in tissues derived from patients with myopathies or other muscular disorders. CaSR could indeed represent an important pharmacological target in pathological conditions.  Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The data presented in this study are available from the corresponding author upon reasonable request.