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Beneficial Effect of H2S-Releasing Molecules in an In Vitro Model of Sarcopenia: Relevance of Glucoraphanin

Sarcopenia is a gradual and generalized skeletal muscle (SKM) syndrome, characterized by the impairment of muscle components and functionality. Hydrogen sulfide (H2S), endogenously formed within the body from the activity of cystathionine-γ-lyase (CSE), cystathionine- β-synthase (CBS), and mercaptopyruvate sulfurtransferase, is involved in SKM function. Here, in an in vitro model of sarcopenia based on damage induced by dexamethasone (DEX, 1 μM, 48 h treatment) in C2C12-derived myotubes, we investigated the protective potential of exogenous and endogenous sources of H2S, i.e., glucoraphanin (30 μM), L-cysteine (150 μM), and 3-mercaptopyruvate (150 μM). DEX impaired the H2S signalling in terms of a reduction in CBS and CSE expression and H2S biosynthesis. Glucoraphanin and 3-mercaptopyruvate but not L-cysteine prevented the apoptotic process induced by DEX. In parallel, the H2S-releasing molecules reduced the oxidative unbalance evoked by DEX, reducing catalase activity, O2− levels, and protein carbonylation. Glucoraphanin, 3-mercaptopyruvate, and L-cysteine avoided the changes in myotubes morphology and morphometrics after DEX treatment. In conclusion, in an in vitro model of sarcopenia, an impairment in CBS/CSE/H2S signalling occurs, whereas glucoraphanin, a natural H2S-releasing molecule, appears more effective for preventing the SKM damage. Therefore, glucoraphanin supplementation could be an innovative therapeutic approach in the management of sarcopenia.


Introduction
Sarcopenia is a skeletal muscle (SKM) disorder characterized by a gradual and generalized impairment in terms of structure and functionality, frequently associated with physical handicap, significantly worsening patient's quality of life and, in worst cases, leading to death [1,2]. In simpler terms, it could be best defined as SKM insufficiency or failure [3]. Sarcopenia is a complex multifactorial condition [4,5], though aging is the major underlying cause [5]. Age-related sarcopenia can be enhanced by various factors, including sedentary lifestyle, poor nutrition, chronic illness, and disturbance in the peripheral and/or central nervous systems. However, even if sarcopenia is usually considered an elderly disorder, its development can also occur in young people and in particular in those suffering from autoimmune diseases [6]. In addition, hormonal changes contribute to the decrease in Importantly, the exogenous replacement of H 2 S improves the molecular features of DMD in terms of inflammation and fibrosis in mdx mice [27].
In the present work, we investigated: (i) the involvement of H 2 S signalling in an in vitro model of sarcopenia based on damage induced by dexamethasone (DEX) on C2C12derived myotubes (ii) the protective potential of an endogenous and exogenous source of H 2 S. For this purpose, glucoraphanin, a glucosinolate occurring exclusively in the botanical order Brassicales, as an exogenous source of H 2 S [28], L-cysteine, as an endogenous source of H 2 S, and 3-mercaptopyruvate, either as an exogenous or endogenous source of H 2 S [29] have been investigated.

Impairment of H 2 S Signalling in DEX-Induced In Vitro Model of Sarcopenia in C2C12 Myotubes
DEX treatment (1 µM) for 48 h induces in vitro model of sarcopenia in C2C12-derived myotubes, as previously reported [30]. Here, we found that treatment with DEX significantly reduced the expression of both CBS and CSE in C2C12 myotubes ( Figure 1A-C, * p < 0.05; ** p < 0.01). Furthermore, a reduction in H 2 S production was observed ( Figure 1D). Indeed, the basal amount of H 2 S was significantly lowered in homogenates of DEX-treated C2C12 myotubes compared with vehicle-treated ones (** p < 0.001, Figure 1D). demonstrates that, in myoblasts of DMD patients, there is an impairment in H2S signalling; indeed, a lowering in the gene expression of the CBS and CSE and H2S production was found [27]. Importantly, the exogenous replacement of H2S improves the molecular features of DMD in terms of inflammation and fibrosis in mdx mice [27].
In the present work, we investigated: (i) the involvement of H2S signalling in an in vitro model of sarcopenia based on damage induced by dexamethasone (DEX) on C2C12derived myotubes (ii) the protective potential of an endogenous and exogenous source of H2S. For this purpose, glucoraphanin, a glucosinolate occurring exclusively in the botanical order Brassicales, as an exogenous source of H2S [28], L-cysteine, as an endogenous source of H2S, and 3-mercaptopyruvate, either as an exogenous or endogenous source of H2S [29] have been investigated.

Impairment of H2S Signalling in DEX-Induced In Vitro Model of Sarcopenia in C2C12 Myotubes
DEX treatment (1 μM) for 48 h induces in vitro model of sarcopenia in C2C12-derived myotubes, as previously reported [30]. Here, we found that treatment with DEX significantly reduced the expression of both CBS and CSE in C2C12 myotubes ( Figure 1A-C, * p < 0.05; ** p < 0.01). Furthermore, a reduction in H2S production was observed ( Figure 1D). Indeed, the basal amount of H2S was significantly lowered in homogenates of DEX-treated C2C12 myotubes compared with vehicle-treated ones (** p < 0.001, Figure 1D).

Protective Effects from Oxidative Stress Induced by DEX
The treatment with DEX (1 µM) evoked oxidative imbalance, enhancing catalase activity ( Figure 3) and increasing up to two folds both levels of O 2 − . (Figure 4) and protein carbonylation ( Figure 5). As shown in Figure 3, glucoraphanin (30 µM), L-cysteine (150 µM), and mercaptopyruvate (150 µM) were effective in reducing catalase activity (ˆˆp < 0.01 versus DEX, for glucoraphanin;ˆp < 0.05 versus DEX for L-cysteine and mercaptopyruvate). The three compounds were also able to prevent the increase in O 2 − levels ( Figure 4); in particular, L-cysteine was able to bring the concentration back to control levels (ˆˆp < 0.01 versus DEX). As shown in Figure 5, protein carbonylation was significantly prevented by all of the three compounds (ˆp < 0.05 versus DEX for L-cysteine;ˆˆp < 0.01 versus DEX for glucoraphanin and mercaptopyruvate). 0.01 versus DEX, for glucoraphanin; ^ p < 0.05 versus DEX for L-cysteine and mer pyruvate). The three compounds were also able to prevent the increase in O2 − level ure 4); in particular, L-cysteine was able to bring the concentration back to control (^^ p < 0.01 versus DEX). As shown in Figure 5, protein carbonylation was signifi prevented by all of the three compounds (^ p < 0.05 versus DEX for L-cysteine; ^^ p versus DEX for glucoraphanin and mercaptopyruvate).   0.01 versus DEX, for glucoraphanin; ^ p < 0.05 versus DEX for L-cysteine and mer pyruvate). The three compounds were also able to prevent the increase in O2 − leve ure 4); in particular, L-cysteine was able to bring the concentration back to control (^^ p < 0.01 versus DEX). As shown in Figure 5, protein carbonylation was signif prevented by all of the three compounds (^ p < 0.05 versus DEX for L-cysteine; ^^ p versus DEX for glucoraphanin and mercaptopyruvate).

Morphology and Morphometrics of C2C12 Myotubes
C2C12 myoblasts completely differentiate in myotubes after 7 days of culture in a differentiation medium (DM). The following 48 h treatment with DEX (1 µM), altered the morphology and morphometry of myotubes ( Figure 6A-E), reducing the diameter of myotube ( Figure 6F) and the quantity of multinucleated myotubes ( Figure 6G) by about 39% and 52%, respectively. The 48 h cotreatment with glucoraphanin (30 µM), Lcysteine (150 µM), and mercaptopyruvate (150 µM) prevented the morphological alterations ( Figure 6A-G). The number of nuclei per myotube was not modified by either DEX or the three compounds ( Figure 6H).

Discussion
Several mechanisms and risk factors, including poor physical activity, smoking, malnutrition, and age-related hormonal changes and cytokine levels, contribute to the development of sarcopenia [4,5]. The aforementioned mechanisms enclose changes in muscle protein turnover, muscle tissue remodelling, muscle cell recruitment, and apoptosis leading to muscle atrophy [9]. Sarcopenia is considered part of the aging process, so much so that it now represents a key point of research and public policy debate for its impact on morbidity, mortality, and healthcare costs. One of the major factors implied in the outbreak of sarcopenia is age-related insulin resistance [31]. Indeed, insulin is an anabolic

Discussion
Several mechanisms and risk factors, including poor physical activity, smoking, malnutrition, and age-related hormonal changes and cytokine levels, contribute to the development of sarcopenia [4,5]. The aforementioned mechanisms enclose changes in muscle protein turnover, muscle tissue remodelling, muscle cell recruitment, and apoptosis leading to muscle atrophy [9]. Sarcopenia is considered part of the aging process, so much so that it now represents a key point of research and public policy debate for its impact on morbidity, mortality, and healthcare costs. One of the major factors implied in the outbreak of sarcopenia is age-related insulin resistance [31]. Indeed, insulin is an anabolic hormone that not only promotes glucose absorption, glycogenesis, and lipogenesis but also stimulates skeletal muscle protein synthesis [32]. In line with this, the association between diabetes and sarcopenia and frailty is well proved by several reports [33]. Older diabetic patients have high incidence of frailty (32-48%) compared with nondiabetic older subjects (5-10%) [34][35][36]. To date, the cause/mechanism by which diabetes often coexists with sarcopenia/frailty remains to be clarified [37,38]. In a cohort of elderly patients suffering both hypertension and frailty, a correlation between physical decline and cognitive impairment was found. This evidence further stresses the role of comorbidities in the context of sarcopenia [39]; thus frailty represents an additional problem to be considered along with the control of blood pressure in older adults. More in general, frailty is frequently accompanied by comorbidities such as diabetes, heart failure, and hypertension in older subjects; therefore, a form of prevention of sarcopenia could result from better management of these populations of patients [40]. Furthermore, there are suggestions of a correlation between chronic kidney disease and muscle impairment, leading to a high incidence of frailty and an increased risk for mortality [41].
In addition, another critical cause implicated in the development of sarcopenia is the use of drugs such as chemotherapies and glucocorticoids [5,9,10]. In fact, a strong association between aging and sarcopenia development exists also following dexamethasone treatment [42]. Indeed, an excess of dexamethasone markedly reduces muscle strength and physical activity in animals [43] and humans [44]. This is not surprising considering that glucocorticoids can trigger muscular atrophy, by both inhibiting synthesis and accelerating degradation of muscle proteins in preclinical models, both in vivo and in vitro [45][46][47][48]. Noteworthy, sarcopenia is not only an age-related disease but it can also hit younger subjects affected by autoimmune diseases, such as rheumatoid arthritis [6]. At last, acute sarcopenia arises in survivors of COVID-19 too [49]. Thus, despite its clinical relevance, sarcopenia is still badly handled in the current clinical practice. In this scenario, H 2 S may offer new pharmacological approaches for the management of sarcopenia. Indeed, it is well accepted the role of H 2 S in the physiopathology of SKM [14,20,27] but also in the pathogenesis of diabetes [50] and not last as an important mediator of inflammation [51][52][53]. Considering this evidence, we assessed the efficacy of endogenous or exogenous sources of H 2 S in an in vitro model of sarcopenia. We evaluated the effects of the following in DEX-induced sarcopenia in C2C12-derived myotubes: glucoraphanin, a natural H 2 S donor; L-cysteine, the endogenous substrate for H 2 S production; and 3-mercaptoyruvate, an endogenous and exogenous source for H 2 S [29]. The potential efficacy of the abovementioned agents was strongly given by the finding that DEX-induced sarcopenia in C2C12 myotubes, both CBS and CSE expression was reduced, coupled with a significant reduction in H 2 S content. Thus, in our experimental condition, DEX negatively impacts the H 2 S biosynthesis in C2C12-derived myotubes. The fact that DEX reduces the CBS and CSE expression has also been observed in animal models such as DEX-induced hypertension and osteoporosis in the rat [54,55] or LPS-induced endotoxic shock [56]. In addition, we also found that in C2C12-derived myotubes DEX increased caspase 3 activity, the canonical biochemical indicator of both early and late phases of apoptosis [57] reinforcing the involvement of the apoptotic process in sarcopenia. Indeed, growing evidence suggests that muscle fibre atrophy is strongly correlated with an increase in muscle cell apoptosis [58,59]. Among the agents tested, glucoraphanin and mercaptopyruvate showed greater efficacy for reducing the increase in caspase 3 activity, while L-cysteine did not affect it. This apparent discrepancy could be related to a different kinetic in releasing H 2 S, based on the fact that either glucoraphanin or 3-mercaptopyruvate releases H 2 S very quickly, i.e., already within 20 min [29,60], while L-cysteine needs to be metabolized, requiring more time to release H 2 S. As reported by studies employing cell-free systems, cytochrome c is able to prime apoptosis-like shift in cytosols derived from several cell types [61][62][63][64]. In particular, cytochrome c, through caspase activation, speeds up apoptotic events in cell extracts, such as degradation of various caspase substrates and nuclear condensation and fragmentation. H 2 S can be placed in this context, acting as a cytochrome c inhibitor [65,66] and thus interfering with the cascade of caspases activation. Therefore, the timely release of H 2 S from glucoraphanin, L-cysteine, or 3-mercaptopyruvate could be crucial for inhibiting cytochrome c to block the caspase activation. The mitochondrial impairment leads to a redox unbalance [67], another important issue of sarcopenia; in our experimental condition, DEX caused an increase in both catalase activity and O 2− levels that resulted in reduced by glucoraphanin, 3-mercaptopyruvate, and L-cysteine. Protein carbonylation, the major hallmark of oxidative-stress-related disorders, also plays a role in labelling damaged proteins during oxidative stress to eliminate them from the biological system [68]. Moreover, we found that protein carbonylation was higher in presence of DEX and reduced by the treatment with all three H 2 S-releasing agents. This effect relies on the unquestionable antioxidant ability of H 2 S. Indeed, it is well known that H 2 S can readily scavenge reactive oxygen and nitrogen species, at higher rates than other canonical antioxidants such as glutathione [69]. The cytoprotective effects of the H 2 S-releasing molecules were also highlighted in myotubes. Our results show that DEX treatment leads to an impairment of morphology, decreasing the diameter and the formation of mature myotubes, whereas glucoraphanin, 3-mercaptopyruvate, and L-cysteine were able to restore the alterations in myotube diameter and number of multinucleated cells. It is well known that an oxidative environment negatively impacts muscle regenerative ability, this is the case of aging. Oxidative intracellular unbalance impairs the differentiation of myoblasts, while myogenesis is promoted by reducing conditions [70]. The same is confirmed also in oxidative conditions related to hyperglycemia [71]. The reason that the reduction in oxidative stress is correlated with the improvement of myotube formation is currently unclear, but in the literature, there is recent pieces of evidence linking calmodulin (CaM) to this phenomenon [72]. CaM is a fundamental regulator protein of muscle physiology, orchestrating functions such as cell proliferation, cell death, and muscle tissue remodelling [73]. CaM is rich in methionine (Met) and therefore particularly sensitive to oxidative triggers [74]. A single amino acid substitution of Met with Met sulfoxide (M109Q) in one or both alleles of the CALM1 gene, which is one of three genes encoding the muscle regulatory protein CaM, strongly impaired C2C12 mouse myoblasts differentiation in myotubes [72]. Therefore, CaM seems to act a redox sensor, blocking myogenesis as consequence of oxidative stress [72].
Taking into account that the modification of the extracellular environment (i.e., the satellite cell niche) could improve the functionality of aged muscle precursor cells [67], our result hints to an important restorative effect elicited by H 2 S able to contrast the muscle atrophy. However, in this scenario, it is important to stress the role of H 2 S in diabetes, while considering the fact that SKM is an important regulator of glucose homeostasis in the human body and that sarcopenia is frequently accompanied by diabetes in older subjects. Indeed, plasma levels of L-cysteine, glutathione, and H 2 S are reduced in diabetic conditions [75,76]; as postulated by in vitro and in vivo studies, a potential connection exists in diabetes between the decreased levels of H 2 S and the impaired glucose homeostasis [77,78]. In addition, it has been reported that a diet with great consumption of organosulfur compounds, such as chives, leeks, garlic, and onions, participates in the recovery of circulating levels of H 2 S positively influencing the metabolic state [79,80]. In addition, as reported in an in vitro study performed on C2C12-derived myotubes, the deletion of CSE leads to a decrement in cellular glutathione concentration and makes the cells susceptible to oxidative stress raising ROS production, while augmentation of GSH levels and a reduction in cellular oxidative stress have been observed after supplementation with NaHS [75]. Similar results have been obtained by Sinha-Hikim and co-workers. They elegantly showed dietary supplementation of a cystine-ameliorated muscle structure and functionality in old mice [22].
In conclusion, this study provides molecular insights into the relevance of the CSE/CBS/H 2 S system in sarcopenia. H 2 S-releasing molecules, targeting diminishment of muscle cell apoptosis, reduction in oxidative stress, and control of glucose homeostasis, may represent a framework for therapeutic intervention in the management of sarcopenia. Furthermore, supplementation with H 2 S-releasing natural compounds, such as glucoraphanin, or alternatively a diet reach in Brassicales, may be also useful in the prevention of sarcopenia. The advantage of using glucoraphanin relies on its H 2 S slow-releasing properties and on its metabolization by intestinal microbiota, resulting in sulforaphane which, in its turn, exerts antioxidant, detoxifying, anti-inflammatory, and antiapoptotic effects. [81,82]. For these reasons, and based on our data, glucoraphanin stands out among other H 2 S donors and could be more effective than L-cysteine or 3-mercaptopyruvate in prevention and treatment of sarcopenia.

Pharmacological Treatments
After confluence attainment myoblasts were plated in proper cell culture plates (Corning Costar, Sigma-Aldrich, Milan, Italy) according to experimental procedures. Once differentiated in myotubes, they were treated with 1 µM DEX (Sigma Aldrich, Milan, Italy) for 48 h. Glucoraphanin (30 µM), purified from Tuscan black kale seeds by Bologna laboratory (CREA-AA; previously CRA-CIN) according to a previously described method [83], L-cysteine (150 µM) and 3-mercaptopyruvate (150 µM) (Sigma-Aldrich, Milan, Italy) were used in the presence of DEX. All treatments were done in DM. Concentration and time of exposure for DEX-induced damage were previously set up [30]; glucoraphanin concentration was chosen based on the ability of the compound to increase the intracellular H 2 S concentration [84]; L-cysteine and 3-mercaptopyruvate concentrations were chosen based on previously obtained results [29].

Cell Viability Assay (MTT Test)
Myotube viability was assessed by evaluating the reduction in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as an index of mitochondrial functionality. Cells were plated into 96-wells cell culture plates (3 × 10 3 cells/well) and grown until 80% confluence. After 7 days of differentiation in DM, cells were treated with 1 µM DEX in absence or presence of glucoraphanin (30 µM), L-cysteine (150 µM), and 3-mercaptopyruvate (150 µM) for 48 h. After abundant washing, 1 mg/mL MTT was added to each well and incubated for 30 min at 37 • C. Upon the formation of formazan, 150 mL of dimethyl sulfoxide were added to each well to dissolve the crystals. The absorbance was measured at 550 nm. Experiments were performed in quadruplicate on at least three different experimental sets.

H 2 S Determination
H 2 S production was measured in homogenates of C2C12 myotube treated with vehicle or DEX. Sample lysis was performed in a modified potassium phosphate buffer (100 mM, pH 7.4, sodium orthovanadate 10 mM, and proteases inhibitors) and Bradford assay was used to determine protein concentration. In order to evaluate the basal content of H 2 S, pyridoxal-5 -phosphate (2 mM) was added to the homogenates. After incubation at 37 • C for 40 min, trichloroacetic acid solution (TCA, 10% wt/vol), zinc acetate (1% wt/vol), N,Ndimethyl-p-phenylenediamine sulphate (DPD; 20 mM) in HCl (7.2 M) and FeCl 3 (30 mM) in HCl (1.2 M) were added to each sample [86]. All samples were performed in duplicate, and H 2 S concentration was measured by optical absorbance at a wavelength of 668 nm and calculated against a standard curve of NaHS (3-250 µM). Data were calculated as nmol/mg protein*min −1 .

Superoxide Dismutase (SOD)-Inhibitable Superoxide Anion (O 2 − ) Production Evaluated by Cytochrome c Assay
Myoblasts were plated in 6-well plates (5 × 10 4 cells/well) and grown until 80% confluence. After 7 days of differentiation in DM, myotubes were then incubated with DEX (1 µM) in absence or presence of glucoraphanin (30 µM), L-cysteine (150 µM), or 3-mercaptopyruvate (150 µM) for 48 h. After treatment, cells were incubated in serum-free DMEM containing cytochrome c (1 mg/mL) for 4 h at 37 • C. Nonspecific cytochrome c reduction was evaluated by performing tests in the presence of bovine SOD (300 mU/mL). After collecting the supernatants, the optical density was spectrophotometrically measured at 550 nm. The SOD-inhibitable O 2 − amount was calculated by subtracting nonspecific absorbance and by using an extinction coefficient of 2.1 × 10 4 M −1 cm −1 and expressed as µM/mg protein/4 h. We chose incubation time based on preliminary experiments, which pointed out poor reliability after longer cellular environment exposure to cytochrome c.

Carbonylated Protein Evaluation
Myoblasts were plated in a 25 cm 2 cell culture flask (7 × 10 4 cells/flask) and cultured upon reaching 80% confluence. Cells were differentiated in myotubes in DM for 7 days. Carbonylated proteins were evaluated after 48 h incubation with 1 µM DEX, both alone and with glucoraphanin (30 µM), L-cysteine (150 µM) or 3-mercaptopyruvate (150 µM). After treatment and PBS washing, myotubes cell cultures were scraped on ice with lysis buffer containing 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, and complete protease inhibitor (Roche, Milan, Italy). Cell suspensions were collected, subjected to a freeze-thaw cycle, and centrifuged at 13,000× g for 10 min at 4 • C. Bicinchoninic acid assay was used to determine protein concentration. Twenty micrograms of proteins from each sample were denatured by 6% SDS and derivatized 10 mM 2,4-dinitrophenyl hydrazine (DNPH; Sigma-Aldrich, Milan, Italy) for 15 min at room temperature. Protein separation was carried out by using a 12% SDS-polyacrylamide gel by electrophoresis. Proteins were transferred onto nitrocellulose membranes (Bio-Rad, Milan, Italy). Membranes were incubated with blocking solution (PBS containing 0.1% Tween 20 (PBST) and 1% BSA; Sigma-Aldrich, Milan, Italy) and then overnight with specific primary antibody versus DNPH (Sigma-Aldrich) (1:5000 in PBST/1% BSA). After washing with PBST, the membranes were incubated for 1 h with horseradish-peroxidase-conjugated secondary antirabbit (1:5000 in PBST; Cell Signaling, Milan, Italy) and washed again. Peroxidase-coated bands were visualized with enhanced chemiluminescence (Pierce, USA). Scion Image analysis software was used to perform densitometric analysis. The density of all the bands for each condition was reported as the mean and normalized to β-Actin expression [87].

Statistical Analysis
Results were expressed as mean ± S.E.M. Analysis of variance (ANOVA) or Student's t-test was performed as needed. A Bonferroni significant difference procedure was used as a post-hoc comparison. All assessments were made by researchers blinded to cell treatments. Data were analysed using the "Origin 8.1" software (OriginLab, Northampton, MA, USA) and GraphPad Software (Prism 8, San Diego, CA, USA). p < 0.05 was considered significant.

Conflicts of Interest:
The authors declare no conflict of interest.