Abstract Ectopic fat deposition in skeletal muscle (SKM) due to obesity leads to biochemical and morphological alterations that deteriorate SKM quality and performance. Here, we show that impaired MPST-derived hydrogen sulfide (H[2]S) signaling contributes to obesity-related SKM dysfunction. Muscle tissues from obese db/db mice exhibit reduced MPST expression, correlating with decreased protein persulfidation and muscle performance in vivo. Mpst^−/− mice show similar deficits as db/db mice, confirming the role of MPST. H[2]S supplementation improves locomotor activity in db/db mice and restores protein persulfidation, including SIRT-1. Myotubes placed in an "obese environment" display a downregulation of MPST, coupled with a reduced SIRT-1 persulfidation leading to an inflammatory state. Exogenous H[2]S exerts beneficial effects recovering SIRT-1 persulfidation/activity. Finally, muscle biopsies from obese individuals show reduced MPST expression, underscoring the translational relevance to human SKM health. Our study unveils a crucial role for MPST-derived H[2]S in obesity-associated SKM dysfunction via SIRT-1 persulfidation, highlighting the importance of the MPST/H[2]S pathway in maintaining healthy SKM function. Keywords: H[2]S donors, Skeletal muscle, Persulfidation, Sodium palmitate, Db/db mice 1. Introduction The contribution of the hydrogen sulfide (H[2]S) pathway to skeletal muscle (SKM) performance has been demonstrated in various human diseases, differing across conditions. For example, in Malignant Hyperthermia (MH), a rare pharmacogenetic disorder, patients exhibit elevated H[2]S levels, contributing to pathological SKM hyper-contractility [[69]1]. Conversely, the H[2]S pathway is impaired in Duchenne Muscular Dystrophy (DMD), a genetic myopathy characterized by rapid SKM weakening and functional failure [[70]2]. H[2]S is a gasotransmitter generated through the reverse transsulfuration pathway (TSP) and l-cysteine catabolism [[71]3] by the action of three enzymes: cystathionine gamma-lyase (CSE), cystathionine beta-synthase (CBS), and 3-mercaptopyruvate sulfurtransferase (MPST), which works in concert with cysteine aminotransferase (CAT) [[72]4,[73]5]. One recently discovered mechanism through which H[2]S exerts its biological effects is protein persulfidation (PSSH), a post-translational modification (PTM) of l-cysteine residues considered an evolutionarily conserved modification associated with enhanced stress resistance and improved health and lifespan in various organisms [[74][6], [75][7], [76][8]]. SKM impairment is also prevalent in obesity. In clinical settings, the increase in whole-body adiposity often correlates with heightened ectopic fat deposition within SKM, a condition known as myosteatosis [[77]9]. Myosteatosis is characterized by mild inflammation, as well as several biochemical and morphological alterations in SKM [[78][10], [79][11], [80][12]]. This chronic low-grade inflammation, termed meta-inflammation, is marked by the accumulation of immune cells in adipose tissue, dysregulation in the synthesis and release of adipokines, and a significant increase in pro-inflammatory cytokines, such as interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and interleukin-1 beta (IL-1β) [[81][13], [82][14], [83][15], [84][16]]. These cytokines adversely affect SKM by reducing muscle mass and strength, stimulating muscle protein breakdown, and inhibiting muscle protein synthesis [[85]17,[86]18]. The situation is exacerbated by diabetes and sedentary behavior associated with obesity, which leads to reduced mobility, further SKM deterioration, impaired whole-body metabolic health, and subsequent weight gain. This vicious cycle ultimately decreases the quality of life [[87]19]. Building on this evidence, the current study aims to investigate whether H[2]S-catalyzed protein persulfidation contributes to obesity-induced SKM dysfunction. To achieve this, we employed in vitro and in vivo approaches, combined with omics platforms. Specifically, we utilized db/db mice, an established genetic model of obesity. This strain lacks the leptin receptor (LEPR), resulting in deficient leptin signaling that fails to suppress appetite and energy expenditure. Consequently, db/db mice develop obesity, insulin resistance, and hyperglycemia [[88]20,[89]21]. For in vitro experiments, we used the murine SKM cell line C2C12 myotubes exposed to sodium palmitate to replicate the hyperlipidemic and obese microenvironment characteristic of db/db mice. To evaluate the role of H[2]S-induced persulfidation, we employed liquid chromatography-mass spectrometry (LC-MS) to identify proteins that undergo persulfidation. Finally, we validated our experimental findings using human quadriceps biopsies harvested from both lean and obese subjects, thereby enhancing the translational relevance of H[2]S in the physiopathology of SKM. 2. Results 2.1. The MPST/H[2]S pathway is defective in db/db mice that display SKM dysfunction To evaluate locomotory impairment in db/db mice, two distinct locomotor activity tests, namely the rotarod and weight tests assessing coordination and muscle strength, respectively, were conducted. As shown in [90]Fig. 1a and b, db/db mice exhibited a significant reduction in muscle activity compared to WT mice in both tests. To probe the potential role of the H[2]S pathway in obesity-induced SKM dysfunction, transcript levels of CBS, CSE, and MPST were measured in the quadriceps (QFA) muscle of 10-week-old WT and db/db mice using quantitative polymerase chain reaction (qPCR) analysis. [91]Fig. 1c revealed a substantial downregulation of MPST expression in QFA of db/db mice, while CBS and CSE exhibited no significant changes among the samples, suggesting that there was no compensatory increase in the expression of the other two H[2]S-generating enzymes. To validate the involvement of MPST in obesity-related SKM dysfunction, in vivo muscle performance was also assessed in Cse^−/− and Mpst^−/− mice. Notably, only Mpst^−/− mice exhibited a locomotor profile akin to that observed in db/db mice. In Mpst^−/− mice, SKM performance was significantly reduced compared to WT in both tests performed ([92]Fig. 1d and e). Conversely, Cse^−/− mice did not show significant changes in SKM activity compared to WT. To investigate if the impaired SKM performance in Mpst^−/− and db/db mice was associated with oxidative stress, markers including malondialdehyde (MDA), hydrogen peroxide (H[2]O[2]), and the glutathione (GSH)/oxidized glutathione (GSSG) ratio were evaluated in QFA of both strains. [93]Fig. 1f and g showed a significant increase in both MDA and H[2]O[2] in QFA of db/db mice compared to WT, with values in Mpst^−/− mice resembling those in db/db mice. Additionally, GSH/GSSG ratios were significantly reduced in both strains compared to the WT, indicative of an elevated oxidative stress environment within SKM ([94]Fig. 1h). These findings strongly indicate the involvement of the MPST/H[2]S pathway in SKM performance and suggest that in db/db mice, this pathway is impaired, resulting in dysregulated SKM function. Fig. 1. [95]Fig. 1 [96]Open in a new tab In vivo evaluation of SKM performance and oxidative stress in QFA of WT, db/db and Mpst^−/− mice. a,b Muscle coordination and strength evaluated by the rotarod (a) and weight (b) tests, were measured in WT (n = 8) and db/db (n = 8) mice at 10 weeks of age. c Quantification of transcripts levels of CBS, CSE, and MPST were evaluated by qPCR in QFA of WT (n = 5) and db/db mice (n = 5) at 10 weeks of age. Data are expressed as 2^∧-ΔΔct relative to β-actin. d,e Muscle coordination and strength evaluated by the rotarod (d) and weight tests (e), were assessed in WT (n = 6), Cse^−/− (n = 6), and Mpst^−/− (n = 5–6) mice at 10 weeks of age. f,h Quantification of oxidative stress evaluated by measuring Malondialdehyde (MDA) levels (f), hydrogen peroxide (H[2]O[2]) levels (g), and glutathione (GSH)/glutathione oxidized (GSSG) ratio (h) was assessed in quadriceps harvested from WT (n = 5–6), db/db (n = 6–7) and Mpst^−/− (n = 5–6) mice at 10 weeks of age. All data are expressed as mean ± SEM, and analyzed using an unpaired-t-test two-tailed for the results in a-c, and a one-way analysis of variance (ANOVA) followed by Dunnett's post hoc test for the results in d-h. Differences are considered statistically significant when p was ≤0.05. A single asterisk (∗), a double asterisk (∗∗), and a triple asterisk (∗∗∗) denote a p-value of ≤0.05, 0.01, and 0.001 vs WT mice, respectively. 2.2. Quadriceps of both db/db and Mpst^−/− mice display reduced levels of protein persulfidation We next compared the persulfidation levels of db/db and Mpst^−/− mice to their WT counterparts. Utilizing the recently developed dimedone-switch method for selective persulfidomic analysis, we quantitatively assessed changes in protein persulfidation in the muscle tissue of these mice [[97]7]. Notably, db/db mice exhibited a distinct persulfidation profile ([98]Fig. 2a, [99]Fig. S1a–b). The decrease in protein persulfidation was not predominantly influenced by alterations in protein expression ([100]Fig. 2b, [101]Fig. S1a), revealing 191 proteins with reduced persulfidation compared to 11 proteins exhibiting an increase. Pathway enrichment analysis of the targets with decreasing persulfidation pointed toward metabolic processes (fatty acid elongation and degradation, glycolysis/gluconeogenesis, and the pentose phosphate pathway, among others), as well as muscle contraction, organization, and translation ([102]Fig. 2c, [103]Fig. S1c). Interestingly, a similar observation emerged from the analysis of Mpst^−/− mice ([104]Fig. 2d–e, [105]Fig. S1d–e). Specifically, 136 proteins displayed significantly reduced protein persulfidation levels, the majority of which were associated with metabolic processes (glycolysis/gluconeogenesis, pentose phosphate pathway, TCA cycle), as well as muscle structure development, translation, and protein folding ([106]Fig. 2f, [107]Fig. S1f). Almost 50 % of proteins exhibiting decreased persulfidation levels in MPST^−/− mice also demonstrated lower persulfidation levels in db/db mice ([108]Fig. 2g). This supports the notion that the impairment of the MPST/H[2]S pathway is accountable for the diminished skeletal muscle (SKM) performance observed in db/db mice. Fig. 2. [109]Fig. 2 [110]Open in a new tab Total protein persulfidation levels in QFA of WT, db/db, and Mpst^−/− mice. a Heatmap showing the significant changes (>1.3-fold change) of PSSH in WT vs db/db mice (n = 4). b PSSH fold change levels normalized to the corresponding protein expression levels (related to A). c GO term enrichment (biological process) of the proteins showing significantly lower PSSH levels in db/db mice. Enrichment was performed using DAVID and the results visualized through REVIGO. Benjamini adjusted p-value <0.01. d Heatmap showing the significant changes (>1.3 fold change) of PSSH in WT vs Mpst^−/− mice (n = 4). e PSSH fold change levels normalized to the corresponding protein expression levels (related to D). f GO term enrichment (biological process) of the proteins showing significantly lower PSSH levels in Mpst^−/− mice. Enrichment was performed using DAVID and the results visualized through REVIGO. Benjamini adjusted p-value <0.01. g Venn diagram comparing protein targets found to have lower PSSH levels in db/db and Mpst^−/− mice. 2.3. Quadriceps of both db/db and Mpst^−/− mice display a similar pattern of metabolites related to the TSP pathway To investigate variations in QFA polar metabolome among WT, db/db, and Mpst^−/− mice, we conducted an unsupervised analysis (Principal Component Analysis - PCA). The PCA model was built considering eight TSP-related metabolites. The relative biplot ([111]Fig. 3a) visually represents the three analyzed groups together with the metabolites (grey dots) that contribute to their separation. Fig. 3. [112]Fig. 3 [113]Open in a new tab Metabolomic analysis in QFA of WT, db/db and Mpst^−/− mice. a PC1 vs PC2 biplot derived from the PCA model performed on SKM pol ar metabolome data including three studied groups and eight metabolites involved in the H[2]S pathway. Keys: Wild type mice (WT, green, squares; n = 5), obese mice (db/db, pink, stars; n = 5), 3-mercaptopyruvate sulfurtransferase knock-out mice (Mpst^−/−, red, diamonds; n = 5), metabolites (grey, dots). b In mammalian cells, the transsulfuration pathway (TSP) is a metabolic pathway that converts the amino acid l-methionine into l-cysteine. This pathway is crucial for the synthesis of sulfur compounds such as hydrogen sulfide (H[2]S), taurine, glutathione and 3-mercaptopyruvate (3-MP). The 3-MP is converted into H[2]S and pyruvate by the action of MPST. The reaction catalyzed by MPST comprises two successive sulfur transfer steps. In the first one, the sulfur atom is transferred from 3-MP to MPST, resulting in a cysteine persulfide intermediate (MPST-cys-S-SH) in the active site. In the second step-reaction, the sulfane sulfur is transferred to a thiophilic acceptor (depicted as R–SH), resulting in the regeneration of the MPST enzyme and a new persulfide product (RS-SH) from which H[2]S can be liberated. Similarly, the persulfidated MPST can react with reduced (thioredoxin) Trx realizing H[2]S and oxidized Trx. Abbreviation: CAT, cysteine aminotransferase; CBS, cystathionine beta-synthase; CDO cysteine dioxygenase; CSAD, cysteine sulfonic acid decarboxylase; CSE, cystathionine-γ-lyase; GS: glutathione synthase; HDD, hypotaurine dehydrogenase; MPST, 3-mercaptopyruvate sulfurtransferase; γ-GCS: Gamma-glutamylcysteine synthetase. Despite the first principal component (PC1) displayed differences among the three experimental groups (WT, db/db and Mpst^−/−), the PC2 revealed a clear separation between WT mice, positioned in the upper part of the plot, and db/db - Mpst^−/− mice, both located in the bottom part. Indeed, db/db and Mpst^−/− groups were characterized by higher levels of taurine, l-glutamine, and glutamate, along with decreased levels of creatine, l-glycine, and pyruvate compared to the WT group. Collectively, these data further confirm the metabolic similarity of db/db mice and Mpst^−/− mice. 2.4. expression is significantly reduced in human SKM of obese subjects To assess the relevance of MPST in SKM dysfunction associated with obesity in humans, we measured mRNA expression levels of CBS, CSE, and MPST in human biopsies of QFA harvested from lean and obese subjects (ranging from 31- to 55 BMI, [114]Table 1). As shown in [115]Fig. 4, obese subjects (n. of samples 10–18) displayed a significantly lower MPST expression than lean subjects (n. of samples 1–9). Instead, CBS and CSE genes showed no significant changes between the two groups. These results have a high translational value confirming the importance of l-cysteine metabolism, specifically of MPST-derived H[2]S, in SKM physiopathology, particularly in obesity. Table 1. Evaluation of weight, height and body mass index in human subjects. Weight (Kg), height (m), and body mass index (BMI; kg/m^2) of the lean (19< BMI<24) and obese (301.3 fold change) of PSSH levels in muscle samples of NaHS-treated mice and WT mice (n = 4). b PSSH fold change levels normalized to the corresponding protein expression levels (related to a). c Kegg pathway enrichment analysis of proteins found to have significantly higher PSSH levels upon NaHS treatment. Benjamini adjusted p-value was used to filter for significant enrichment. d Heatmap showing the significant changes (>1.3 fold change) of PSSH levels in muscle samples of Erucin-treated mice and WT mice (n = 4). e PSSH fold change levels normalized to the corresponding protein expression levels (related to d). f Kegg pathway enrichment analysis of proteins found to have significantly higher PSSH levels upon Erucin treatment. Benjamini adjusted p-value was used to filter for significant enrichment. g Venn diagram comparing protein targets found to have higher PSSH levels in NaHS vs Erucin treatment. Finally, histological analysis and relative qualitative evaluation of lipid droplet deposition have been assessed conducted in murine SKM from all groups tested (wild-type, db/db mice, db/db mice treated with NaHS, and db/db mice treated with Erucin) using three different staining to evaluate: i) the integrity of myofibers composing the tissue morphological structure (Hematoxylin and Eosin, H&E); ii) the collagen fiber deposition, indicative of fibrotic infiltrate (Masson Trichrome, MT); iii) and the amount of lipid stored as lipid droplets deposition (Oil Red O, ORO). As expected, db/db mice exhibit a slight alteration of muscle architecture, evaluable as the area covered by myofibers, and both H[2]S donors do not affect it significantly ([135]Fig. S6a). Similarly, MT staining reveals no significant difference among groups ([136]Fig. S6b), excluding a fibrotic process within SKM of db/db mice. Conversely, QFA of db/db mice exhibit a significant increase in the amount of lipids store as lipid droplets area qualitatively evaluated by ORO staining, which is ameliorated by both H[2]S donors ([137]Fig. S6c). Collectively, the histological analysis does not reveal marked alterations of SKM among groups, either in morphological structure or in fibrotic areas; this finding is not surprising since obesity is not an SKM disease but rather a physiopathology status contributing to reduced SKM performance. Therefore, it is feasible that no significant difference among groups has been detected. Lipid deposition, instead, was found to be increased in obese mice and ameliorated by H[2]S donors’ treatments. 2.6. H[2]S donors recover the sodium palmitate-induced inflammation and insulin resistance in C2C12 myotubes Sodium Palmitate (SP), a saturated free fatty acid, is widely employed to replicate the "obese state" in vitro due to its ability to induce lipid droplet accumulation, insulin resistance, inflammation, and mitochondrial dysfunction, recapitulating key features of obesity-associated metabolic syndrome [[138][24], [139][25], [140][26], [141][27]]. Starting from this finding, we exposed murine C2C12 myotubes to SP to simulate a hyperlipidemic environment in vitro. To assess if SP-exposed C2C12 cells exhibited classical hallmarks of insulin resistance, we initially measured the expression levels of pAKT/AKT ratio, pAMPK/AMPK ratio, and GLUT-4 [[142]28,[143]29]. As shown in [144]Fig. 7a–c, C2C12 myotubes exposed to SP for 18 h exhibited a significant reduction in both pAKT/AKT and pAMPK/AMPK ratios compared to the vehicle, consistent with previous studies [[145]30,[146]31]. qPCR analysis of GLUT-4 expression, a well-known insulin-regulated glucose transporter in SKM [[147]32,[148]33], also revealed a significant downregulation in SP-exposed cells compared to the vehicle. Additionally, exposure of C2C12 cells to SP significantly increased mRNA expression levels of pro-inflammatory markers such as TNFα and iNOS ([149]Fig. 7d and e) compared to the vehicle. Evaluation of H[2]S-generating enzyme mRNA levels in C2C12 myotubes exposed to SP showed a significant reduction of CBS and MPST compared to the vehicle, confirming the impairment of the MPST/H[2]S pathway in SKM cells under hyperlipidemic conditions ([150]Fig. 7f). To investigate if the beneficial effects of H[2]S donors' treatment also occurred in an in vitro setting, C2C12 cells were pre-treated with Erucin (1 μM) or NaHS (30 μM) for 2 h, followed by SP addition. As shown in [151]Fig. 7a–c, Erucin not only ameliorated the phosphorylation of both AKT and AMPK ([152]Fig. 7a and b), increasing the values of the ratio but also prevented GLUT-4 downregulation, confirming the data obtained from db/db mice. However, despite NaHS treatment inducing a trend of improvement, it did not reach statistical significance. Additionally, H[2]S-donors treatment significantly reduced the elevated levels of iNOS and TNF expression, restoring them to the levels observed in control cells ([153]Fig. 7d and e). These results suggest that H[2]S donors, by resolving inflammation and improving insulin sensitivity, protect SKM from damage caused by a hyperlipidemic condition. Fig. 7. [154]Fig. 7 [155]Open in a new tab Effect of H[2]S donors on insulin resistance and inflammation in vitro in C2C12 myotubes exposed to the hyperlipidaemic environment. a-d Western blot analysis showing the expression and/or phosphorylation of AKT a,b and AMPK c,d in vehicle and SP-exposed C2C12 cells treated or not with NaHS or Erucin and stimulated with insulin. C2C12 unstimulated were used as basal control (CTL). Fold change data represent the mean ± SEM of four separate experiments. The blots shown are representative of four independent experiments with similar outcomes. The GAPDH bands confirm that similar amounts of proteins were loaded on the gel for each sample. e Bar graphs showing the mRNA expression levels of GLUT-4 in vehicle (n = 7) and SP-exposed C2C12 cells treated or not (n = 7) with NaHS (n = 4) or Erucin (n = 4) and then stimulated with insulin. f,g Bar graphs showing the mRNA expression levels of iNOS (f) and TNFα (g) in control myotubes (n = 4) and SP-exposed myotubes treated or not (n = 4) with NaHS (n = 4) or Erucin (n = 4) and then stimulated with insulin. h Bar graphs showing the mRNA expression levels of CBS, CSE and MPST in control myotubes and SP-exposed myotubes (n = 5) then stimulated with insulin. Data are expressed as 2^∧−ΔΔct relative to β-actin for the results in e-h. All data are expressed as mean ± SEM and analyzed using one-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test for the results in a-g and an unpaired-t-test two-tailed for the results in g. Differences are considered statistically significant when p was ≤0.05. Single asterisk (∗), double asterisk (∗∗) and triple asterisk (∗∗∗) denote a p-value of ≤0.05, 0.01, 0.001 vs vehicle, respectively. Single circle (^○), double circle (^○○) and triple circle (^○○○) denote a p-value of ≤0.05, 0.01, 0.001 vs SP-exposed C2C12 group, respectively. Single ash (^#) and triple ash (^###) denote a p-value ≤0.05, 0.01, 0.001 vs. unstimulated C2C12 (CTL). 2.7. H[2]S donors positively modulates SIRT-1 activity through persulfidation Silent information regulator-1 (SIRT-1), a member of the mammalian sirtuin protein family categorized as a class III histone protein deacetylase, plays a pivotal role in the regulation of muscle metabolism [[156][34], [157][35], [158][36], [159][37]]. Recent literature has highlighted SIRT-1 as a potential target for persulfidation, positively influencing its activity [[160][38], [161][39], [162][40]]. Here, we explored the prospect that SIRT-1 could represent a molecular target through which H[2]S donors exert their beneficial effects in SKM. SIRT-1 expression, in terms of both mRNA and protein levels, remained unaffected in all experimental conditions ([163]Fig. 8b and c). Using the biotin switch method, we determined the persulfidation levels of SIRT-1 in both physiological and hyperlipidemic conditions in vitro. Intriguingly, the data indicated that under normal conditions, SIRT-1 is endogenously persulfidated in C2C12 myotubes. Conversely, in SP-induced hyperlipidemia, SIRT-1 persulfidation was undetectable ([164]Fig. 8d). Importantly, treatment with NaHS or Erucin restored SIRT-1 persulfidation in SP-exposed C2C12 myotubes ([165]Fig. 8d). Specifically, while NaHS significantly improved SIRT-1 persulfidation, Erucin completely restored it to the levels observed in physiological conditions. This was further confirmed by the persulfidomic analysis where both NaHS and Erucin restored the PSSH levels of C354 of SIRT-1 that was undetectable under hyperlipidemic conditions ([166]Fig. 8e; [167]Fig. S4). Fig. 8. [168]Fig. 8 [169]Open in a new tab Effect of H[2]S donors on SIRT-1 expression and activity in vitro in C2C12 myotubes exposed to hyperlipidemic environment. a Schematic representation of experimental design performed in C2C12 myotubes. b Bar graphs showing the mRNA expression levels of SIRT-1 in control myotubes (n = 6) and SP-exposed myotubes treated or not (n = 4) with NaHS (n = 4) or Erucin (n = 4) and then stimulated with insulin. Data are expressed as 2^∧−ΔΔct relative to β-actin. c Western blot analysis showing the changes in the expression of SIRT-1 in control myotubes (n = 4) and SP-exposed myotubes treated or not (n = 4) with NaHS (n = 4) or Erucin (n = 4) and then stimulated with insulin. Fold change data represent the mean ± SEM of four separate experiments. The blots shown are representative of four independent experiments with similar outcomes. The GAPDH bands confirm that similar amounts of proteins were loaded on the gel for each sample. d Persulfidation levels of SIRT-1 in control myotubes (n = 5) and SP-exposed myotubes treated or not (n = 6) with NaHS (n = 5) or Erucin (n = 5) and then stimulated with insulin. e Quantification of C354 PSSH levels of SIRT-1 in control myotubes (n = 4) and SP-exposed myotubes treated or not (n = 4) with NaHS (n = 3) or Erucin (n = 3) and then stimulated with insulin. f, g Bar graphs showing the mRNA expression levels of iNOS (f) and TNFα (g) in SP-exposed myotubes pre-treated with the selective SIRT-1 inhibitor, EX-527, following the addition of vehicle or NaHS or Erucin (n = 4). Data are expressed as 2^∧−ΔΔct relative to β-actin. h Evaluation of SIRT-1 activity by cell-free assay. Data are expressed as relative fluorescence intensity (RFU). i Illustrative cartoon of the signaling pathways involved in SIRT-1 persulfidation. All data are expressed as mean ± SEM and analyzed using one-way analysis of variance (ANOVA) followed by Dunnett's and Bonferroni's post hoc test for the results in b-e and f, g respectively, and two-way analysis of variance (ANOVA) followed by Dunnett's post hoc test for the results in h. Differences are considered statistically significant when p was ≤0.05. Single asterisk (∗), double asterisk (∗∗) and triple asterisk (∗∗∗) denote a p-value of ≤0.05, 0.01, 0.001 vs vehicle, respectively. Single circle (^○) and triple circle (^○○○) denote a p-value of ≤0.05, 0.001 vs SP-exposed C2C12 group, respectively. Single dollar (^§) and double dollar (^§§) denote a p-value ≤0.05, 0.01, vs. NaHS or Erucin respectively. To further assert that the beneficial effects of H[2]S donors were mediated by SIRT-1 activation, we evaluated the mRNA levels of iNOS and TNF-α in SP-exposed cells pre-treated with a selective inhibitor of SIRT-1, EX527 (100 μM), followed by the addition of NaHS or Erucin. As depicted in [170]Fig. 8f and g, pre-treatment of cells with EX527 significantly increased mRNA expression levels of pro-inflammatory markers compared to the vehicle, highlighting the role of SIRT-1 in the protective action of H[2]S. Finally, to substantiate H[2]S as a positive modulator of SIRT-1 activity through persulfidation, a cell-free assay for evaluating SIRT-1 activity was employed. As shown in [171]Fig. 8h, both H[2]S donors (100 μM) significantly increased SIRT-1 activity compared to the vehicle. Specifically, within a 10-min timeframe of the assay, NaHS and Erucin enhanced SIRT-1 activity comparably to resveratrol, a well-known SIRT-1 activator used as a positive control [[172]38]. In summary, these results demonstrate that: i) H[2]S-derived persulfidation occurs physiologically in SKM, modulating SIRT-1 activity; ii) under hyperlipidemic conditions in vitro, a notable reduction of SIRT-1 persulfidation takes place; iii) H[2]S donors, by restoring SIRT-1 persulfidation, positively modulate SIRT-1 activity. 3. Discussion Recent literature has shown that impaired H[2]S metabolism is involved in obesity and adipose tissue disturbance [[173][41], [174][42], [175][43]], as well as in SKM diseases [[176]1,[177]2,[178]44]. Indeed, in Mpst^−/− mice, an increase in body weight associated with a reduced metabolic rate was found coupled to an impaired glucose tolerance [[179]45,[180]46] suggesting a specific role of MPST, among the H[2]S-generating enzymes, in metabolic health and obesity. Here, by using in vivo, in vitro, and translational approaches, we have demonstrated that MPST-derived H[2]S plays a crucial role in obesity-associated SKM dysfunction through protein persulfidation. We first defined that MPST is downregulated in QFA of db/db mice. Based on this finding, we tested whether the observed reduced expression of MPST could affect SKM activity in vivo. The db/db mice displayed a decrease in SKM performance in both locomotor tests, indicating a decline in balance, coordination, motor skills, and muscular strength compared to healthy mice. These findings suggest that the MPST/H[2]S pathway is involved in SKM dysfunction related to obesity and propose a primary role for MPST in SKM activity. This hypothesis is confirmed by the locomotor tests performed in Mpst^−/− mice. Indeed, the lack of MPST caused a reduced SKM activity, almost indistinguishable from that observed in db/db mice, reinforcing the concept that MPST-derived H[2]S is involved in SKM homeostasis. The finding that mice lacking CSE gene (Cse^−/−), an alternative source of H[2]S, do not display any impaired locomotory activity, strongly indicates a specific role of MPST in SKM function. These results have been further supported by the molecular data revealing that the expression of the gene encoding for the Cse was not different in SKM of db/db and WT mice. In agreement with this finding, Lu and colleagues reported equal CSE expression in SKM harvested from db/db mice and WT mice at the 6th and 12th weeks of age [[181]47]. Jointly, our data strongly suggest the previous findings that H[2]S endogenously produced from different sources exerts distinct biological effects on SKM activity [[182]1,[183]48]. MPST has been shown to participate in redox cycling and contributes to detoxification from cellular oxidants through the production of persulfidated species such as l-cysteine- and glutathione-persulfide [[184]49,[185]50]. The assessment of the cellular redox state in the quadriceps of db/db and MPST^−/− mice unequivocally revealed heightened levels of ROS, escalated lipid peroxidation, and diminished glutathione levels, conclusively affirming the involvement of the MPST/H[2]S pathway. Furthermore, the data presented support the well-established connection between obesity and oxidative stress [[186]51,[187]52] and confirm our previous discovery regarding the antioxidant properties of MPST [[188]45,[189]50]. To gain further insights on the impact of either MPST impairment/lack on TSP-related products, we performed a metabolomic analysis on QFA harvested from WT, db/db, and MPST^−/− mice. Also in this case, in both MPST^−/− and db/db mice the levels of pyruvate, methionine, l-glycine, l-glutamate, and l-taurine (all TSP-related products) were significantly altered. Physiologically MPST produces H[2]S and pyruvate [[190]53] see [191]Fig. 3b. Therefore, the absence of MPST leads to a reduced amount of these two products. Furthermore, the significant reduction of pyruvate in db/db mice provides compelling support for the pivotal role of MPST in SKM, as previously postulated. The decreased levels of l-glycine showed by db/db mice could be considered as an indirect index of SKM damage, as l-glycine is known to be involved in signaling pathways that protect SKM from wasting and loss of function [[192]2,[193]54]. The lack of MPST also impacts the levels of upstream metabolites of TSP, such as methionine, which are augmented. Finally, the increased levels of l-taurine, and l-glutamate, could be ascribed to a rearrangement of the pathway due to the absence of MPST (see [194]Fig. 3b) [[195]55], as a compensatory mechanism aimed at rebalancing the cellular redox state. The antioxidant properties of l-taurine have been widely investigated in various model systems. It was concluded that l-taurine's protective actions could be related to the maintenance/preservation of mitochondrial integrity and functions in various stress conditions [[196]56,[197]57]. The increase in glutamate level was also observed (to a lesser extent) in db/db group. Overall, these data, taken together with the in vivo and molecular findings, support a key role for MPST in SKM homeostasis. MPST also acts as a protein persulfidase contributing to overall protein persulfidation [[198]23]. Specifically, Pedre and coauthors reported that MPST displays an intrinsic ability to persulfidate a broad range of proteins under physiological conditions through direct protein-to-protein transpersulfidation reaction. Consistent with these findings, we found diminished levels of protein persulfidation in both db/db and Mpst^−/− mice. Thus, the impairment of the MPST-derived H[2]S pathway leads to a decreased persulfidation activity, which translates into a reduced SKM performance. To provide a human translational insight into the role of MPST in SKM physiopathology, we analyzed human biopsies of QFA obtained from both lean and obese subjects. In agreement with experimental data, we found a significant reduction of MPST in obese SKM, reinforcing the concept that MPST/H[2]S signaling plays a pivotal role in SKM physiopathology and conferring strength to the overall results. Several studies refer to MPST as a “mitochondrial enzyme,” as it is a major H[2]S-generating enzyme within the mitochondria [[199]50,[200][58], [201][59], [202][60]]. In our study, we observed an exclusive impairment of MPST expression in SKM tissues from both human and murine models of obesity. This reduction in MPST expression negatively impacts SKM performance, highlighting the relationship between H[2]S and SKM function. Indeed, it is feasible that, by producing H[2]S in the mitochondria, MPST helps mitigate the effects of oxidative stress in muscle cells, thereby protecting mitochondrial function and improving overall muscle health. Recent work from Banerjee's group has shown that by modulating mitochondrial complex IV activity, H[2]S can exert long-lasting regulation of metabolism [[203]61]. According to this hypothesis, in db/db mice we found reduced levels of sulfide quinone oxidoreductase (SQR) persulfide ([204]Supplementary Table 1), an intermediate formed during sulfide oxidation, whose steady-state levels can be considered a measure of the enzyme's activity [[205]62]. Then, we attempted to rescue SKM functionality by treating mice with exogenous sources of H[2]S. For this purpose, we used two H[2]S donors that differ in source and kinetics: a fast sodium salt donor (NaHS), and Erucin, a naturally occurring slow releaser of H[2]S [[206]49]. This choice was driven by the intention to assess whether there were differences also attributable to the release kinetics of the donors. Fast donors are effective for short-term effects, as they rapidly release a substantial amount of H[2]S, leading to a swift modulation of cell metabolism and reduced inflammation in experimental animal models. Conversely, slow donors release smaller amounts over a prolonged period, promoting long-term effects on muscle recovery and tissue integrity [[207]63]. The donors were administered starting from 6 up to 10 weeks of age, a timeframe in which no muscle atrophy was present ([208]Figure S5 a-b). Both donors prevented the progressive loss of locomotor activity observed in db/db mice. Specifically, the H[2]S donors rescued muscle strength, and significantly improved coordination. Similar results were obtained in the high-fat diet (HFD) mice model of obesity-related disorders [[209]42,[210]46,[211]64]. However, in these studies HFD mice were treated with H[2]S donors for at least 8 weeks, and an anti-obesity and hypoglycaemic action was observed. Therefore, these findings do not conclusively establish whether the benefits of H[2]S donors are directly on SKM or if they are due to the compounds ‘ability to reduce hyperglycemia and body weight. For this reason, we have opted for a shorter treatment period (i.e 4 weeks). Despite not detecting any significant effect of H[2]S donors on glycemia or body weight ([212]Figure S2 c-d), we found a significant improvement in locomotor activity. This demonstrates a direct beneficial effect of H[2]S donors on SKM. The next step was to evaluate if the benefits of H[2]S donors were due to the antioxidant activity and free radical scavenging capacity of H[2]S [[213]65] since several studies have shown that exogenous H[2]S exerts antioxidant activity in physiological systems exposed to ROS and RNS. However, it is more readily accepted that the observed antioxidant effects of H[2]S are broader indirect signaling rather than direct scavenging action, modulating the expression and activity of the classical endogenous antioxidants, GSH included [[214][66], [215][67], [216][68]]. Our data clearly showed that H[2]S donors did not affect the SKM oxidative stress in db/db mice, ruling out the antioxidant activity as the main molecular mechanism. Instead, what was significantly changed by both treatments was protein persulfidation compared to the vehicle. In this context, our results show that several subunits of complex I and complex II are significantly less persulfidated in db/db mice—an effect that is reversed by H[2]S donors’ treatment. Furthermore, the persulfidation of subunits in complexes III, IV, and V is also increased in H[2]S donors-treated db/db mice ([217]Supplementary Tables 3–4). In line with these results, it has been shown that H[2]S-induced protein persulfidation of complex I controls its ability to oxidize NADH, thereby maintaining the proper NAD^+/NADH ratio within cells [[218]69]. Although the functional consequences of these changes on mitochondrial activity remain unclear, requiring future studies, our data strongly indicate that the MPST-derived H[2]S is specifically responsible for the persulfidation of those targets, rather than direct persulfidase activity as recently suggested [[219]23]. The latest experimental evidence demonstrated that SIRT-1 persulfidation is involved in several beneficial health outcomes [[220]38,[221]40,[222]70]. SIRT-1 is a NAD^+-dependent enzyme with deacetylase activity, involved in cellular energy metabolism and redox status [[223]71] as well as in inflammation, fibrosis, lipid and glucose metabolism [[224][72], [225][73], [226][74], [227][75]]. To evaluate the possible involvement of SIRT-1 in the beneficial effect of H[2]S donors we switched from in vivo to in vitro approach. To this purpose, we used C2C12 cell lines myotubes exposed to Sodium Palmitate (SP), a saturated free fatty acid widely employed to replicate the "obese state" in vitro [[228][24], [229][25], [230][26], [231][27],[232]76]. Our experimental data reveal that SP-exposed C2C12 myotubes displayed a significant reduction in MPST gene expression. Thus, the in vitro model mimics the molecular mechanism hypothesized in vivo. Starting from this evidence, we have explored the role of SIRT-1, specifically its persulfidation. The basal level of persulfidated SIRT-1, detected in C2C12 placed in a physiological environment, was completely abrogated following exposure to SP, strongly suggesting a detrimental role of fatty acids on SIRT-1 activity. These molecular effects translated into an increase of iNOS and TNFα expression and a reduction in the phosphorylation of Akt and AMPK, used as a molecular readout of the damage. While the increase of pAKT/AKT ratio is a classical marker of insulin activated signaling, the pAMPK/AMPK ratio is an indirect marker of insulin pathway activation, being part of the metabolite-sensing protein kinase family that monitors cellular energy levels [[233]77]. Additionally, AMPK can improve insulin action by inhibiting the mTOR/S6K1 pathway, which is involved in inflammation and insulin resistance [[234]78]. The observed reduced values of both pAKT/AKT and pAMPK/AMPK ratio are widely recognized as an index of insulin resistance [[235]79]. To understand the impact of persulfidation on SIRT1 activity, we treated cells with H[2]S donors before adding SP. Our findings show that Erucin treatment fully recovered the SIRT-1 persulfidation meanwhile NaHS partially increased it. Remarkably, the H[2]S donors blunted the TNFα and iNOS expression but only Erucin fully rescued Akt and AMPK phosphorylation, which represents an index of insulin signaling activation. This discrepancy is likely due to the different H[2]S-kinetic releases between the H[2]S donors, as also demonstrated by others [[236]46]. These results were also confirmed by using a SIRT-1 inhibitor, EX-527. As expected, EX-527 treatment abolished the protection of Erucin and/or NaHS against SP-induced inflammation demonstrating that SIRT-1 plays a crucial role in the benefits induced by H[2]S. Taken together this evidence strongly suggests that SIRT-1 persulfidation is a physiological phenomenon contributing to SKM function. But does SIRT-1 directly interact with H[2]S? To obtain direct evidence for the activation of SIRT-1 by H[2]S donors, a commercially available cell-free assay assessing SIRT-1 activity was employed. Specifically, in the time frame between 3 and 9 min, SIRT-1 deacetylation activity induced by H[2]S donors was almost triplicate compared to the vehicle, like the effect observed with Resveratrol, a well-known SIRT-1 activator [[237]80,[238]81]. Interestingly, NaHS displayed the same SIRT-1 activity as Erucin in the assay showing that the release kinetic does not affect the interaction with a freely available substrate. Our results were also in accordance with Du and coauthors who demonstrated that NaHS increased SIRT-1 activity dose-dependently in both cell free-assay and in transfected human wild-type SIRT-1 HEK-293 cells [[239]38]. In conclusion, MPST and MPST-derived H[2]S play a key role in SKM disorders related to obesity/metabolic syndrome ([240]Fig. 9). The molecular mechanisms involve persulfidation of proteins, specifically SIRT-1, ameliorating the molecular features of obesity (inflammation, insulin resistance). This improvement is clearly demonstrated in vivo by the restoration of muscular strength as depicted in the graphical abstract. Additionally, our study suggests that sulfide donors could represent a promising new additive therapeutic approach in treating SKM dysfunction related to obesity/metabolic syndrome. Fig. 9. [241]Fig. 9 [242]Open in a new tab Graphical abstract was created with [243]BioRender.com. 4. Materials and methods 4.1. Animal model and drug treatment Animal care and experimental procedures in this study adhere to specific guidelines outlined by the Italian and European Council laws for experiments involving animals. All procedures were approved by the local animal care office (Centro Servizi Veterinari, Università degli Studi di Napoli, Federico II) and were carried out following the recommendations for experimental design and analysis in pharmacology care (Ministero della Salute, n. 97/2020). For this study, male db/db mice or their lean db/+ littermates, 5 weeks of age, were obtained from Charles River Laboratories (Milan, Italy). All mice were housed in pathogen-free cages (three mice per cage) with a 12-h light-dark cycle, at a temperature of 23 ± 2 °C and humidity of 60 %, and were provided with free access to dry feed and water. NaHS (3 mg/kg), Erucin (3 mg/kg), or vehicle (potassium phosphate buffer pH 7.4) was administered orally to the mice once a day for four weeks (from 6 to 10 weeks). Animals within each cage were randomly assigned to different experimental groups, with each group including at least five mice. The experimenters conducting the behavioral testing were blinded to the genotype and treatment. 4.1.1. Prof. Andreas Papapetropoulos from the University of Athens, Athens, Greece, kindly provided CSE^−/− and MPST^−/− mice, which were bred in our facility. 4.2. Locomotor tests 4.2.1. Rotarod test Motor coordination and balance were assessed using an accelerating rotarod (Ugo Basile). For wt and db/db mice, the rotarod was performed immediately before the beginning of the pharmacological treatment (6 weeks) and at the end (10 weeks) of treatment with vehicle, NaHS, or Erucin. Briefly, the rotarod was settled with a start speed of 5 rpm, and the mice were placed on the rotating rod for 30 s. The speed was then gradually increased to 40 rpm over 300 s. The time (s) when mice dropped from the rod was recorded. The results were expressed as an average of two different trials, and the interval time of each trial was 30 min. For wt, CSE^/-, and MPST^−/− the rotarod was performed at 10 weeks of age. In brief, each animal was initially habituated and then performed 3 trials. During the habituation session, the mice were placed on the rotarod for 1 min at a constant speed of 4 rpm (revolutions per minute). If they fell, they were put back on the rotarod. During the trial session, each mouse was placed on the rotating rod at a constant speed of 4 rpm, and the speed gradually increased (012 rpm/s) to 40 rpm. Each mouse performed three trials, and the latency to fall was recorded and averaged for each animal. The maximum trial time was limited to 300s/trial. 4.2.2. Muscle strength test Forelimb strength was evaluated by using four weights of 20, 33, 46, and 59 g. Mice were handled by the base of the tail and were allowed to grip the first weight (20 g) and to hold 3 s was the criterion. If the mouse dropped the weight in less than 3 s, the same weight was tried again for a maximum of three times. If the mouse held it for 3 s, then the next heaviest weight was tried. The mouse was assigned the maximum time/weight achieved. The final total score is calculated as the product of the number of links in the heaviest chain held for the full 3 s, multiplied by the time (s) it is held. 4.3. Determination of oxidative stress in SKM Biomarkers of oxidative stress were determined by measuring malondialdehyde (MDA) levels, hydrogen peroxide (H[2]O[2]) content, and GSH/GSSG ratio in SKM from several experimental groups. For MDA measurement, SKM samples (∼10 mg) were lysed according to the manufacturer's instructions by using a commercially available kit (MAK085, Sigma-Aldrich, Milan, Italy). H[2]O[2] production was evaluated using Amplex Red Hydrogen Peroxide/peroxidase assay kit (cat. n. A22188; Invitrogen). The fluorescence was measured by using GloMax Explorer (Promega) under the following settings Ex = 530 nm, Em = 590 nm. Tissue weight was used for normalization. For the measurement of GSH/GSSG ratio, powdered tissues (∼20 mg) were lysed with the RIPA buffer and then GSH/GSSG ratio was determined using a commercially available kit (arbor assays cat. n. K006–H1, Ann Arbor, Michigan, USA) according to the manufacturer's instructions. Tissue weight was used for normalization. 4.4. ^1H-NMR-based skeletal muscle analysis To extract metabolites of interest, a dual phase extraction was employed as reported elsewhere [[244]82]. A total of fifteen frozen skeletal muscle samples were thawed at room temperature and processed as follows. About 40 mg of skeletal muscle was mixed with 100 μL of cold water and 320 μL of cold methanol and the mixture was homogenized by employing an UltraTurrax. Then 320 μL of cold chloroform was added and the mixture was vortexed for 1 min, kept on ice for 10 min, and finally centrifuged for 15 min at 15000 rpm at 4 °C. This procedure generated a two-phase extract: the aqueous upper phase contained hydrophilic metabolites, while non-polar metabolites, as lipid molecules, moved into the organic lower phase. Proteins and macromolecules were trapped, instead, in the thin skin-like layer between the two phases. For each sample, the aqueous and organic phases were separated and carefully transferred into Eppendorf tubes and glass vials, respectively. Both phases were firstly dried by using a SpeedVac Concentrator for 5 h and then lyophilized overnight. In this study, only the hydrophilic phase was considered, however the organic phase has been stored at −80 °C for future analysis. The dried hydrophilic phases were dissolved in 630 μL of sodium phosphate buffer (pH 7.4) and 70 μL of D[2]O (volume ratio of 9:1), briefly vortexed and then centrifuged for 15 min at 15000 rpm at 4 °C. Both phases were transferred into 5-mm NMR tubes. All one-dimensional ^1H NMR spectra were acquired at 298 K on a Bruker Avance NEO 600 MHz spectrometer (Bruker Biospin Gmbh, Rheinstetten, Germany) equipped with a QCI cryo-probe set for 5 mm sample tubes and a SampleJet autosampler. The ^1H NMR spectra of hydrophilic skeletal muscle extracts were acquired with Topspin 4.1 (Bruker Biospin GmbH, Rheinstetten, Germany), using the ‘noesygppr1d’ pulse sequence allowing for a quantitative evaluation even close to the water signal [[245]83], which was presatured at 4.698 ppm. All the experiments were acquired with an acquisition time of 2.62 s, a relaxation delay of 4 s, receiver gain of 101, 128 scans, 4 dummy scans and a spectral width of 12,500 Hz (20.828 ppm). All samples were automatically tuned, matched and shimmed. Prior to Fourier transformation, the free induction decays were multiplied by an exponential function equivalent to a 0.3-Hz line-broadening factor. Then, the transformed spectra were automatically corrected for phase and baseline distortions and calibrated using TopSpin built-in processing tools. The assignment of the hydrophilic metabolites was achieved by (i) analysis of literature data [[246]84,[247]85]; (ii) comparison with the chemical shifts of the metabolites in the Human Metabolome Database (HMDB); (iii) peak fitting routine within the spectral database in Chenomx NMR Suite 8.8 software package (Chenomx, AB, Canada) in its evaluation version. NMR spectra were then imported into MATLAB (R2015b; Mathworks, Natick, MA) where the spectral regions above 10 ppm and below 0 ppm were removed because they contained only noise. To correct for spectral misalignment, an interval-based alignment step was carried out using the icoshift algorithm [[248]86] and choosing the lactate doublet at 1.33 ppm as reference signal. Then, to reduce the model complexity, the peak areas of the well-separated and safely assigned resonances of selected metabolites (belonging to the transsulfuration pathway) were manually integrated and submitted to the data analysis as a data matrix made of 15 rows (samples) x 8 columns (metabolites). Such data matrix was then submitted to the PLS toolbox version 8.6.1 (Eigenvector Research, Manson, USA) under MATLAB environment, version R2015b (MathWorks Inc., Massachusetts, USA) to perform Principal Components Analysis (PCA). Prior to the analysis, data was normalized, according to the total area (1-norm) and then autoscaled. Autoscaling employs both the standard deviation as a scaling factor thus giving all metabolites the same chance to affect the model and the mean-centering, which is needed to compute PCA. PCA is an unsupervised pattern recognition method that allows to identify trends and clusters in the dataset under investigation. 4.5. Histological analysis on mice QFA 4.5.1. ORO staining QFA samples were cut on a cryostat in 20-μm-thick serial sections and stored at −80 °C until processed for the ORO staining, by adapting the protocol reported in a previous study [[249]87]. Briefly QFA sections were stained with Oil Red-O solution (Sigma-Aldrich) at 0,6 % in isopropyl alcohol for 10 min at room temperature and then cover-slipped with Fluoromount (Sigma-Aldrich). 4.5.2. H&E staining QFA samples were cut on a cryostat in 20-μm-thick serial sections and stored at −80 °C until processed for the H&E staining, by adapting the protocol reported in a previous study [[250]88]. Briefly, QFA sections were fixed in 4 % paraformaldehyde in PBS (pH 7.4) for 20 min at 4 °C; after several washes, sections were dipped in Mayers Hematoxylin solution (VWR Chemicals); then sections were gently dipped in increasingly concentrated ethanol solutions and counterstained with alcoholic-eosin (AppliChem). The sections were then dehydrated in graded ethanol (AppliChem), immersed in xylene and cover-slipped with Eukitt (Sigma-Aldrich). 4.5.3. MT staining QFA samples were cut on a cryostat in 20-μm-thick serial sections and stored at −80 °C until processed for the MT staining, by adapting the protocol reported in a previous study [[251]89]. Briefly, QFA sections were fixed in 4 % paraformaldehyde in PBS (pH 7.4) for 1 h at RT and then in Bouin solution (Sigma-Aldrich) for 1 h at 56 °C. Then different dyes were used to stain the various cellular components: Weigert's iron hematoxylin solution (Abcam) for staining in black the nuclei; Biebrich scarlet-acid fuchsin solution (Sigma-Aldrich) for staining in red the cytoplasm; Aniline blue solution (Sigma-Aldrich) for staining in blue the collagen fibers. The sections were then dehydrated in graded ethanol, immersed in xylene and cover-slipped with Eukitt (Sigma-Aldrich). All the H&E− and ORO-stained slides were observed under an Eclipse 80i microscope (Nikon, Tokyo, Japan) controlled by the software NIS-Elements-BR (Nikon). For the MT staining, slides were observed under a NanoZoomer S60 scanner (Hamamatsu) controlled by the software NanoZoomer Digital Pathology (Hamamatsu). Three 20X images were taken randomly from each section for all the staining. 4.6. Human skeletal muscle collection 18 frozen biopsies of quadriceps from healthy subjects, (9 lean and 9 obese), were taken from the bank tissues of Centro di Biotecnologie, Cardarelli Hospital, Naples (Italy). The biopsies were used for the evaluation of H[2]S-generating enzymes (CBS, CSE, MPST) by RT-PCR analysis. The research was carried out following the Code of Ethics of the World Medical Association (Declaration of Helsinki) and approved by the Ethical Committee of the Institution (Cardarelli Hospital Centre for the Study of Malignant Hyperthermia; 4/13 prot. 358). The subjects gave written informed consent. The parameter used to select lean versus obese subjects was the body mass index (BMI). In detail, the lean subjects fell in 19< BMI<24 range, while the obese subjects in 313′) REVERSE Sequence (5'->3′) Mouse CBS CCAGGCACCTGTGGTCAAC GGTCTCGTGATTGGATCTGCT CSE TTCCTGCCTAGTTTCCAGCAT GGAAGTCCTGCTTAAATGTGGTG MPST GAACCTGCCAATCAGCTCC CCGGGCATCCAAGTTCTCC TNFα AGGAGGAGTCTGCGAAGAAGA GGCAGTGGACCATCTAACTCG iNOS GAGACAGGGAAGTCTGAAGCAC CCAGCAGTAGTTGCTCCTCTTC SIRT-1 TGATTGGCACCGATCCTCG CCACAGCGTCATATCATCCAG Glut-4 GGTGTGTCAATACGGTCTTCAC AGCAGAGCCACGGTCATCAAGA ACTIN GGCTGTATTCCCCTCCATCG CCAGTTGGTAACAATGCCATGT Human CBS GGCCAAGTGTGAGTTCTTCAA GGCTCGATAATCGTGTCCCC CSE AGGTTTAGCAGCCACTGTAAC GGGGTTTCGATCCAAACAAGC MPST CATTTCGCGGAGTACGCAG GCTGGCGTCGTAGATCACG ACTIN CACCATTGGCAATGAGCGGTTC AGGTCTTTGCGGATGTCCACGT [255]Open in a new tab 4.14. Persulfidation assay Persulfidation was detected using a modified biotin switch assay as described previously [[256]76,[257]90]. Samples were precipitated with 20 % trichloroacetic acid and stored at −80 °C for 24 h. Precipitates were washed with 10 % and 5 % trichloroacetic acid and then resuspended in HENS buffer (250‐mM HEPES–NaOH, 1 mM EDTA, 0.1‐mM neocuproine, 100‐μM deferoxamine, and 2.5 % SDS) containing 20 mM methanethiosulfonate and protease and phosphatase inhibitors to block free thiols. Samples were then incubated for 20 min at 56 °C with rotation. Acetone precipitation was performed, and pellets were resuspended in 150‐μl qPerS‐SID lysis buffer (6‐M urea, 100‐mM NaCl, 2 % SDS, 5‐mM EDTA, and 200‐mM Tris, pH 8.2, 50‐mM iodoacetyl–PEG2–biotin, and 2.5‐mM dimedone), sonicated, and incubated for 2 h at room temperature in the dark. A negative control was generated for each sample by adding DTT (1 mM) during biotin cross‐linking. 10 % of the sample was boiled at 95 °C by the addition of 3 % SDS, 1 % β‐mercaptoethanol, 8‐M urea, and 0.005 % bromophenol blue and used for the identification of the levels of SIRT-1 among the samples (input). 90 % of the samples were used for biotin immunoprecipitation overnight (4 °C) using a high‐capacity streptavidin resin. Elution was performed by the addition of 3 % SDS, 1 % β‐mercaptoethanol, 8‐M urea, and 0.005 % bromophenol blue in PBS for 15 min at room temperature followed by 15 min at 95 °C. Persulfidated proteins were then detected by SDS‐PAGE and Western blotting with a specific antibody against SIRT-1. 4.15. Data analysis All data were expressed as means ± standard error of the mean (SEM) and “n” refers to the number of samples for each set of experiments. Statistical analysis was performed by using one-way analysis of variance (ANOVA), followed by Dunnett's post-test or two-way ANOVA followed by Bonferroni's post-test or one-sample t-test where appropriate. All data were statistically analyzed using GraphPad Prism software version 8. P < 0.05 was considered significant. CRediT authorship contribution statement M. Smimmo: Methodology, Investigation, Data curation. V. Casale: Methodology, Investigation, Data curation. D. D'Andrea: Methodology, Data curation. I. Bello: Methodology. N. Iaccarino: Methodology, Formal analysis. F. Romano: Methodology. V. Brancaleone: Software. E. Panza: Software, Formal analysis. R. d’Emmanuele di Villa Bianca: Writing – review & editing, Software. A. Katsouda: Methodology. E. Mitidieri: Formal analysis. I. Antoniadou: Methodology. A. Papapetropoulos: Supervision. F. Maione: Formal analysis. S. Castaldo: Software. M. Friuli: Methodology. A. Romano: Methodology. S. Gaetani: Methodology. R. Sorrentino: Writing – review & editing, Validation. A. Randazzo: Writing – review & editing, Validation. G. Cirino: Supervision, Conceptualization. M. Bucci: Writing – review & editing, Project administration, Funding acquisition, Conceptualization. M. Filipovic: Writing – review & editing, Writing – original draft, Funding acquisition. V. Vellecco: Writing – review & editing, Writing – original draft, Funding acquisition, Conceptualization. Fundings This research was cofinanced by: Ministero dell’Università e della Ricerca (MUR)—PRIN 2022 (project numbers: 202275M25F to M. Bucci and n. 2022AT7P9T to V. Vellecco) “Finanziato dall'Unione europea – Next Generation EU”; European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (Grant Agreement No. 864921 to M.R.F.). Declaration of competing interest The authors declare no conflict of interest. Acknowledgments