Background: During aging, skeletal muscle mass constantly diminishes and myogenic potential declines. At the cellular level, a decline in mitochondrial function is a hallmark of the aging process and the deficiency of the mitochondrial network contributes to a progressive reduction in muscle mass. Autophagic clearance of mitochondria through the process of mitophagy is required to remove impaired or damaged mitochondria, while mitophagy is a key regulator of muscle maintenance. Dysfunctional degradation of mitochondria is increasingly associated with aging (mitophaging), while mechanical stimuli have been shown to ameliorate the aging-induced impaired muscle mass and function; however, less is known about the potential effects of mechanical loading on mitophaging. The aim of the present study was to investigate the effect of mechanical stretching on mitophagy in aged myoblasts, in vitro. Methods: Cell senescence was replicated using a multiple cell division model of C2C12 myoblasts. The control and aged cells were cultured on elastic membranes and underwent passive stretching using a mechanical loading protocol of 15% elongation for 12 h at a frequency of 1 Hz. Cell signaling and gene expression responses of mitophagy-associated and myogenic regulatory factors (MRFs) were assessed through immunoblotting and qRT-PCR of the cell lysates derived from stretched and non-stretched control and aged myoblasts. Results: Mitophagy factor AMP-activated protein kinase (AMPK), mitochondrial biogenesis stimulator peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1a), and mitophagy/mitochondrial biogenesis factor Parkin were downregulated in control stretched myoblasts compared to non-stretched cells, while the specific mechanical loading protocol used also reduced the phosphorylation of unc-51-like autophagy-activating kinase 1 (p-ULK1) (p < 0.05), as well as the expression of myogenic factor 5 (Myf5) and myogenic factor 4 (myogenin) (p < 0.001). Interestingly, this mechanical loading resulted in increased PGC-1a and Parkin expression (p < 0.05) and induced the previously undetected BCL2 interacting protein 3-like (BNIP3L/NIX) and AMPK expression and p-ULK1 activation in the aged myoblasts. In addition, mechanical stretching differentially affected the expression of MRFs in aged cells, upregulating the early differentiation factor, Myf5 (p < 0.01), while downregulating the late differentiation factor myogenin (p < 0.001). Conclusions: These findings suggest the beneficial effects of mechanical loading on the impaired mitophagy and early differentiation in aged myoblasts, as indicated by the mitophagy initiation and the promotion of mitochondrial biogenesis in these cells. The mechanical loading-induced downregulation of mitophagy and myogenesis in the control myoblasts might indicate their loading-specific differential responses compared to the aged cells.

Background The present study aims at deciphering the individual or combined benefits of aerobic exercise and dietary restriction on liver senescence, a state characterized by cell cycle arrest and simultaneous resistance to apoptosis, which is considered an established hallmark of metabolic dysfunction-associated steatotic liver disease (MASLD). Methods C57BL6 mice were subjected to a normal diet (ND) for 20 weeks or a high-fat diet (HFD) supplemented with 5 % High-fructose Corn Syrup (HFCS) for 12 weeks, followed by eight-week interventions, including dietary restriction (DR), aerobic exercise (EX), a combination of both (DREX) or continuation of a HFD-HFCS diet without intervention. Biomarkers of senescence were analyzed in terms of their liver mRNA expression levels, while GL13 and p21WAF1/CIP1 immunohistochemical stainings were conducted to examine the levels of senescence-associated lipofuscin and p21WAF1/CIP1 respectively, to finally investigate their relationship with the grade of hepatic steatosis and fibrosis observed in the studied mice. Results DR and DREX groups exhibited significantly reduced features of obesity and MASLD-related hepatic steatosis and fibrosis, to a greater extent than the respective amelioration driven by aerobic exercise only in HFDEX animals. A statistically significant increase of mRNA expression was detected for cyclin-dependent kinase p21WAF1/CIP1 in HFD livers as compared to ND, which was also reversed upon DR-inclusive interventions. Immunohistochemical stainings for GL13 and p21WAF1/CIP1, as well as for p16INK4A confirmed the aforementioned alterations of p21WAF1/CIP1 at the tissular level while also revealed a p16INK4A elevation in HFD livers which was reversed only upon DR/DREX. Conclusion Liver senescence is responsive both to exercise and dietary restriction, but its amelioration in the context of MASLD is more robust upon DR-inclusive interventions.

Physical activity exerts systemic anti-inflammatory effects and reduces the risk for multiple non-communicable diseases, with 7.2% of all-cause deaths globally being attributed to physical inactivity. However, the cellular and molecular components of the exercise-induced anti-inflammatory effects remain only partly understood. Herein we show that moderate-intensity exercise promotes anti-inflammatory reprograming of macrophages orchestrated by the skeletal muscle cells secretome. Primary bone marrow-derived macrophages (BMDMs) exposed to the secretome of mechanically-loaded myotubes (exercise-conditioned medium, exCM) acquire an anti-inflammatory transcriptional profile and increased reliance on oxidative phosphorylation, as shown by Seahorse real-time cell metabolic analysis, compatible with an M2-like phenotypic switch. Using an unbiased proteomic analysis of the exCM we identify the chaperonin Hsp60 as a key mediator of the anti-inflammatory effects of exercise. Hsp60 expression increases in mechanically loaded myotubes in vitro, in the quadriceps muscle and serum of mice following an 8-week program of moderate-intensity aerobic exercise, as well as in human muscle after resistance training. Importantly, treatment of BMDMs with Hsp60 in vitro recapitulates the exCM-induced transcriptional reprograming, promoting an M2-like phenotype. Taken together, our data highlight Hsp60 as a novel component of the skeletal muscle cell-macrophage crosstalk, providing mechanistic insights into the anti-inflammatory effects of exercise. ### Competing Interest Statement The authors have declared no competing interest.

Background/Objectives: There is a growing interest in the research of wound healing mechanisms worldwide. Particular attention has been paid to the expression of tissue remodeling- and inflammation-related factors. Herein, we investigate the expression patterns of TGF-β1, IL-6, TNF-a, uPA, uPA receptors, MMP-2, and MMP-9 through the four phases of the normal wound-healing process in humans. Methods: Twenty-two individuals presenting with a first episode of pilonidal sinus underwent surgical excision and the wound was left to heal by secondary intention. Sequential biopsies were collected on day 0 (operation), day 2 (inflammatory phase), day 9 (proliferative phase), and day 14 (tissue remodeling phase). The expression levels of the proteins were evaluated using reverse transcription-quantitative PCR. Statistical analyses were performed using GraphPad Prism software. One-way analysis of variance (ANOVA) with Dunn's Multiple Comparison post hoc test was used. Results: The results showed statistically significant differences in the expressions of the factors during wound healing (p < 0.05). TGF-b1 increased on days 2 and 9. TNF-a increased on day 2 and then decreased on day 9. Il-6 was increased on day 2 and decreased on days 9 and 14. uPa mRNA increased up to day 9 but its receptor exhibited high expression throughout the observation time. Finally, MMP-2 mRNA expression increased on day 2 and declined on days 9 and 14, while MMP-9 was highly expressed until the 14th postoperative day. Conclusions: Each factor investigated in this study has an important and distinct role in the normal wound repair procedure. Further investigation is required to evaluate the tissue-specific regulation of these factors and their potential use as therapeutic targets or prognostic biomarkers in wound healing.

BACKGROUND:Regular exercise training provides significant health benefits among cancer survivors and is associated with lower breast cancer mortality and reduced risk of recurrence. Both exercise-induced factors secreted into circulation (exerkines) and bioactive molecules contained in skeletal muscle secretome have been proposed to affect the tumor microenvironment and mediate some of the anti-carcinogenic effects of exercise. This study utilized exercise-conditioned human serum obtained from breast cancer patients during chemotherapy and skeletal myotubes' secretome after mechanical loading to investigate their effects on breast cancer cells in vitro. METHODS:Breast cancer patients participated in a 12-week exercise training program during their chemotherapy, and blood serum was collected immediately before and after an exercise session in the 2nd and 12th weeks of training. Skeletal myoblasts were differentiated into myotubes and subjected to mechanical stretching to collect their secretome (stretch medium (SM)). Hormone-sensitive Michigan Cancer Foundation-7 (MCF-7) and triple-negative M.D. Anderson-Metastatic Breast-231 (MDA-MB-231) breast cancer cells were treated with either human serum or with the skeletal myotubes' secretome to examine their metabolic activity, migration, cytotoxicity levels and apoptosis regulation. RESULTS:The exercise-conditioned serum obtained from breast cancer patients who were subjected to the 12-week training during chemotherapy resulted in reduced metabolic activity (p < 0.001) and increased lactate dehydrogenase activity (cytotoxicity) (p < 0.001) in both MCF-7 and MDA-MB-231 breast cancer cells when compared with the control condition. Moreover, incubation of breast cancer cells with the post-exercise serum induced apoptosis in MCF-7 and MDA-MB-231 cells, as indicated by increase in DNA damage and the percentage of necrotic cells (p < 0.05) when compared to pre-exercise condition. Similarly, a significant decrease (p < 0.001) was observed in the metabolic activity of MCF-7 cells treated with the SM, along with increased cytotoxicity (p < 0.05), compared to the cells cultured with the regular growth media. Comparable though not as profound effects were observed in MDA-MB-231 cells when treated with the SM secretome. Furthermore, the expression of apoptosis-inducing Caspase-7 (p < 0.001) and Caspase-8 (p < 0.01) proteins was increased, whereas cell survival-regulating factors interleukin-8 (IL-8) (p < 0.001), superoxide dismutase-2 (p < 0.05), Fas cell surface death receptor (p < 0.05), and vascular endothelial growth factor (p < 0.01) were downregulated in the SM-treated MCF-7 cells. In addition, the migrating behavior of MCF-7 cells was diminished, and higher levels of DNA damage were observed in cells treated with either SM or non-stretch media. CONCLUSION:Both exercise-conditioned serum of breast cancer patients and skeletal myotubes secretome after mechanical loading can reduce the metabolic activity, promote cell toxicity and DNA damage, modulate the protein expression of crucial cell survival-regulating factors, and lead to apoptosis in breast cancer cells. These findings suggest that even after cancer diagnosis, exercise may exert beneficial effects additive to chemotherapy against breast cancer prognosis.

Purpose: High-intensity interval training (HIIT) has emerged as a time-efficient alternative to traditional endurance training. This study investigated the molecular, systemic, and physiological adaptations induced by an 8-week HIIT program in national-level flatwater kayak athletes. Methods: Six trained male kayakers completed an 8-week HIIT intervention. Skeletal muscle biopsies and venous blood samples were collected before and after training to assess markers related to endocrine function, growth and remodeling, angiogenesis, and inflammation. Physiological and performance measures were evaluated using a maximal oxygen uptake (VO2max) test and kayak ergometer trials. Results: HIIT elicited significant improvements across molecular, systemic, and performance parameters. Post-training analyses showed increased expression of IGF-1R, MMP-4, MMP-9, and TNF-α (p < 0.05), along with elevated serum testosterone concentrations (p < 0.05). Notable performance gains were observed in paddling speed at the second ventilatory threshold (PSVT2; p < 0.05) and in 1000 m (p < 0.01) and 200 m (p < 0.001) time-trial performances. Conclusions: An 8-week HIIT program effectively enhanced molecular signaling, systemic adaptation, and sport-specific performance in elite flatwater kayak athletes. The concurrent upregulation of anabolic, remodeling, and inflammatory pathways suggests that HIIT facilitates coordinated muscular and systemic adaptations beneficial for kayak performance.

Background: The present study aims at deciphering the potential benefits of aerobic exercise and dietary restriction on liver senescence, which is an established hallmark of metabolic dysfunction-associated steatotic liver disease (MASLD), a condition with limited therapeutic options. Methods: C57BL6 mice were subjected to normal diet (ND, 10% of kilocalories from fat) or a high-fat diet (HFD, 60% of kilocalories deriving from fat and water supplemented with 5% High-fructose Corn Syrup, HFCS) for 12 weeks. Then, for additional 8 weeks, the ND group continued with the same diet, while the HFD group was divided into four subgroups: a)mice that continued with the same HFD-5% HFCS in water scheme (HFD), b)mice that continued with the same HFD-5% HFCS in water scheme and underwent supervised aerobic exercise 3-times/week (HFDEX), c)mice that were switched to ND (dietary restriction, DR) and d)mice that were switched to ND while undergoing supervised aerobic exercise 3-times/week (DREX). Phenotypic and histological characterization of obesity and MASLD were performed in all groups. Biomarkers of senescence were analyzed in terms of their mRNA expression levels to assess the impact of all interventions on MASLD-related senescence in the liver. GL13 and p21 immunohistochemical stainings were conducted to examine the protein levels of the presence of senescence-associated lipofuscin and p21CIP/WAF respectively, so as to finally investigate their relationship with the grade of steatosis observed in each individual animal. Results: DR and DREX groups exhibited significantly reduced features of obesity and MASLD-related hepatic steatosis, to a greater extent than the respective amelioration driven by aerobic exercise-only in HFDEX animals. A statistically significant increase of the mRNA expression of cyclin-dependent kinase p21CIP/WAF was detected in HFD livers as compared to ND, which was also reversed upon DR-inclusive interventions. In contrast, the gene expression levels of cyclin-dependent kinase p16INK4a remained similar in all groups even after a combined intervention. Increased hepatic expression of the p27 and p53 components of the p53-p21 CIP/WAF-driven axis of cellular senescence as well as their restoration to ND-like levels upon DR and DREX, suggest an active participation of the p21CIP/WAF mechanism of senescence in the emergence of MASLD, but also in its reversal through DR or/and EX interventions. Immunohistochemical stainings for GL13 and p21 confirmed the aforementioned alterations of p21CIP/WAF at the tissular level. Conclusion: Liver senescence is responsive both to exercise and dietary restriction, but its amelioration in the context of MASLD is more robust upon DR-inclusive interventions. ### Competing Interest Statement The authors have declared no competing interest.

Endometriosis is a benign, estrogen-dependent gynecological condition with an uncertain exact pathogenetic mechanism. The aim of this study was to evaluate the potential differential expression of Insulin Growth Factor 1 (IGF-1) isoforms in deeply infiltrating endometriotic (DIE) lesions, in ovarian endometriomas, and in the eutopic endometrium of the same endometriosis patients and to compare their expression with that in the eutopic endometrium of women without endometriosis. A total of 39 patients were included: 28 with endometriosis, of whom 15 had endometriomas only, 7 had DIE nodules only, and 6 had both DIE and endometriomas, and 11 without endometriosis served as controls. We noticed a similar pattern of expression between IGF-1Ea and IGF-1Ec, which differed from that of the IGF-1Eb isoform, possibly implying differential biological actions of different isoforms in DIE subtypes. We observed a tendency of lower expression of IGF-1Ea and IGF-1Ec in endometriomas without DIE compared to endometriomas with concurrent DIE or in DIE nodules. In conclusion, differential expression of IGF-1 isoforms may indicate that DIE with its associated ovarian lesions and simple ovarian endometriosis should be considered as two forms of the disease developing under different molecular pathways.

Uremic toxins have been linked to muscle pathology (uremic myopathy) observed in chronic kidney disease patients. Among prominent uremic toxins, indoxyl sulfate (IS) has been shown to negatively affect various tissues. However, there is little understanding of its role in uremic myopathy. PURPOSE: To study the effects of IS on anabolic and atrophy-related genes of myoblasts and myotubes. METHODS: C2C12 myoblasts were cultured under standard conditions, treated with 0.1, 0.5, 1 mM IS and collected after 48 h. In two separate experimental set ups, myoblasts were allowed to differentiate and treated with 0.1, 0.5, 1 mM IS for 48 h before their harvest at day3, or day5 of differentiation (myotubes). Real-time PCR was used to detect expression changes of IGF-1 and Atrogin-1. Comparisons between conditions were made using one-way ANOVA. Data are shown as mean ± SEM of three independent experiments performed in triplicate. RESULTS: The anabolic factor IGF-1 was found to be downregulated by IS. Specifically, its fold changes after the treatment of the cells with 0.1, 0.5 and 1 mM IS were as follows: Myoblasts: 0.47 ± 0.19, 0.56 ± 0.12, 0.60 ± 0.15 respectively with a significant difference observed between control and 0.1 mM IS (p = 0.022); Day3 myotubes: 0.62 ± 0.05, 0.85 ± 0.18, 0.34 ± 0.04 respectively with IGF-1 being significantly downregulated in 0.1 mM IS and 1 mM compared to control (p = 0.043 and p < 0.001 respectively); Day5 myotubes: 0.99 ± 0.29, 0.57 ± 0.10, 0.90 ± 0.19 respectively (p > 0.05). The atrophy factor Atrogin-1 was found to be mainly upregulated by IS with its fold changes after 48 h treatment with 0.1, 0.5 and 1 mM IS being as follows: Myoblasts: 0.92 ± 0.29, 1.43 ± 0.40, 1.29 ± 0.25 respectively (p > 0.05); Day3 myotubes: 0.90 ± 0.15, 1.42 ± 0.14, 1.07 ± 0.17 respectively, with a significant difference found between 0.1 mM IS and 0.5 mM IS (p = 0.044); Day5 myotubes: 0.85 ± 0.19, 0.97 ± 0.2, 1.27 ± 0.09 respectively (p > 0.05). CONCLUSIONS: IS appears to have a negative effect on proliferation and early differentiation of muscle cells even in low concentrations, indicating that it could lead to low muscle regeneration capacity in renal patients. Future experiments will examine additional anabolic and atrophy-related genes as well as myogenic factors as linked also to mature muscle samples. Greek State Scholarships Foundation-MIS5033021

Endometriosis, defined as the development of endometrial tissue outside of the uterine cavity, is a common gynecological disorder. The prevalence of pelvic endometriosis approaches 6%-10% in the general female population, and in women with pain, infertility, or both, the frequency is 35%-50%. The gold standard recommended process for diagnosing endometriosis is laparoscopy, an invasive surgical procedure, with or without histologic verification. The currently available nonsurgical treatments include oral contraceptives (estrogen-progestogen preparations), progestogen preparations (containing progesterone derivatives), androgenic hormones (danazol), and gonadotropin-releasing hormone (GnRH) agonists and antagonists. Two GnRH types have been discovered in mammals, GnRH I and GnRH II. In particular, GnRH I is released by the hypothalamus; however, it can be present in various tissues and organs of the body, including neural tissue, where it exerts neuroendocrine, autocrine, and paracrine actions in the peripheral and central nervous system (CNS). Interestingly, another GnRH isoform, GnRH III, has been identified, which has 60% similarity with GnRH I from which it varies by four amino acids. This peptide has been shown to have a significant role in reproduction, specifically in gametogenesis and steroidogenesis. Further research is needed to identify innovative treatment options for endometriosis, such as the therapeutic exogenous administration of GnRH II or antagonists of the GnRH I receptor. In this review, we examined the role of GnRH in endometriosis, outlining the specific actions of GnRH and GnRH receptors (GnRHRs). The innovative use of GnRH analogs and antagonists in the treatment of endometriosis is also discussed.

CHCHD2 is an antiapoptotic mitochondrial protein acting through the BCL2/BAX pathway in various cancers. However, data on the regulatory role of CHCHD2 in adrenal tumourigenesis are scarce. We studied the expression of CHCHD2, BCL2, and BAX in human adrenocortical tissues and SW13 cells. mRNA and protein levels were analyzed through qPCR and immunoblotting, respectively, in 16 benign adrenocortical neoplasms (BANs), along with their adjacent normal adrenal tissues (controls), and 10 adrenocortical carcinomas (ACCs). BCL2/BAX mRNA expression was also analyzed in SW13 cells after CHCHD2 silencing. MTS, flow cytometry and scratch assays were performed to assess cell viability, apoptosis, and invasion, respectively. BCL2 and CHCHCD2 mRNA and protein expression was increased in BANs compared to normal adrenal tissues whereas BAX was decreased. BAX and CHCHD2 mRNA and protein levels were significantly downregulated and upregulated, respectively, in ACCs compared with either BANs or controls. Expression of the studied genes was not different among cortisol-secreting and nonfunctional ACAs. No significant association was found between genes’ expression and other established prognostic markers of ACCs patients. In vitro analysis showed that CHCHD2 silencing resulted in reduced cell viability and invasion as well as increased SW13 cells apoptosis. CHCHD2 expression seems to be implicated in adrenal tumourigenesis and its absence resulted to increased apoptosis in vitro. However, the exact mechanism of action and particularly its association with the BAX/BCL2 pathway needs to be further studied and evaluate whether it could be a protentional therapeutic target.

Cardiomyocytes possess the ability to respond to mechanical stimuli by adapting their biological functions. This study investigated cellular and molecular events in cardiomyocyte-like H9C2 cells during differentiation as well as the signalling and gene expression responses of the differentiated cells under various mechanical stretching protocols in vitro. Immunofluorescence was used to monitor MyHC expression and structural changes during cardiomyoblast differentiation. Moreover, alterations in the expression of cardiac-specific markers, cell cycle regulatory factors, MRFs, hypertrophic, apoptotic, atrophy and inflammatory factors, as well as the activation of major intracellular signalling pathways were evaluated during differentiation and under mechanical stretching of the differentiated H9C2 cells. Compared to undifferentiated cells, advanced-differentiation cardiomyoblasts exhibited increased expression of cardiac-specific markers, MyHC, MRFs, and IGF-1 isoforms. Moreover, differentiated cells that underwent a low strain/frequency mechanical loading protocol of intermediate duration showed enhanced expression of MRFs and hypertrophic factors, along with a decreased expression of apoptotic, atrophy, and inflammatory factors compared to both high-strain/frequency loading protocols and to unloaded cells. These findings suggest that altering the strain and frequency of mechanical loading applied on differentiated H9C2 cardiomyoblasts can regulate their anabolic/survival program, with a low-strain/frequency stretching being, overall, most effective at inducing beneficial responses.

BACKGROUND:During aging, muscle cell apoptosis increases and myogenesis gradually declines. The impaired myogenic and survival potential of the aged skeletal muscle can be ameliorated by its mechanical loading. However, the molecular responses of aged muscle cells to mechanical loading remain unclear. This study examined the effect of mechanical loading of aged, proliferating, and differentiated myoblasts on the gene expression and signaling responses associated with their myogenic lineage progression and survival.METHODS:Control and aged C2C12 cells were cultured on elastic membranes and underwent passive stretching for 12 h at a low frequency (0.25 Hz) and different elongations, varying the strain on days 0 and 10 of myoblast differentiation. Activation of ERK1/2 and Akt, and the expression of focal adhesion kinase (FAK) and key myogenic regulatory factors (MRFs), MyoD and Myogenin, were determined by immunoblotting of the cell lysates derived from stretched and non-stretched myoblasts. Changes in the expression levels of the MRFs, muscle growth, atrophy, and pro-apoptotic factors in response to mechanical loading of the aged and control cells were quantified by real-time qRT-PCR.RESULTS:Mechanical stretching applied on myoblasts resulted in the upregulation of FAK both in proliferating (day 0) and differentiated (day 10) cells, as well as in increased phosphorylation of ERK1/2 in both control and aged cells. Moreover, Akt activation and the expression of early differentiation factor MyoD increased significantly after stretching only in the control myoblasts, while the late differentiation factor Myogenin was upregulated in both the control and aged myoblasts. At the transcriptional level, mechanical loading of the proliferating myoblasts led to an increased expression of IGF-1 isoforms and MRFs, and to downregulation of muscle atrophy factors mainly in control cells, as well as in the upregulation of pro-apoptotic factors both in control and aged cells. In differentiated cells, mechanical loading resulted in an increased expression of the IGF-1Ea isoform and Myogenin, and in the downregulation of atrophy and pro-apoptotic factors in both the control and aged cells.CONCLUSIONS:This study revealed a diminished beneficial effect of mechanical loading on the myogenic and survival ability of the senescent muscle cells compared with the controls, with a low strain (2%) loading being most effective in upregulating myogenic/anabolic factors and downregulating atrophy and pro-apoptotic genes mainly in the aged myotubes.

Wheelchair basketball (WB) is one of the most popular sports among people with varying physical disabilities, characterized by intermittent moderate- and high-intensity efforts. Exercise can modulate the Hypothalamus-Pituitary-Adrenal (HPA) axis, levels of circulating steroids and cytokines however, little is known about the salivary hormonal and cytokine concentrations in response to a wheelchair basketball match. PURPOSE: The present study investigated potential changes in salivary cortisol (CORT), testosterone (TESTO) and interleukin (IL)-6 after a WB match. METHODS: Eight male WB players (age: 40.50±3.12 yrs; injury functional classification: 1.0-4.5), members of the winning team of the official national championship, participated in this study. Unstimulated saliva samples were collected from the athletes using specialized salivette cotton swabs, immediately before and after the completion of the match and stored at -80oC until analysis. The collected saliva samples were assayed for CORT, TESTO and IL-6 using commercially available ELISA kits. Wilcoxon signed-rank test was used for statistics and results are presented as mean ± SE. RESULTS: Cortisol levels exhibited a trend to increase (5.83±2.15 ng/ml vs 11.25±4.54 ng/ml) and those of testosterone to decrease (30.68±8.73 ng/ml vs 18.55±4.57 ng/ml) after the match compared to baseline, though without reaching statistical significance in both hormones (p>0.05). Similarly, there were no significant changes in cortisol to testosterone ratio post-match compared to pre-match values (0.14±0.04 vs 0.50±0.25; p>0.05). In addition, IL-6 concentrations decreased after the game (2.22±0.87 pg/ml vs 1.72±0.32 pg/ml), though these changes were not significant (p>0.05). CONCLUSIONS: Our findings suggest that although a high-level WB match may acutely trigger only mild changes in saliva CORT and TESTO, as well as in IL-6 concentrations in those athletes, these salivary changes may not reflect the systemic hormonal and/or cytokine responses to the exercise-induced stress and the frequently excessive efforts of elite athletes with neurological impairments during a match.

The process of myogenesis gradually deteriorates as the skeletal muscle ages, contributing to muscle mass loss. The aim of this study is to investigate the effect of senescence/aging on skeletal myogenesis, in vitro. A model of multiple cell divisions of C2C12 myoblasts was used to replicate cell senescence. Control and aged myoblasts were investigated during myogenesis, i.e., at days 0, 2, and 6of differentiation. SA-β-gal activity and comet assay were used as markers of aging and DNA damage. Flow cytometry was performed to characterize potential differences in cell cycle between control and aged cells. Alterations in the mRNA and/or protein expression of myogenic regulatory factors (MRFs), IGF-1 isoforms, apoptotic, atrophy, inflammatory, metabolic and aging-related factors were evaluated. Compared with the control cells, aged myoblasts exhibited G0/G1 cell cycle arrest, DNA damage, increased SA-β-gal activity, and increased expression of aging-related factors p16 and p21 during differentiation. Moreover, aged myoblasts showed a reduction in the expression of MRFs and metabolic/anabolic factors, along with an increased expression of apoptotic, atrophy and inflammatory factors. A diminished differentiation capacity characterized the aged myoblasts which, in combination with the induction of apoptotic and atrophy factors, indicated a disrupted myogenic lineage in the senescent muscle cells.

Cardiomyocytes possess the ability to respond to mechanical stimuli by reprogramming their gene expression. This study investigated the effects of different loading protocols on signaling and expression responses of myogenic, anabolic, inflammatory, atrophy and pro-apoptotic genes in cardiomyocyte-like H9C2 cells. Differentiated H9C2 cells underwent various stretching protocols by altering their elongation, frequency and duration, utilizing an in vitro cell tension system. The loading-induced expression changes of MyoD, Myogenin, MRF4, IGF-1 isoforms, Atrogin-1, Foxo1, Fuca and IL-6 were measured by Real Time-PCR. The stretching-induced activation of Akt and Erk 1/2 was also evaluated by Western blot analysis. Low strain (2.7% elongation), low frequency (0.25 Hz) and intermediate duration (12 h) stretching protocol was overall the most effective in inducing beneficial responses, i.e., protein synthesis along with the suppression of apoptosis, inflammation and atrophy, in the differentiated cardiomyocytes. These findings demonstrated that varying the characteristics of mechanical loading applied on H9C2 cells in vitro can regulate their anabolic/survival program.

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Skeletal muscle can adapt to mechanical loading by changing its mass and overall contractile phenotype via the activation of mechanotransduction and intracellular signaling mechanisms. In vitro mechanical loading of differentiated myoblasts (myotubes) has been utilized for mimicking the mechanical loading conditions of skeletal muscle in vivo. PURPOSE: This study investigated the effects of mechanical loading of myotubes on their gene expression responses associated with various aspects of cellular function, such as differentiation, hypertrophy and apoptosis. METHODS: C2C12 myoblasts were cultured on elastic membranes up to day 9 of their differentiation and then underwent a passive, cyclic stretching (15% elongation, at a frequency of 0.25 Hz, for 12 hours). Myotubes were harvested and lysed 12 hours after the completion of the stretching protocol. Real Time-PCR was utilized to measure changes in mRNA expression levels of myogenic regulatory factors (MRFs: MyoD, Myogenin, MRF4), as well as growth (IGF-1 isoforms: IGF-1Ea, IGF-1Eb), atrophy (Murf1, Atrogin, Myostatin), apoptotic (Foxo, Fuca, p53) and inflammatory factors (IL-6, IL-1b) in response to mechanical loading of the differentiated myoblasts. RESULTS: Mechanical loading of the myotubes resulted in increased expression of MyoD (1.5-fold; p<0.05) and MRF4 (2.0-fold; p<0.05) while Myogenin expression) decreased by 0.4-fold (p<0.05). Expression of muscle atrophy factors Atrogin(0.5-fold), Myostatin (0.4-fold), and Murf1 (0.4-fold), and of the inflammatory factor IL-1b (0.5-fold) was significantly decreased (p<0.05). No significant changes were revealed in the expression levels of IGF-1 isoforms (IGF-1Ea: 0.9-fold, IGF-1Eb: 1.1-fold) and apoptotic factors (Foxo: 0.8-fold, Fuca: 1.1-fold, p53: 1.0-fold), as well as of IL-6 (0.8-fold) in response to the selected stretching protocol of the differentiated myoblasts. CONCLUSIONS: These findings suggest that the specific mechanical loading protocol can further affect the myogenic differentiation program and protein synthesis of skeletal myotubes by influencing the expression of myogenic factors and downregulating muscle atrophy genes.