Search

The production of a cellularized silk fibroin scaffold is very difficult because it is actually impossible to differentiate cells into a well-organized cardiac tissue. Without vascularization, not only do cell masses fail to grow, but they may also exhibit an area of necrosis, indicating a lack of oxygen and nutrients. In the present study, we used the so-called tyrosine protein kinase kit (c-kit)-positive cardiac progenitor cells (CPCs) to generate cardiac cellularized silk fibroin scaffolds, multipotent cells isolated from the adult heart to date that can show some degree of differentiation toward the cardiac phenotype. To test their ability to differentiate into the cardiac phenotype in vivo as well, CPC and collagen organoid-like masses were implanted into nude mice and their behavior observed. Since the three-dimensional structure of cardiac tissue can be preserved by scaffolds, we prepared in parallel different silk fibroin scaffolds with three different geometries and tested their behavior in three different models of immunosuppressed animals. Unfortunately, CPC cellularized silk fibroin scaffolds cannot be used in vivo. CPCs implanted alone or in collagen type I gel were destroyed by CD3+ lymphocytes’ aggregates, whereas the porous and partially oriented scaffolds elicited a consistent foreign body response characterized by giant cells. Only the electrospun meshes were resistant to the foreign body reaction. In conclusion, c-kit-positive CPCs, although expressing a good level of cardiac differentiation markers in vitro with or without fibroin meshes, are not suitable for an in vivo model of cardiac organoids because they are degraded by a T-cell-mediated immune response. Even scaffolds, which may preserve the survival of these cells in vivo, also induced a host response. However, among the tested scaffolds, the electrospun meshes (F-scaffold) induced a lower response compared to all the other tested structures.

This study was aimed at evaluating the prevalence of use of dietary supplements (DSs) among gym users and gym instructors involved in body shaping-oriented fitness training. Furthermore, this study aimed to verify whether differences existed in the prevalence and in the types of DSs used in both gym users and gym instructors involved in body shaping-oriented fitness competitions vs. those not involved in fitness competitions. A survey was distributed to 316 participants, composed of 89 gym instructors and 227 gym users of both genders aged 27.3 ± 7.7. Among these participants, 52 were involved in competitions and 248 were not, while 16 participants did not specify either way. The results showed a high prevalence in the use of DSs in the population considered, with 85.4% of the participants declaring they used DSs, with high heterogeneity in the numbers and in the combinations used. No differences were found between gym instructors and gym users, or between participants involved and those not involved in competitions. The results indicate that DSs are widely used by persons involved in body shaping-oriented fitness training. The results also suggest that the majority of the participants decided individually which DSs to use.

In recent years, there has been a shift from tissue engineering to the production of organoids. The latter are useful tools to study many biochemical aspects and cellular reactions while avoiding the excessive use of laboratory animals. Organoids are very interesting tools because they can replicate the cellular and extracellular environment of an organ and retain some of the properties of the organ itself. However, without an adequate network of vessels, cell masses not only fail to grow, but they may exhibit an area of necrosis, indicating a lack of oxygen and nutrients. For this reason, scientific researchers are looking for ways to create organoids that can also mimic the vascular network of the organ from which they originate. One possibility is to implant the organoids in immunocompromised animals.In the present study, we generated cardiac organoids ex vivo by seeding tyrosine protein kinase kit (c-kit)-positive cardiac progenitor cells (CPC cells) from fresh rat hearts into a rat collagen I gel. We then implanted these patches into immunosuppressed animals and compared the suitability of different silk fibroin scaffolds with three different geometries. We demonstrated that CPC cells were destroyed by CD3+ lymphocytes, that the porous and partially oriented scaffolds induced a consistent foreign body response compared to the electrospun meshes, and that CPC cells were degraded by a T-cell-mediated immune response, although the latter may be suitable for generating rat cardiac organoids.

In the last few years, there has been emerging interest in developing treatments against human diseases using natural bioactive content. Here, the powder of the edible mushroom Pleurotus eryngii var. eryngii was mixed with the normal diet of mice bearing C26 colon carcinoma. Interestingly, it was evidenced by a significant increase in the survival rate of C26 tumor-bearing mice accompanied by a significant increase in Hsp90 and Hsp27 protein levels in the tumors. These data were paralleled by a decrease in Hsp60 levels. The mushroom introduced in the diet induced the inhibition of the transcription of the pro-inflammatory cytokines IL-6 and IL-1 exerting an anti-inflammatory action. The effects of the mushroom were mediated by the activation of c-Jun NH2-terminal kinases as a result of metabolic stress induced by the micronutrients introduced in the diet. In the tumors of C26 bearing mice fed with Pleurotus eryngii there was also a decreased expression of the mitotic regulator survivin and the anti-apoptotic factor Bcl-xL as well as an increase in the expression levels of Atg7, a protein that drives autophagy. In our hypothesis the interplay of these molecules favored the survival of the mice fed with the mushroom. These data are promising for the introduction of Pleurotus eryngii as a dietary supplement or as an adjuvant in anti-cancer therapy.

An investigation into the histological changes in skeletal muscle fibres and jump performance indicators after 8 weeks of plyometric squat jump training was conducted. Healthy inactive participants (n = 13; age: 21.5 ± 1.7 year.; height: 173.6 ± 10.7 cm; weight: 68.5 ± 18.4 kg; BMI 22.4 ± 3.8 kg/m2) were recruited, where eight participants completed plyometric squat jump training and five control participants refrained from performing any jumping activities. Blood samples, vastus lateralis muscle biopsies and functional testing (peak and average power, peak and average velocity, maximal jump height) were collected/recorded 10 days prior to and 3 days after the training/rest period. Participants completed 1644 squat jumps over an 8-week training period of 24 sessions with a progressive increase in the number of squat jumps. The trained group significantly increased their jumping average and peak power (mean increases in average power: 16.7 ± 1.2% and peak power: 8.2% ± 0.1) and velocity (mean increases in average velocity: 13.7 ± 0.1% and peak velocity: 5.2% ± 0.03), resulting in a 25% improvement in vertical jump height. No muscle morphological changes in terms of the cross-sectional area (CSA) or muscle-fibre-type transition were observed after the plyometric training. Improvements in the functional performance indicators following training may more likely be explained by sarcomere ultrastructural adaptation, which did not directly affect myosin heavy chain or CSA.

The αB-crystallin (HSPB5) protein is modulated in response to a wide variety of stressors generated by multiple physio-pathological conditions, sustained by reactive oxygen species (ROS) production. In cardiac muscle tissue, this protein regulates various cellular processes, such as protein degradation, apoptosis and the stabilization of cytoskeletal elements. In this work, we studied the role of HSPB5 expression, activation and localization in HL-1 murine cardiomyocytes exposed to pro-oxidant and non-cytotoxic H2O2 concentration, as well as in cardiac tissue isolated from mice following an acute, non-damaging endurance exercise. Our results demonstrated that HSPB5 is the most abundant HSP in both cardiac muscle tissue and HL-1 cells when compared to HSPB1 or HSPA1A (≈3–8 fold higher protein concentrations, p < 0.01). The acute exposure of cardiac muscle cells to sustainable level of H2O2 “in vitro” or to aerobic non-damaging exercise “in vivo” determined a fast and specific increase of HSPB5 phosphorylation (from 3 up to 25 fold increase, p < 0.01) correlated to an increase in lipid peroxidation (p < 0.05). In both experimental models, p-HSPB5 likely facilitated both the interaction with β-actin, desmin, and α-Filamin 1, the last one identified as new HSPB5 substrate in cardiac cells, as well as the sub-localization of HSPB5 within the same cellular compartment or the re-localization between compartments (i.e., nucleus and cytosol). Taken together, these data point out the role of “oxidative eustress” induced by physiological conditions in activating the molecular machinery devoted to cardiomyocytes’ protection and candidate HSPB5 as a putative molecular mediator for the health benefits induced in cardiac tissue by exercise training.

BAC KGROU ND: Estimates indicate that more than one third of European adults are inactive, despite the known benefits of physical activity. In 2015 the European Commission launched the European Week of Sport (EWoS), to encourage people to engage in sport and physical activity. The aim of this study was to evaluate if participation in the EWoS could motivate children to participate in physical activity in future. METHOD S: A total of 10,892 children (aged 6-14), from 6 EU cities (Palermo, Italy; Ankara, Turkey; Lousada, Portugal; Gardabaer, Iceland; Rijeka, Croatia; Albacete, Spain), were enrolled in sport activities (running sport event, extra hours of physical activity, seminars on physical activity, and a family sport festival during the weekend) during the EWoS 2016. A questionnaire was set up and distributed amongst participants to identify the physical activity habits of schoolchildren and whether the activities conducted during the project were able to establish the desire to participate in physical activity. RE SUL TS: Data has shown that 15% of the individuals (respondents from the 6 countries) did not practice sport, although large variability among participating countries exists. The majority (15%) of these children showed an interest in practicing sport in ensuing months following EWoS. CO NCLU SIO NS: The results suggest that the participation in sport activities during the EWoS encouraged inactive European children to practice physical activity in the months that followed. Future researchers should however investigate whether the motivation to participate in sport observed in this study in fact became reality.

Besides its substantial role in eye lens, αB-crystallin (HSPB5) retains fundamental function in striated muscle during physiological or pathological modifications. In this study, we aimed to analyse the cellular and molecular factors driving the functional response of HSPB5 protein in different muscles from mice subjected to an acute bout of non-damaging endurance exercise or in C2C12 myocytes upon exposure to pro-oxidant environment, chosen as “in vivo” and “in vitro” models of a physiological stressing conditions, respectively.To this end, red (GR) and white gastrocnemius (GW), as sources of slow-oxidative and fast-glycolytic/oxidative fibers, as well as the soleus (SOL), mainly composed of slow-oxidative type fibers, were obtained from BALB/c mice, before (CTRL) and at different times (0′, 15′, 30′ 120′) following 1-h of running. Although the total level of HSPB5 protein was not affected by exercise, we found a significantly increase of phosphorylated HSPB5 (p-HSPB5) only in GR and SOL skeletal muscle with a higher amount of type I and IIA/X myofibers. The fiber-specific activation of HSPB5 was correlated to its interaction with the actin filaments, as well as to an increased level of lipid peroxidation and carbonylated proteins. The role of the pro-oxidant environment in HSPB5 response was investigated in terminally differentiated C2C12 myotubes, where most of HSPB5/pHSPB5 pool was present in the cytosolic compartment in standard culture conditions. As a result of exposure to pro-oxidizing, but not cytotoxic, H2O2 concentration, the p-38MAPK-mediated phosphorylation of HSPB5 resulted functional to promote its interaction with the myofibrillar components, such as β-actin, desmin and filamin 1.This study provides novel information on the molecular pathway underlying the HSPB5 physiological function in skeletal muscle, confirming the contribution of the pro-oxidant environment in HSPB5 activation and interaction with substrate/client myofibrillar proteins, offering new insights for the study of myofibrillar myopathies and cardiomyopathies. Keywords: αB-crystallin phosphorylation, Endurance exercise, Oxidative stress, Skeletal muscle

Heat shock proteins (Hsps) are highly expressed in a variety of cancer types contributing to tumor cell propagation and protection against apoptosis [1]. The current anti-cancer therapy is not always target specific and often is associated with complications for patients, Therefore new effective, specific and less toxic therapeutic approaches are needed. Medicinal mushrooms have emerged as wonderful source of nutraceuticals, anti-oxidants, anticancer, prebiotic, anti-inflammatory, cardiovascular, anti-microbial, and anti-diabetic. The ongoing research projects are aimed to promote mushrooms as new generation ‘‘biotherapeutics’’[2]. The aim of this study was to evaluate whether the cold-water extracts of Pleurotus eryngii var. eryngii can affect Hsp90, 70, 60 and 27 levels in an in vitro model of colon cancer (C26 cells). Cell viability was evaluated using MTT assay after treating the cells with different concentrations of extracts (0-1.9 µg/µl) in the culture medium for 24 and 48 hours. Hsp90, 70, 60 and 27 levels were measured using western blotting and immunofluorescence analysis. Moreover, we evaluated the anticancer effect of the P. eryngii var. eryngii extract in an animal model of ectopically-implanted C26 colon carcinoma, widely used as an experimental model of cancer cachexia. We prepared a mixture of lyophilized P. eryngii var. eryngii with the mice standard diet and the animals were daily fed with ~4g of the mix until they died to draw a survival curve. We sampled the neoformations grown after implantation e on these we performed an immunohistochemistry for Hsp60. Our results showed that the extract significantly decreased cells viability at 0.48 µg/µl after both 24 and 48 hours of treatments. Western blotting analysis and immunofluorescence showed that Hsp60 protein levels were down-regulate at 24h of treatment but increased after 48h. On the contrary, Hsp90, 70 and 27 protein levels did not changed. In the in vivo model, P. eryngii var. eryngii in the diet significantly extended the median survival compared to untreated mice. The immunoistochemical experiments suggested that Pleuery significantly affected the increase of Hsp60 protein levels. These preliminary results are promising for further studies to better understand the potential effects of P. eryngii var. eryngii on cancer progression especially regarding Hsp60 role.

Conjugated linoleic acid (CLA) has been reported to improve muscle hypertrophy, steroidogenesis, physical activity, and endurance capacity in mice, although the molecular mechanisms of its actions are not completely understood. The aim of the present study was to identify whether CLA alters the expression of any of the peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α) isoforms, and to evaluate the possible existence of fibre-type-specific hypertrophy in the gastrocnemius and plantaris muscles. Mice were randomly assigned to one of four groups: placebo sedentary, CLA sedentary, placebo trained, or CLA trained. The CLA groups were gavaged with 35 μl per day of Tonalin® FFA 80 food supplement containing CLA throughout the 6-week experimental period, whereas the placebo groups were gavaged with 35 μl sunflower oil each day. Each administered dose of CLA corresponded to approximately 0.7 g/kg or 0.5%, of the dietary daily intake. Trained groups ran 5 days per week on a Rota-Rod for 6 weeks at increasing speeds and durations. Mice were sacrificed by cervical dislocation and hind limb posterior muscle groups were dissected and used for histological and molecular analyses. Endurance training stimulated mitochondrial biogenesis by PGC1α isoforms (tot, α1, α2, and α3) but CLA supplementation did not stimulate PGC1α isoforms or mitochondrial biogenesis in trained or sedentary mice. In the plantaris muscle, CLA supplementation induced a fibre-type-specific hypertrophy of type IIx muscle fibres, which was associated with increased capillary density and was different from the fibre-type-specific hypertrophy induced by endurance exercise (of types I and IIb muscle fibres). J. Cell. Physiol. 232: 1086-1094, 2017. © 2016 Wiley Periodicals, Inc.

Purpose: The purpose of this study was to investigate skeletal muscle changes induced by an acute bout of plyometric exercise (PlyEx) both before and after PlyEx training, to understand if titin is affected differently after PlyEx training. Methods: Healthy untrained individuals (N=11) completed the 1stPlyEx (10x10 squat-jumps, 1min rest). Thereafter, 6 subjects completed 8 weeks of PlyEx, while 5 controls abstained from any jumping activity. Seven days after the last training session all subjects completed the 2ndPlyEx. Blood samples were collected before, 6 hours and 1, 2, 3 and 4 days after each acute bout of PlyEx, and muscle biopsies 4 days before and 3 days after each acute bout of PlyEx. Results: The 1stPlyEx induced an increase in circulating myoglobin concentration. Muscle sample analysis revealed Z-disk streaming, a stretch or a fragmentation of titin (immunogold), and increased calpain-3 autolysis. After training, 2ndPlyEx did not induce Z-disk streaming, or calpain-3 activation. The previously observed post-1stPlyEx positional change of the titin c-terminus was still present pre-2ndPlyEx, in all trained and all control subjects. Only 2 controls presented with Z-disk streaming after 2ndPlyEx, while calpain-3 activation was absent in all controls. Discussion: Eccentric explosive exercise induced a stretch or fragmentation of titin, which presented as a positional change of the c-terminus. Calpain-3 activation does not occur when titin is already stretched before explosive jumping. Enzymatic digestion results in titin fragmentation, but since an increase in calpain-3 autolysis was visible only after the 1stPlyEx acute bout, fragmentation cannot explain the prolonged positional change.

Introduction: We investigated changes in satellite cell (SC) pool size after an acute bout of strenuous exercise and evaluated the influence of baseline SC count and fiber type. Methods: Participants completed a downhill running (DHR) intervention (5 × 8 min, 2-min rest; 80% VO2max; −10% gradient). Muscle biopsies were taken 7 days before VO2max and 7–9 days after the DHR intervention. Delayed-onset muscle soreness (DOMS) and creatine kinase activity (CK) were measured on days 1, 2, 7, and 9 post-DHR. SCs were identified by Pax7 and laminin staining. Relative distribution of MHC isoforms was determined by electrophoresis. Results: DOMS and CK peaked on day 1 post-DHR (P < 0.01). The SC pool increased (26%) after DHR (P = 0.005). SCs/total myonuclei after recovery correlated with baseline SCs (r = 0.979, P = 0.003) and VO2max (r = 0.956, P = 0.011), whereas change in SC pool (Pax7+ cells/total myonuclei: recovery minus baseline) tended to correlate with percent MHC II (r = 0.848; P = 0.06). Conclusion: Interindividual physiological characteristics affect SC pool expansion after a single bout of DHR and are influenced by VO2max. Muscle Nerve, 2013

Fish oil and conjugated linoleic acid (CLA) belong to a popular class of food supplements known as "fat supplements", which are claimed to reduce muscle glycogen breakdown, reduce body mass, as well as reduce muscle damage and inflammatory responses. Sport athletes consume fish oil and CLA mainly to increase lean body mass and reduce body fat. Recent evidence indicates that this kind of supplementation may have other side-effects and a new role has been identified in steroidogenensis. Preliminary findings demonstrate that fish oil and CLA may induce a physiological increase in testosterone synthesis. The aim of this review is to describe the effects of fish oil and CLA on physical performance (endurance and resistance exercise), and highlight the new results on the effects on testosterone biosynthesis. In view of these new data, we can hypothesize that fat supplements may improve the anabolic effect of exercise.

Context: Plyometric training has been successfully used in different sporting contexts. Studies that investigated the effect of plyometric training on muscle morphology are limited, and results are controversial with regard to which muscle fiber type is mainly affected. Objective: To analyze the skeletal muscle structural and ultrastructural change induced by an acute bout of plyometric exercise to determine which type of muscle fibers is predominantly damaged. Design: Descriptive laboratory study. Setting: Research laboratory. Patients or Other Participants: Eight healthy, untrained individuals (age = 22 ± 1 years, height = 179.2 ± 6.4 cm, weight = 78.9 ± 5.9 kg). Intervention(s): Participants completed an acute bout of plyometric exercise (10 sets of 10 squat-jumps with a 1-minute rest between sets). Main Outcome Measure(s): Blood samples were collected 9 days and immediately before and 6 hours and 1, 2, and 3 days after the acute intervention. Muscle samples were collected 9 days before and 3 days after the exercise intervention. Blood samples were analyzed for creatine kinase activity. Muscle biopsies were analyzed for damage using fluorescent and electron transmission microscopy. Results: Creatine kinase activity peaked 1 day after the exercise bout (529.0 ± 317.8 U/L). Immunofluorescence revealed sarcolemmal damage in 155 of 1616 fibers analyzed. Mainly fast-twitch fibers were damaged. Within subgroups, 7.6% of type I fibers, 10.3% of type IIa fibers, and 14.3% of type IIx fibers were damaged as assessed by losses in dystrophin staining. Similar damage was prevalent in IIx and IIa fibers. Electron microscopy revealed clearly distinguishable moderate and severe sarcomere damage, with damage quantifiably predominant in type II muscle fibers of both the glycolytic and oxidative subtypes (86% and 84%, respectively, versus only 27% of slow-twitch fibers). Conclusions: We provide direct evidence that a single bout of plyometric exercise affected mainly type II muscle fibers.

The purposes of the present study were to investigate the effect of conjugated linoleic acid (CLA) supplementation on testosterone levels in vitro on a cell line derived from Leydig cells (R2C) and in vivo in the blood of physically active subjects before and after a resistance exercise bout. In vitro R2C cells were treated with different CLA concentrations (0-30 μM) for 24 and 48 hours. After treatment, supernatant media were tested to determine testosterone secretion. The CLA increased the testosterone secretion only after 48 hours. In vivo, 10 resistance-trained male subjects, in a double-blind placebo-controlled and crossover study design were randomized for 3 weeks of either 6 g·d⁻¹ CLA or placebo. Blood was drawn pre and post each resistance exercise bout to determine the total testosterone and sex hormone-binding globulin (SHBG) levels. No significant differences were observed for total testosterone or SHBG pre and post each resistance exercise bout; although after the resistance exercise bouts, total testosterone increased moderately (effect size = moderate), whereas after CLA supplementation, there was a large increase in total testosterone (effect size = large). CLA supplementation induced an increase in testosterone levels in Leydig cells in vitro after 48 hours but not in vivo before and after a resistance exercise bout. These findings suggest that CLA supplementation may promote testosterone synthesis through a molecular pathway that should be investigated in the future, although this effect did not have an anabolic relevance in our in vivo model.

Summary Aims The purpose of this study was to evaluate the effects of three different water temperatures on physiological responses (dehydration, sweat rate, urine output, rectal temperature and plasma electrolytes) of competitive athletes during a “simulated” race of 5 km in an indoor swimming pool. Methods Nine male competitive master swimmers swam 5 km with the water at temperatures of 23, 27 and 32 C. Immediately before (Pre) and after (Post) each trial, samples of blood and urine were collected, body weight was recorded and rectal temperature was measured. The dehydration percentage and sweat rate were the highest at 32 C and the lowest at 23 C (23 C: −0.9 ± 0.5; 27 C: −1.3 ± 0.6; 32 C: −2.2 ± 0.7% and 23 C: 0.48 ± 0.28; 27 C: 0.76 ± 0.36; 32 C: 1.25 ± 0.37 l/h). The Post urine volume output was not significantly different in the three trials (23 C: 122.6 ± 62.4; 27 C: 78.2 ± 24.9; 32 C 81.4 ± 37.0 mL). The 27 and 32 C water increased the rectal temperature (Pre: 37.0 ± 0.3; Post: 37.9 ± 0.5 C–Pre: 36.9 ± 0.4; Post: 38.0 ± 0.4 C, respectively). Results This study shows that dehydration, sweat rate and body temperatures simultaneously increase with the rise of water temperature during the shortest open water swimming event distance (5 km) performed at race intensity.

Satellite cells (SCs) are responsible for muscle repair following strenuous exercise or injury. SC responses to intervention have been studied, but most studies do not discuss or take into account the substantial variability in SC number among young individuals. We hypothesized that an active lifestyle reflected in higher VO(2max) may be associated with greater SC number. As training alters basal p38-mitogen-activated protein kinase (MAPK) activity, which is associated with SC proliferation, SC count may also correlate with this stress signaling kinase. Muscle biopsies from vastus lateralis of eight male participants were analyzed for fiber type, myogenin, and p38/phospho-p38 MAPK using SDS-PAGE and Western blotting. Immunofluorescence was used to detect Pax7(+) SCs. Two weeks following the biopsy, subjects underwent an incremental treadmill test to determine VO(2max) . A strong positive correlation (P = 0.0087) was found between the number of Pax7(+) nuclei and VO(2max) . Pax7(+) cell number correlated negatively with phospho-p38/p38 MAPK (P = 0.0006), but had no correlation with fiber type or myogenin. SC number is proportional to VO(2max) , and hence it can be postulated that higher levels of physical activity activate SC proliferation but not fusion, underlining the relevance of exercise in stimulating SC pool size even without injury.

CRYAB is a small Heat Shock Protein, expressed in various tissues such as skeletal and cardiac muscles, activated as phosphorylated CRYAB (pCRYAB) and involved in several pathophysiological processes. In mammals there are no reports to date on CRYAB activation following an acute endurance exercise, so the aim of my study was to explore in mouse cardiac tissue the pCRYAB levels as effect of this exercise at 0’, 15’ and 120’ of recovery. H2O2 - treated HL-1 cardiomyocytes have been utilized as in vitro model to identify the underlying molecular mechanism/s. Both in vivo and in vitro models showed no changes in CRYAB protein expression level but its phosphorylation state was significantly increased at short time, paralleled by oxidative stress markers’ modulation (i.e. 4-HNE), indicating a correlation between CRYAB activation and redox perturbation. Our previous results in skeletal muscles where exercise modulated CRYAB only in oxidative fiber types supported these data. All together these results suggest a role for CRYAB in the defense system against ROS production. Further experiments are in progress to study the possible interactors, the cellular localization and the eventual release of CRYAB in extracellular vesicles as a mean for a cross talk between tissues

In the last few years, a major goal of cardiac research has been to drive stem cell differentiation to replace damaged myocardium. Several research groups have attempted to differentiate potential cardiac stem cells (CSCs) using bi- or three-dimensional systems supplemented with growth factors or molecules acting as differentiating substances. We hypothesize that these systems failed to induce a complete differentiation because they lacked an architectural space. In the present study, we isolated a pool of small proliferating and fibroblast-like cells from adult rat myocardium. The phenotype of these cells was assessed and the characterized cells were cultured in a collagen I/OPLA scaffold with horse serum to obtain fine myocardial differentiation. C-Kit(POS)/Sca-1(POS) CSCs fully differentiated in vitro when an environment more similar to the CSC niche was created. These experiments demonstrated an important model for the study of the biology of CSCs and the biochemical pathways that lead to myocardial differentiation. The results pave the way for a new surgical approach.

Heart disease is the leading cause of death in the industrialized world, and stem cell therapy seems to be a promising treatment for injured cardiac tissue. To reach this goal, the scientific community needs to find a good source of stem cells that can be used to obtain new myocardium in a very period range of time. Since there are many ethical and technical problems with using embryonic stem cells as a source of cells with cardiogenic potential, many laboratories have attempted to isolate potential cardiac stem cells from several tissues. The best candidates seem to be cardiac "progenitor" and/or "stem" cells, which can be isolated from subendocardial biopsies from the same patient or from embryonic and/or fetal myocardium. Regardless of the technique used to isolate and characterize these cells, it appears that the different cells isolated from adult myocardium to date are all phenotypic variations of a unique cell type that expresses several markers, such as c-Kit, CD34, MDR-1, Sca-1, CD45, nestin, or Isl-1, in various combinations.

Heat shock protein 60 (Hsp60) is a chaperone localizing in skeletal muscle mitochondria, whose role is poorly understood. In the present study, the levels of Hsp60 in fibres of the entire posterior group of hindlimb muscles (gastrocnemius, soleus and plantaris) were evaluated in mice after completing a 6-week endurance training program. The correlation between Hsp60 levels and the expression of four isoforms of peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1α) were investigated only in soleus. Short-term overexpression of hsp60, achieved by in vitro plasmid transfection, was then performed to determine whether this chaperone could have a role in the activation of the expression levels of PGC1α isoforms. The levels of Hsp60 protein were fibre-type specific in the posterior muscles and endurance training increased its content in type I muscle fibers. Concomitantly with the increased levels of Hsp60 released in the blood stream of trained mice, mitochondrial copy number and the expression of three isoforms of PGC1α increased. Overexpressing hsp60 in cultured myoblasts induced only the expression of PGC1 1α, suggesting a correlation between Hsp60 overexpression and PGC1 1 α activation.

Objectives Alcoholism is one of the most devastating diseases with high incidence, but knowledge of its pathology and treatment is still plagued with gaps mostly because of the inherent limitations of research with patients. We developed an animal model for studying liver histopathology, Hsp (heat-shock protein)-chaperones involvement, and response to treatment. Methods The system was standardized using mice to which ethanol was orally administered alone or in combination with Lactobacillus fermentum following a precise schedule over time and applying, at predetermined intervals, a battery of techniques (histology, immunohistochemistry, western blotting, real-time PCR, immunoprecipitation, 3-nitrotyrosine labeling) to assess liver pathology (e.g., steatosis, fibrosis), and Hsp60 and iNOS (inducible form of nitric oxide synthase) gene expression and protein levels, and post-translational modifications. Results Typical ethanol-induced liver pathology occurred and the effect of the probiotic could be reliably monitored. Steatosis score, iNOS levels, and nitrated proteins (e.g., Hsp60) decreased after probiotic intake. Conclusions We describe a mouse model useful for studying liver disease induced by chronic ethanol intake and for testing pertinent therapeutic agents, e.g., probiotics. We tested L. fermentum, which reduced considerably ethanol-induced tissue damage and deleterious post-translational modifications of the chaperone Hsp60. The model is available to test other agents and probiotics with therapeutic potential in alcoholic liver disease.

Recent studies have correlated physical activity with a better prognosis in cachectic patients, although the underlying mechanisms are not yet understood. In order to identify the pathways involved in the physical activity-mediated rescue of skeletal muscle mass and function, we investigated the effects of voluntary exercise on cachexia in colon carcinoma (C26)-bearing mice. Voluntary exercise prevented loss of muscle mass and function, ultimately increasing survival of C26-bearing mice. We found that the autophagic flux is overloaded in skeletal muscle of both colon carcinoma murine models and patients, but not in running C26-bearing mice, thus suggesting that exercise may release the autophagic flux and ultimately rescue muscle homeostasis. Treatment of C26-bearing mice with either AICAR or rapamycin, two drugs that trigger the autophagic flux, also rescued muscle mass and prevented atrogene induction. Similar effects were reproduced on myotubes in vitro, which displayed atrophy following exposure to C26-conditioned medium, a phenomenon that was rescued by AICAR or rapamycin treatment and relies on autophagosome-lysosome fusion (inhibited by chloroquine). Since AICAR, rapamycin and exercise equally affect the autophagic system and counteract cachexia, we believe autophagy-triggering drugs may be exploited to treat cachexia in conditions in which exercise cannot be prescribed. Recent studies have correlated physical activity with a better prognosis in cachectic patients, although the underlying mechanisms are not yet understood. In order to identify the pathways involved in the physical activity-mediated rescue of skeletal muscle mass and function, we investigated the effects of voluntary exercise on cachexia in colon carcinoma (C26)-bearing mice. Voluntary exercise prevented loss of muscle mass and function, ultimately increasing survival of C26-bearing mice. We found that the autophagic flux is overloaded in skeletal muscle of both colon carcinoma murine models and patients, but not in running C26-bearing mice, thus suggesting that exercise may release the autophagic flux and ultimately rescue muscle homeostasis. Treatment of C26-bearing mice with either AICAR or rapamycin, two drugs that trigger the autophagic flux, also rescued muscle mass and prevented atrogene induction. Similar effects were reproduced on myotubes in vitro, which displayed atrophy following exposure to C26-conditioned medium, a phenomenon that was rescued by AICAR or rapamycin treatment and relies on autophagosome-lysosome fusion (inhibited by chloroquine). Since AICAR, rapamycin and exercise equally affect the autophagic system and counteract cachexia, we believe autophagy-triggering drugs may be exploited to treat cachexia in conditions in which exercise cannot be prescribed.

Several studies focused on the macroscopic architecture of increased cardiac wall induced by exercise training. Our goal was to evaluate myocardiocyte, interstitial and vascular component, and connexin-43 expression in endurance-trained mouse hearts. Sixty-three 10-week-old male Swiss mice were divided into four sedentary groups (C0, C15, C30 and C45) and three groups exercised respectively for 15 (T15-D; running intensity [RI]: 3.18 m/min; running duration [RD]: 75 min/first week and 150 min/second week), 30 (T30-D; RI: 3.96 m/min; RD: 150 min/third week and 225 min/fourth week) and 45 days (T45-D; RI: 3.96 m/min and 4.8 m/min, respectively for the fifth and sixth week; RD: 300 min) on a treadmill. Morphometric analyses were performed to quantify myocardiocyte size and number, and the capillary and interstitial connective tissue (ICT) area. We assessed the expression of ventricle myosin light chain-II, vimentin and connexin-43 by western blot analyses. Our results showed a hypertrophy of the interventricular septum and left ventricle in T30-D and T45-D mice that was not due to variations in myofibrillar content, myocardiocyte size and number, or ICT quantity but to a significant increase in the capillary area. The microvascular remodeling was associated with vimentin increased expression in ICT cells and connexin-43 upregulation. The first phenomenon might be related to an enhanced request of remodeling and growth factors; the second suggests a new role of connexin-43 in cardiac angiogenesis.

αB-crystallin (CRYAB) is a member of the small heat shock proteins implicated in various biological functions, particularly in skeletal muscle where it is involved in adaptive remodelling processes, activation of gene transcription and stabilization of nascent proteins.In this research we analysed αB-crystallin' response in mouse gastrocnemius at 15' and 30' of recovery from an acute aerobic exercise (1hour), correlating its modulation with oxidative stress level and fiber composition, red (RG) and white gastrocnemius (WG).We found for the first time that the acute exercise lead to a short term, specific increase of phospho-αB-crystallin level (pCRYAB) in the RG, while no changes were observed in the WG. Moreover, this induction was correlated with increased level of 4-hydroxynonenal (HNE),suggesting a putative role for oxidative stress in driving CRYAB, but not hsp70 or hsp27, activity during exercise. Any increased level of αB-crystallin' protein was observed neither in RG nor in WG. These data were also supported by our in vitro experiments showing a significant enhancement of pCRYAB in H2O2-treated C2C12 myotubes.Although our results seem suggest a fiber-dependent role of CRYAB, further experiments are in progress to clarify both the molecular pathway driving CRYAB phosphorylation and its fiber-specific induction after exercise -induced oxidative stress.This work was supported by MIUR - PRIN 2012 grant.

Alpha B-crystallin (CRYAB) is a member of the small heat shock proteins implicated in various biological functions, particularly in skeletal muscle tissue [1], where it results to be modulated following exercise-induced reactive oxygen species (ROS) [2]. In this work we aimed to analyse the CRYAB response to acute exercise with respect muscle fiber composition and to identify the underlying molecular mechanism by the utilization of the C2C12 “in vitro” cellular model. Our results highlighted as acute exercise determines a specific increase of phospho-CRYAB both in the red, but not white, gastrocnemius (GS), with an higher amount of oxidative and oxidative-glycolytic fibers, and in soleus (SOL), mainly composed of fibers Ia and IIa/x. Confocal microscopy analysis showed a localization of pCRYAB only at cytoskeletal level of these fibers. Moreover, pCRYAB activazion was correlated with increased level of 4-hydroxynonenal (HNE) with absence of apoptotic activation, suggesting a putative role of mild oxidative stress in pCRYAB protective response [3, 4]. H 2 O 2- treatment of C2C12 myotubes, whose MHC composition resembles the Ia-IIa/x fibers, confirmed that the ROS-dependent pCRYAB activation was paralleled by the upregulation of ROCK1 and ROCK2, two serine/threonine kinases known to mediate the cytoskeletal stability. We hypothesize that the activation of CRYAB and the subsequent stabilization of the cytoskeleton might represent a specific adaptive response of skeletal muscle oxidative fibers to exercise. References [1] Mercatelli N, et al. Free Radic Biol Med. 2010 Vol. 49(3):374-82. [2] Dimauro I, et al. Free Radic Biol Med. 2016 Vol. 98:46-55. [3] Dimauro I, et al. Free Radic Biol Med. 2012 Vol. 53(11):2017-27. [4] Fittipaldi S, et al. Free Radic Biol Med. 2014 Vol. 48(1):52-70

Alpha-B-Crystallin (CRYAB), a Small Heat Shock Protein sensitive to oxidative stress, is implicated in various biological processes in many tissues. In cardiac muscle, CRYAB exerts a cardio protective role in ischemia-induced damage preventing apoptosis and necrosis. We aimed to study αB-crystallin’ response in mouse cardiac tissue (H), at different time of recovery from an acute aerobic exercise (1 hour), correlating its modulation with oxidative stress level. We found that a single bout exercise lead to a specific short-term increase of phospho-αB-crystallin level (pCRYAB), without changes of its total expression. Further, the level of 4-hydroxynonenal, a marker of lipidic peroxidation, has shown a similar trend of pCRYAB enhancement. This may indicate that CRYAB in cardiac muscle is activated and it has a putative role in oxidative stress during exercise. These results are supported by our previous data obtained in mouse skeletal tissues (i.e. gastrocnemius, soleus) and in H₂O₂-treated C2C12 myotubes. In particular, we observed not only a fiber-dependent response of pCRYAB, but also its translocation into myofibrillar compartment. Experiments are in progress to further investigate on CRYAB role during exercise and its interactions with cytoskeletal structures.

Open-water swimming is a rapidly growing sport discipline worldwide, and clinical problems associated with long-distance swimming are now better recognized and managed more effectively. The most prevalent medical risk associated with an open-water swimming event is hypothermia; therefore, the Federation Internationale De Natation (FINA) has instituted 2 rules to reduce this occurrence related to the minimum water temperature and the time taken to complete the race. Another medical risk that is relevant to open-water swimmers is heat stroke, a condition that can easily go unnoticed. The purpose of this review is to shed light on this physiological phenomenon by examining the physiological response of swimmers during long-distance events, to define a maximum water temperature limit for competitions. We conclude that competing in water temperatures exceeding 33°C should be avoided.

The lack of dystrophin in mdx mice leads to cycles of muscle degeneration and regeneration processes. Various strategies have been proposed in order to reduce the muscle-wasting component of muscular dystrophy, including implementation of an exercise programme. The aim of this study was to examine how low-intensity endurance exercise affects the degeneration-regeneration process in dystrophic muscle of male mdx mice. Mice were subjected to low-intensity endurance exercise by running on a motorized Rota-Rod for 5 days/week for 6 weeks. Histomorphological analysis showed a signifi cant reduction of measured inflammatory-necrotic areas in both gastrocnemius and quadriceps muscle of exercised mdx mice as compared to matched sedentary mdx mice. The degenerative-regenerative process was also evaluated by examining the protein levels of connexin 39 (Cx39), a specifi c gene expressed in injured muscles. Cx39 was not detected in sedentary wild type mice, whereas it was found markedly increased in sedentary mdx mice, revealing active muscle degeneration-regeneration process. These Cx39 protein levels were signifi cantly reduced in muscles of mdx mice exercised for 30 and 40 days, revealing together with histomorphological analysis a strong reduction of degeneration process in mice subjected to low-intensity endurance exercise. Muscles of exercised mdx mice did not show significant changes in force and fatigue resistance as compared to sedentary mdx mice. Overall in this study we found that specifi c lowintensity endurance exercise induces a beneficial effect probably by reducing the degeneration of dystrophic muscle.

We investigated changes in satellite cell (SC) pool size after an acute bout of strenuous exercise and evaluated the influence of baseline SC count and fiber type.Participants completed a downhill running (DHR) intervention (5 × 8 min, 2-min rest; 80% VO2max ; -10% gradient). Muscle biopsies were taken 7 days before VO₂max and 7-9 days after the DHR intervention. Delayed-onset muscle soreness (DOMS) and creatine kinase activity (CK) were measured on days 1, 2, 7, and 9 post-DHR. SCs were identified by Pax7 and laminin staining. Relative distribution of MHC isoforms was determined by electrophoresis.DOMS and CK peaked on day 1 post-DHR (P0.01). The SC pool increased (26%) after DHR (P = 0.005). SCs/total myonuclei after recovery correlated with baseline SCs (r = 0.979, P = 0.003) and VO₂max (r = 0.956, P = 0.011), whereas change in SC pool (Pax7(+) cells/total myonuclei: recovery minus baseline) tended to correlate with percent MHC II (r = 0.848; P = 0.06).Interindividual physiological characteristics affect SC pool expansion after a single bout of DHR and are influenced by VO₂max .

The number of adult stem cells (ASCs) is very small, limiting the regenerative potential of tissues. One of the most studied ASCs in humans is the satellite cell (SC), which proliferates and increases pool size under exercise stress and muscle damage. This review examines the growth factor response to specific types of exercise to show the potential of exercise to stimulate not only SC self-renewal, but also other ASCs. We postulate that the same factors that stimulate a high proliferation of SCs in skeletal muscle after physical exercise should also stimulate the proliferation of ASCs in the tissue in which they reside, such as heart, bone, liver and etc. Regular exercise should be promoted, not only for disease prevention, but to maintain a high ASCs reserve and progenitor cell potential for rapid activation in response to future stressors and damage.

Rosario Barone1, Filippo Macaluso2, Marcello Traina3,4, Vincenza Leonardi4,5, Felicia Farina1, Valentina Di Felice11Human Anatomy Section 'E. Luna', BioNeC, University of Palermo, Palermo, Italy; 2Department of Physiological Science, Stellenbosch University, Stellenbosch, South Africa; 3Department of Internal Medicine, Cardiovascular and Renal Diseases, 4Methods and Didactics of Motory Activities, DISMOT, 5Department of General Surgery, Emergency and Organ Transplants (GENURTO), University of Palermo, Palermo, Italy Rosario Barone and Filippo Macaluso contributed equally to the workAbstract: The purpose of this study was to analyze the differences in standing balance during dominant and nondominant one-legged stance among athletes of different sports and sedentary subjects. The right-footed subjects of four groups (sedentary, n = 20; soccer, n = 20; basketball, n = 20; windsurfer n = 20) underwent 5-sec unipedal (left and right foot) stabilometric analysis with open eyes and closed eyes to measure center of pressure (COP) sway path and COP velocity (mean value, anteroposterior, and laterolateral in millimeters per second). The soccer group showed better standing balance on the left leg than the sedentary group (P < 0.05). No other significant differences were observed within and amongst groups. The soccer players have a better standing balance on the nondominant leg because of soccer activity.Keywords: body sway, bipedal stance, center of pressure, sport practice