In vitro susceptibility of thioredoxins and glutathione to redox modification and aging-related changes in skeletal muscle

AbstractThioredoxins (Trx's) regulate redox signaling and are localized to various cellular compartments. Specific redox-regulated pathways for adaptation of skeletal muscle to contractions are attenuated during aging, but little is known about the roles of Trx's in regulating these pathways. This study investigated the susceptibility of Trx1 and Trx2 in skeletal muscle to oxidation and reduction in vitro and the effects of aging and contractions on Trx1, Trx2, and thioredoxin reductase (TrxR) 1 and 2 contents and nuclear and cytosolic Trx1 and mitochondrial Trx2 redox potentials in vivo. The proportions of cytosolic and nuclear Trx1 and mitochondrial Trx2 in the oxidized or reduced forms were analyzed using redox Western blotting. In myotubes, the mean redox potentials were nuclear Trx1, −251mV; cytosolic Trx1, −242mV; mitochondrial Trx2, −346mV, data supporting the occurrence of differing redox potentials between cell compartments. Exogenous treatment of myoblasts and myotubes with hydrogen peroxide or dithiothreitol modified glutathione redox status and nuclear and cytosolic Trx1, but mitochondrial Trx2 was unchanged. Tibialis anterior muscles from young and old mice were exposed to isometric muscle contractions in vivo. Aging increased muscle contents of Trx1, Trx2, and TrxR2, but neither aging nor endogenous ROS generated during contractions modified Trx redox potentials, although oxidation of glutathione and other thiols occurred. We conclude that glutathione redox couples in skeletal muscle are more susceptible to oxidation than Trx and that Trx proteins are upregulated during aging, but do not appear to modulate redox-regulated adaptations to contractions that fail during aging

MiR-23-TrxR1 as a novel molecular axis in skeletal muscle differentiation

Thioredoxin reductase 1 (TrxR1) is a selenocysteine-containing protein involved in cellular redox homeostasis which is downregulated in skeletal muscle differentiation. Here we show that TrxR1 decrease occurring during myogenesis is functionally involved in the coordination of this cellular process. Indeed, TrxR1 depletion reduces myoblasts growth by inducing an early myogenesis -related gene expression pattern which includes myogenin and Myf5 up-regulation and Cyclin D1 decrease. On the contrary, the overexpression of TrxR1 during differentiation delays myogenic process, by negatively affecting the expression of Myogenin and MyHC. Moreover, we found that miR-23a and miR-23b - whose expression was increased in the early stage of C2C12 differentiation - are involved in the regulation of TrxR1 expression through their direct binding to the 3′ UTR of TrxR1 mRNA. Interestingly, the forced inhibition of miR-23a and miR-23b during C2C12 differentiation partially rescues TrxR1 levels and delays the expression of myogenic markers, suggesting the involvement of miR-23 in myogenesis via TrxR1 repression. Taken together, our results depict for the first time a novel molecular axis, which functionally acts in skeletal muscle differentiation through the modulation of TrxR1 by miR-23

Explosive type of moderate-resistance training induces functional, cardiovascular, and molecular adaptations in the elderly

Current recommendations aimed at reducing neuromuscular and functional loss in aged muscle have identified muscle power as a key target for intervention trials, although little is known about the biological and cardiovascular systemic response in the elderly. This study investigated the effects of 12 weeks of low-frequency, moderate-intensity, explosive-type resistance training (EMRT) on muscle strength and powerin oldcommunity-dwellingpeople(70–75years), monitoring functional performance linked to daily liv- ing activities (ADL) and cardiovascular response, as well as biomarkers of muscle damage, cardiovascular risk, and cellular stress response. The present study provides the first evidence that EMRT was highly effective in achieving a significant enhancement in muscular strength and power as well as in functional performance without causing any detrimental modification in cardiovascular, inflammatory, and damage parameters. Moreover, trained elderly subjects showed an adaptive response at both systemic and cellular levels by modulation of antioxidant and stress-induced markers such as myeloperoxidase (MPO), heat shock protein 70 (Hsp70) and 27 (Hsp27), and thioredoxin reductase 1 (TrxR1)

An animal and cellular study on αB-crystallin activation in cardiac muscle by acute exercise

Alpha-B-Crystallin (CRYAB), a Small Heat Shock Protein sensitive to oxidative stress, is expressed in many tissues and implicated in various biological processes. In cardiac muscle, CRYAB exerts a cardio protective role in ischemia-induced damage preventing apoptosis and necrosis. In the present study we used forty young (7-weeks old) healthy male mice (BALB/c AnNHsd), which after 1 week of acclimatization to the new housing environment, runned 2 days per 10 minutes. The TR mice ran for 60 min at a speed of 5.5 m/min. Mice were sacrificed immediately after, 15 and 120 minutes after the end of the acute bout of endurance exercise (TR-0’, TR-15’ and TR-120’, respectively). We prepared samples from the heart and from the group of posterior muscles study αB-crystallin’ response 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

Alpha B-Crystallin in Muscle Disease Prevention: The Role of Physical Activity

HSPB5 or alpha B-crystallin (CRYAB), originally identified as lens protein, is one of the most widespread and represented of the human small heat shock proteins (sHSPs). It is greatly expressed in tissue with high rates of oxidative metabolism, such as skeletal and cardiac muscles, where HSPB5 dysfunction is associated with a plethora of human diseases. Since HSPB5 has a major role in protecting muscle tissues from the alterations of protein stability (i.e., microfilaments, microtubules, and intermediate filament components), it is not surprising that this sHSP is specifically modulated by exercise. Considering the robust content and the protective function of HSPB5 in striated muscle tissues, as well as its specific response to muscle contraction, it is then realistic to predict a specific role for exercise-induced modulation of HSPB5 in the prevention of muscle diseases caused by protein misfolding. After offering an overview of the current knowledge on HSPB5 structure and function in muscle, this review aims to introduce the reader to the capacity that different exercise modalities have to induce and/or activate HSPB5 to levels sufficient to confer protection, with the potential to prevent or delay skeletal and cardiac muscle disorders

A simple protocol for the subcellular fractionation of skeletal muscle cells and tissue

<p>Abstract</p> <p>Background</p> <p>We describe a method for subcellular fractionation of mouse skeletal muscle, myoblast and myotubes to obtain relatively pure fractions of nuclear, cytosolic and mitochondrial compartments. Fractionation allows the analysis of a protein of interest (or other cellular component) based on its subcellular compartmental distribution and can also generate molecular information about the state of a cell and/or tissue and how the distribution of a protein may differ between different cellular compartments, tissues or cell types, in response to treatments or ageing.</p> <p>Findings</p> <p>The described method was specifically developed for skeletal muscle and proliferating/differentiated muscle cells. The purity of the different fractions, representing the cytoplasmic, mitochondrial and nuclear subcellular compartments was validated by western blot analysis of “house-keeper” marker proteins specific for each cellular compartment.</p> <p>Conclusion</p> <p>This low cost method allowed the mitochondrial, cytoplasmic and nuclear subcellular compartments from the same starting muscle samples to be rapidly and simultaneously isolated with good purity and without the use of an ultracentrifuge. This method permits samples to be frozen at −80°C for future analysis and/or additional processing at a later date.</p

The early response of αB-crystallin to a single bout of aerobic exercise in mouse skeletal muscles depends upon fiber oxidative features

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 fiberspecific 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

New Strategy of Home-Based Exercise during Pandemic COVID-19 in Breast Cancer Patients: A Case Study

The COVID-19 pandemic has posed several challenges for the oncology health care system. The need to improve patients&rsquo; Quality of Life (QoL) through exercise, which is related to survival and healing, has increased, especially during lockdowns. Technologies are often used to help with patient care as well as to monitor exercise training. This case study, developed during the pandemic period, aims to evaluate the effectiveness of a proposed home-based combined training (CT) regimen, supervised through online lessons, in increasing QoL and fatigue in breast cancer patients undergoing adjuvant therapy. Additionally, we evaluated the effect of exercise on psychological and functional parameters. Methods: Two breast cancer (BC) survivors were required to participate in 2 h/week of supervised and home-based CT for 16 weeks. Results: Improvements were found in the emotional function of QoL (10% in patient A; 70% in patient B) and in all variables of fatigue (physical fatigue 66% in patient A; 33% in patient B). Conclusion: The findings from this study revealed positive effects of CT on QoL and fatigue perception in BC women undergoing therapy. Both patients attended all training sessions with no adverse events, showing the sustainability of this training as an alternative and affordable method that is capable of improving patients&rsquo; wellbeing

Physical Exercise and Redox Balance in Type 2 Diabetics: Effects of Moderate Training on Biomarkers of Oxidative Stress and DNA Damage Evaluated through Comet Assay

Objective. Hyperglycemia leads to increased production of reactive oxygen species (ROS) in type 2 diabetes, which reduces cellular antioxidant defenses and induces DNA lesions. The aim of this study was to investigate the effects on redox homeostasis and DNA oxidative damage of exercise training in patients with type 2 diabetes compared with nondiabetic individuals. Methods and Results. 12 sedentary type 2 diabetic males (62.1 ± 4.3 yrs) and 12 sedentary healthy males (61.7 ± 3.9 yrs) were exposed to 4-month moderate training, 3 times per week, to evaluate the effect on plasma biomarkers of oxidative stress malondialdehyde and antioxidant status (GSSG, GSH/GSSG, and ascorbic acid) as well as basal and H2O2-induced DNA damage trough alkaline comet assay in peripheral blood lymphocytes. After training, glutathione and ascorbic acid levels increased in both groups, but only in diabetics the malondialdehyde as well as the DNA damage decreased. Conclusion. Our study demonstrates for the first time that moderate exercise training is not only effective in improving the redox homeostasis, through an increase of the endogenous antioxidant defences in healthy as well as in diabetic patients, but also, specifically in diabetic patients, effective in lowering the susceptibility to oxidative DNA damage and the lipid peroxidation levels