Resveratrol, a Multitasking Molecule That Improves Skeletal Muscle Health.

Resveratrol is a natural polyphenol utilized in Chinese traditional medicine and thought to be one of the determinants of the "French Paradox". More recently, some groups evidenced its properties as a calorie-restriction mimetic, suggesting that its action passes through the modulation of skeletal muscle metabolism. Accordingly, the number of studies reporting the beneficial effects of resveratrol on skeletal muscle form and function, in both experimental models and humans, is steadily increasing. Although studies on animal models confer to resveratrol a good potential to ameliorate skeletal muscle structure, function and performance, clinical trials still do not provide clear-cut information. Here, we first summarize the effects of resveratrol on the distinct components of the skeletal muscle, such as myofibers, the neuromuscular junction, tendons, connective sheaths and the capillary bed. Second, we review clinical trials focused on the analysis of skeletal muscle parameters. We suggest that the heterogeneity in the response to resveratrol in humans could depend on sample characteristics, treatment modalities and parameters analyzed; as well, this heterogeneity could possibly reside in the complexity of skeletal muscle physiology. A systematic programming of treatment protocols and analyses could be helpful to obtain consistent results in clinical trials involving resveratrol administration.

Upregulation of Sarcolemmal Hemichannels and Inflammatory Transcripts with Neuromuscular Junction Instability during Lower Limb Unloading in Humans.

Human skeletal muscle atrophy and a disproportionate force loss occur within a few days of unloading in space and on Earth, but the underlying mechanisms are not fully understood. Disruption of neuromuscular junction homeostasis has been proposed as one of the possible causes. Here, we investigated the potential mechanisms involved in this neuromuscular disruption induced by a 10-day unilateral lower limb suspension (ULLS) in humans. Specifically, we investigated hemichannels' upregulation, neuromuscular junction and axonal damage, neurotrophins' receptor downregulation and inflammatory transcriptional signatures. Biomarkers were evaluated at local and systemic levels. At the sarcolemmal level, changes were found to be associated with an increased expression of connexin 43 and pannexin-1. Upregulation of the inflammatory transcripts revealed by deep transcriptomics was found after 10 days of ULLS. The destabilisation of the neuromuscular junction was not accompanied by changes in the secretion of the brain-derived neurotrophic factor and neurotrophin-4, while their receptor, BDNF/NT growth factors receptor (TrkB), decreased. Furthermore, at 5 days of ULLS, there was already a significant upregulation of the serum neurofilament light chain concentration, an established clinical biomarker of axonal injury. At 10 days of ULLS, other biomarkers of early denervation processes appeared. Hence, short periods of muscle unloading induce sarcolemmal hemichannels upregulation, inflammatory transcripts upregulation, neuromuscular junction instability and axonal damage.

Experimental models for ageing research.

Ageing is a biological process caused by the malfunctioning of multiple cellular mechanisms, ascribable to nine hallmarks: genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, deregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and altered intercellular communication. These ageing pillars have three common traits: (i) they appear during normal ageing; (ii) their experimental intensification accelerates ageing; and (iii) their experimental reduction delays ageing. The evidence that the elderly are more prone to develop pathologies such as cancer, diabetes and degenerative diseases, together with data showing that the elderly population is steadily increasing, has stimulated an important effort to find specific countermeasures to physiological ageing. Unfortunately, the investigation of ageing processes and the search for countermeasures in humans is very difficult. Therefore, researchers must rely on a wide range of experimental models that span from unicellular to more complex organisms. Unfortunately, experimental models are not devoid of pitfalls, flaws or obstacles that can have an impact in ageing research. In the present review we describe the most exploited experimental models in the field, such as in vitro, animal and human models, highlighting the characteristics that justify their application in the laboratory routine, and translation to human research.

Golgi Complex form and Function: A Potential Hub Role Also in Skeletal Muscle Pathologies?

A growing number of disorders has been associated with mutations in the components of the vesicular transport machinery. The early secretory pathway consists of Endoplasmic Reticulum, numerous vesicles, and the Golgi Complex (GC), which work together to modify and package proteins to deliver them to their destination. The GC is a hub organelle, crucial for organization of the other secretory pathway components. As a consequence, GC's form and function are key players in the pathogenesis of several disorders. Skeletal muscle (SKM) damage can be caused by defective protein modifications and traffic, as observed in some Limb girdle muscular dystrophies. Interestingly, in turn, muscle damage in Duchenne dystrophic SKM cells also includes the alteration of GC morphology. Based on the correlation between GC's form and function described in non-muscle diseases, we suggest a key role for this hub organelle also in the onset and progression of some SKM disorders. An altered GC could affect the secretory pathway via primary (e.g., mutation of a glycosylation enzyme), or secondary mechanisms (e.g., GC mis-localization in Duchenne muscles), which converge in SKM cell failure. This evidence induces considering the secretory pathway as a potential therapeutic target in the treatment of muscular dystrophies.

Skeletal Muscle Mitochondrial and Perilipin Content in a Cohort of Obese Subjects Undergoing Moderate and High Intensity Training.

Obesity is a complex condition characterized by abnormal and excessive fat accumulation, resulting in an increased risk for severe health problems. Skeletal muscles play a major role in movement and fat catabolism, but the insulin resistance that comes with obesity makes it difficult to fulfill these tasks. In this study, we analyse two types of training protocols, moderate intensity continuous training (MICT) versus high intensity interval training (HIIT), in a cohort of obese subjects to establish which muscle adaptations favour fat consumption in response to exercise. Mitochondria play a role in fat oxidation. We found protein upregulation of mitochondrial biomarkers, TOMM20 and Cox-4, in HIIT but not in MICT, without detecting any shifts in fibre composition phenotype of the vastus lateralis in both training groups. Interestingly, both MICT and HIIT protocols showed increased protein levels of perilipin PLIN2, which is involved in the delivery and consumption of fats. HIIT also augmented perilipin PLIN5. Perilipins are involved in fat storage in skeletal muscles and their upregulation, along with the analysis of circulatory lipid profiles reported in the present study, suggest important adaptations induced by the two types of training protocols that favour fat consumption and weight loss in obese subjects.

The Contribution of Genetics to Muscle Disuse, Retraining, and Aging.

Genetic background may partly explain differences in muscle responses to internal or external stimuli. Muscle disuse involves various degrees of skeletal muscle atrophy due to inactivity and mechanical unloading. Whether and to which extent genetic background impacts disuse atrophy and retraining in individuals of different ages are currently unclear. Here, we provide a brief overview of relevant literature on the contribution of genetics to muscle disuse, retraining, and aging, and offer a perspective on unanswered questions on the subject that may open new venues for research.

A short-term treatment with resveratrol improves the inflammatory conditions of Middle-aged mice skeletal muscles.

Sarcopenia starts around the age of 40, causes the loss of 8% of muscle mass every 10 years, and is accompanied by functional deficit, chronic low-grade inflammation, and can result in several negative health outcomes. Considering the early and gradual onset of sarcopenia, the time window of the potential interventions could be crucial for the exertion of a beneficial effect. We recently showed that the long-term supplementation with Resveratrol contrasts sarcopenia in naturally ageing C57BL/6 mice. Aiming to understand the effects of a short term treatment, we administered intraperitoneally middle aged male mice with 20 mg/kg body weight Resveratrol daily for 5 weeks. Although we could not observe major differences in the histological properties of SKMs, we detected a significant decrease of Cox-2 in RES-treated muscles, confirming the anti-inflammatory action of Resveratrol, and suggesting that its anti-inflammatory action precedes modifications to SKM fibres.

Signatures of muscle disuse in spaceflight and bed rest revealed by single muscle fiber proteomics.

Astronauts experience dramatic loss of muscle mass, decreased strength, and insulin resistance, despite performing daily intense physical exercise that would lead to muscle growth on Earth. Partially mimicking spaceflight, prolonged bed rest causes muscle atrophy, loss of force, and glucose intolerance. To unravel the underlying mechanisms, we employed highly sensitive single fiber proteomics to detail the molecular remodeling caused by unloading and inactivity during bed rest and changes of the muscle proteome of astronauts before and after a mission on the International Space Station. Muscle focal adhesions, involved in fiber-matrix interaction and insulin receptor stabilization, are prominently downregulated in both bed rest and spaceflight and restored upon reloading. Pathways of antioxidant response increased strongly in slow but not in fast muscle fibers. Unloading alone upregulated markers of neuromuscular damage and the pathway controlling EIF5A hypusination. These proteomic signatures of mechanical unloading in muscle fiber subtypes contribute to disentangle the effect of microgravity from the pleiotropic challenges of spaceflight.

The Potential of Calorie Restriction and Calorie Restriction Mimetics in Delaying Aging: Focus on Experimental Models.

Aging is a biological process determined by multiple cellular mechanisms, such as genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, deregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and altered intercellular communication, that ultimately concur in the functional decline of the individual. The evidence that the old population is steadily increasing and will triplicate in the next 50 years, together with the fact the elderlies are more prone to develop pathologies such as cancer, diabetes, and degenerative disorders, stimulates an important effort in finding specific countermeasures. Calorie restriction (CR) has been demonstrated to modulate nutrient sensing mechanisms, inducing a better metabolic profile, enhanced stress resistance, reduced oxidative stress, and improved inflammatory response. Therefore, CR and CR-mimetics have been suggested as powerful means to slow aging and extend healthy life-span in experimental models and humans. Taking into consideration the difficulties and ethical issues in performing aging research and testing anti-aging interventions in humans, researchers initially need to work with experimental models. The present review reports the major experimental models utilized in the study of CR and CR-mimetics, highlighting their application in the laboratory routine, and their translation to human research.

The effect of leg preference on mechanical efficiency during single-leg extension exercise.

To investigate how leg preference affects net efficiency (ηnet), we examined central and peripheral hemodynamics, muscle fiber type, activation and force of preferred (PL) and nonpreferred (NPL) leg. Our hypothesis was that PL greater efficiency could be explained by adaptations and interactions between central, peripheral factors, and force. Fifteen young participants performed single-leg extension exercise at absolute (35 W) and relative [50% peak power-output (Wpeak)] workloads with PL and NPL. Oxygen uptake, photoplethysmography, Doppler ultrasound, near-infrared-spectroscopy deoxyhemoglobin [HHb], integrated electromyography (iEMG), maximal isometric force (MVC), rate of force development (RFD50-100), and muscle biopsies of both vastus lateralis were studied to assess central and peripheral determinants of ηnet. During exercise executed at 35 W, ηnet was 17.5 ± 5.1% and 11.9 ± 2.1% (P < 0.01) in PL and NPL respectively, whereas during exercise at the 50% of Wpeak was in PL = 18.1 ± 5.1% and in NPL = 12.5 ± 1.9 (P < 0.01). The only parameter correlated with ηnet was iEMG, which showed an inverse correlation for absolute (r = -0.83 and -0.69 for PL and NPL) and relative workloads (r = -0.92 and -0.79 for PL and NPL). MVC and RFD50-100 were higher in PL than in NPL but not correlated to ηnet. This study identified a critical role of leg preference in the efficiency during single-leg extension exercise. The whole spectrum of the central and peripheral, circulatory, and muscular determinants of ηnet did not explain the difference between PL and NPL efficiency. Therefore, the lower muscle activation exhibited by the PL is likely the primary determinant of this physiological phenomenon.NEW & NOTEWORTHY This study examined the impact of leg preference on efficiency during single-leg exercise. The results revealed lower efficiency of the nonpreferred leg during exercises performed at absolute and relative workloads. Central (cardiac output) and peripheral (fiber typing) determinants of efficiency did not explain the difference between the legs. However, the lower muscle activation of the preferred leg that was inversely correlated with efficiency is likely the primary determinant of this physiological feature.

Neuromuscular junction instability and altered intracellular calcium handling as early determinants of force loss during unloading in humans.

KEY POINTS: Few days of unloading are sufficient to induce a decline of skeletal muscle mass and function; notably, contractile force is lost at a faster rate than muscle mass. The reasons behind this disproportionate loss of muscle force are still poorly understood. We provide strong evidence of two mechanisms only hypothesized until now for the rapid muscle force loss in only 10 days of bed rest. Our results show that an initial neuromuscular junction instability, accompanied by alterations in the innervation status and impairment of single fibre sarcoplasmic reticulum function contribute to the loss of contractile force in front of a preserved myofibrillar function and central activation capacity. Early onset of neuromuscular junction instability and impairment in calcium dynamics involved in excitation-contraction coupling are proposed as eligible determinants to the greater decline in muscle force than in muscle size during unloading. ABSTRACT: Unloading induces rapid skeletal muscle atrophy and functional decline. Importantly, force is lost at a much higher rate than muscle mass. We aimed to investigate the early determinants of the disproportionate loss of force compared to that of muscle mass in response to unloading. Ten young participants underwent 10 days of bed rest (BR). At baseline (BR0) and at 10 days (BR10), quadriceps femoris (QF) volume (VOL) and isometric maximum voluntary contraction (MVC) were assessed. At BR0 and BR10 blood samples and biopsies of vastus lateralis (VL) muscle were collected. Neuromuscular junction (NMJ) stability and myofibre innervation status were assessed, together with single fibre mechanical properties and sarcoplasmic reticulum (SR) calcium handling. From BR0 to BR10, QFVOL and MVC decreased by 5.2% (P = 0.003) and 14.3% (P < 0.001), respectively. Initial and partial denervation was detected from increased neural cell adhesion molecule (NCAM)-positive myofibres at BR10 compared with BR0 (+3.4%, P = 0.016). NMJ instability was further inferred from increased C-terminal agrin fragment concentration in serum (+19.2% at BR10, P = 0.031). Fast fibre cross-sectional area (CSA) showed a trend to decrease by 15% (P = 0.055) at BR10, while single fibre maximal tension (force/CSA) was unchanged. However, at BR10 SR Ca2+ release in response to caffeine decreased by 35.1% (P < 0.002) and 30.2% (P < 0.001) in fast and slow fibres, respectively, pointing to an impaired excitation-contraction coupling. These findings support the view that the early onset of NMJ instability and impairment in SR function are eligible mechanisms contributing to the greater decline in muscle force than in muscle size during unloading.

Nutrition, Diet and Healthy Aging.

The current increase in life expectancy is confirmed by data from different sources (i.e.,The World Population Prospects 2019 issued by the United Nations; https://population.un.org/wpp/ (accessed on 20 December 2021)), which predict that, in the near future, individ-uals who are over 65 and over 80 will be the fastest-growing portion of the population [...].

Long-term resveratrol treatment improves the capillarization in the skeletal muscles of ageing C57BL/6J mice.

We recently showed that the treatment with Resveratrol (RES) contrasts the effects of ageing on the skeletal muscle (SKM), reduces the appearance of tubular aggregates (TAs), and improves the fatigue resistance. Since fatigue resistance depends on the SKM capillary network, and RES has been described to improve vascularisation, we analysed the SKM capillarization in naturally ageing C57BL/6J male mice, fed with 0.04% RES in the diet for 6 months, which showed a better fatigue resistance in a previous work. Our data show an inverse correlation between the number of capillaries per fibre (CAF) and TAs in both control and treated type IIB fibres, and an increase of CAF in ageing SKM upon RES-treatment. The present work suggests that capillarization is one of the determinants of the development of TAs and fatigue resistance, and that RES can be considered a good candidate to counteract capillary rarefaction in the SKM tissue.

Are muscle fibres of body builders intrinsically weaker? A comparison with single fibres of aged-matched controls.

AIM: Skeletal muscles of Body Builders (BB) represent an interesting model to study muscle mass gains in response to high volume resistance training. It is debated whether muscle contractile performance improves in proportion to mass. Here, we aim to assess whether muscle hypertrophy does not occur at the expense of performance. METHODS: Six BB and Six untrained controls (CTRL) were recruited. Cross-sectional area (CSA) and maximum voluntary contraction (MVC) of quadriceps femoris muscle (QF) and CSA and architecture of vastus lateralis (VL) were determined. Moreover, a biopsy was taken from VL mid-portion and single fibres were analysed. RESULTS: QF CSA and MVC were 32% (n.s., P = .052) and 58% (P = .009) higher in BB than in CTRL, respectively. VL CSA was 37% higher in BB (P = .030). Fast 2A fibres CSA was 24% (P = .048) greater in BB than in CTRL, when determined in immunostained sections of biopsy samples. Single permeabilized fast fibres CSA was 37% (n.s., P = .052) higher in BB than in CTRL, and their force was slightly higher in BB (n.s.), while specific tension (P0 ) was 19% (P = .024) lower. The lower P0 was not explained either by lower myosin content or by impaired calcium diffusion. Conversely, the swelling caused by skinning-induced permeabilization was different and, when used to correct P0 , differences between populations disappeared. CONCLUSIONS: The results show that high degree of muscle hypertrophy is not detrimental for force generation capacity, as increases in fibre size and force are strictly proportional once the differential swelling response is accounted for.

Age Dependent Modification of the Metabolic Profile of the Tibialis Anterior Muscle Fibers in C57BL/6J Mice.

Skeletal muscle aging is accompanied by mass reduction and functional decline, as a result of multiple factors, such as protein expression, morphology of organelles, metabolic equilibria, and neural communication. Skeletal muscles are formed by multiple fibers that express different Myosin Heavy Chains (MyHCs) and have different metabolic properties and different blood supply, with the purpose to adapt their contraction to the functional need. The fine interplay between the different fibers composing a muscle and its architectural organization determine its functional properties. Immunohistochemical and histochemical analyses of the skeletal muscle tissue, besides evidencing morphological characteristics, allow for the precise determination of protein expression and metabolic properties, providing essential information at the single-fiber level. Aiming to gain further knowledge on the influence of aging on skeletal muscles, we investigated the expression of the MyHCs, the Succinate Dehydrogenase (SDH) activity, and the presence of capillaries and Tubular Aggregates (TAs) in the tibialis anterior muscles of physiologically aging C57BL/6J mice aged 8 (adult), 18 (middle aged), and 24 months (old). We observed an increase of type-IIB fast-contracting fibers, an increase of the oxidative capacity of type-IIX and -IIA fibers, a general decrease of the capillarization, and the onset of TAs in type-IIB fibers. These data suggest that aging entails a selective modification of the muscle fiber profiles.

Corrigendum: Skeletal Muscle Fiber Size and Gene Expression in the Oldest-Old With Differing Degrees of Mobility.

[This corrects the article DOI: 10.3389/fphys.2019.00313.].

Musculoskeletal adaptations to strength training in frail elderly: a matter of quantity or quality?

BACKGROUND: The improvement in muscle strength generally exceeds the increase in muscle size following strength training in frail elderly, highlighting the complex aetiology of strength deficit in aging. The aim of this study was to investigate the effect of heavy-load strength training on a broad number of factors related to specific strength in frail elderly. METHODS: Thirty-four frail elderly men (n = 18) and women (n = 16) aged 67 to 98 (86 ± 7 years) were randomized to either a group performing strength training twice a week for 10 weeks (ST) or a non-exercising control group (CON). Knee extensor muscle strength was tested as one-repetition maximum (1RM) and isometric maximal voluntary contraction (MVC) torque. Muscle activation was assessed by the interpolated twitch technique, and muscle density [mean Hounsfield units (HU)] and intermuscular adipose tissue (IMAT) by computed tomography scans of the quadriceps femoris. Muscle biopsies from the vastus lateralis were obtained to investigate changes in intramyocellular lipids and single-fibre specific tension. RESULTS: In ST, knee extension 1RM and MVC improved by 17 and 7%, respectively. Muscle cross-sectional area of the quadriceps femoris increased by 7%, accompanied by a 4% increase of muscle density. No changes in IMAT, voluntary activation level, single-fibre specific tension, or lipid content were observed. CONCLUSIONS: In contrast to several previous reports, the improvements in isometric muscle strength and muscle area were in good agreement in the present study. The training-induced increase in muscle density was not due to changes in skeletal muscle lipid content. Instead, the increase in muscle density may reflect increased packing of contractile material or simply an increased ratio of muscle tissue relative to IMAT.

Neuromuscular Electrical Stimulation Induces Skeletal Muscle Fiber Remodeling and Specific Gene Expression Profile in Healthy Elderly.

Skeletal muscle aging is a multifactorial process strictly related to progressive weakness. One of the results that were focused on was the fiber phenotype modification and their loss. The physiological muscle recruitment to contraction, basically prosecuted under volitional control, can also be engaged by means of Neuromuscular Electrical Stimulation (NMES). Knowing that the NMES is effective in improving muscle strength in active healthy elderly, the aim was to investigate which physiological modifications were able to produce in the Vastus lateralis muscle and the pathways involved. It was found that NMES increased the cross sectional area and the isometric strength of type II myofibers together with the activated myogenic pathway in order to shift glycolytic toward the oxidative phenotype II myofibers, at a molecular level and with an increase of maximal voluntary contraction (MVC) at a functional level. Using the TaqMan low density array on 48 different genes, we found that NMES specific gene regulation highlighted: (i) increased protein synthesis with respect to protein degradation; (ii) the activation of an apoptotic pathway involved in the differentiation process; (iii) increased regeneration signals; (iv) oxidative enzyme regulation. These pathways were validated via confirmatory RT-PCR for genes involved in the regeneration process as well as Myosin isoforms. We also investigated the oxidative stress status analyzing superoxide anion levels, the protein expression of two different superoxide dismutase and the activity of both catalase and superoxide anion dismutase, being two main antioxidant enzymes. In conclusion, data demonstrates that NMES is effective in producing physiological adaptation on Vastus Lateralis of active healthy elderly as well as providing new insights for further research on elderly who experienced muscle detriment for periodic or permanent immobility.

Skeletal Muscle Fiber Size and Gene Expression in the Oldest-Old With Differing Degrees of Mobility.

The oldest-old, in the ninth and tenth decades of their life, represent a population characterized by neuromuscular impairment, which often implies a loss of mobility and independence. As recently documented by us and others, muscle atrophy and weakness are accompanied by an unexpected preservation of the size and contractile function of skeletal muscle fibers. This suggests that, while most fibers are likely lost with their respective motoneurons, the surviving fibers are well preserved. Here, we investigated the mechanisms behind this fiber preservation and the relevance of physical activity, by comparing a group of 6 young healthy controls (YG: 22-28 years) with two groups of oldest-old (81-96 years), one able to walk (OW: n = 6, average 86 years) and one confined to a wheelchair (ONW n = 9, average 88 years). We confirmed previous results of fiber preservation and, additionally, observed a shift in fiber type, toward slow predominance in OW and fast predominance in ONW. Myonuclear density was increased in muscles of ONW, compared to YG and OW, potentially indicative of an ongoing atrophy process. We analyzed, by RT-qPCR, the expression of genes relevant for fiber size and type regulation in a biopsy sample from the vastus lateralis. In all oldest-old both myostatin and IGF-1 expression were attenuated compared to YG, however, in ONW two specific IGF-1 isoforms, IGF-1EA and MGF, demonstrated a further significant decrease compared to OW. Surprisingly, atrogenes (MURF1 and atrogin) expression was also significantly reduced compared to YG and this was accompanied by a close to statistically significantly attenuated marker of autophagy, LC3. Among the determinants of the metabolic fiber type, PGC1α was significantly reduced in both OW and ONW compared to YG, while AMPK was down-regulated only in ONW. We conclude that, in contrast to the shift of the balance in favor of pro-atrophy factors found by other studies in older adults (decreased IGF-1, increase of myostatin, increase of atrogenes), in the oldest-old the pro-atrophy factors also appear to be down-regulated, allowing a partial recovery of the proteostasis balance. Furthermore, the impact of muscle activity, as a consequence of lost or preserved walking ability, is limited.