Impaired myoblast differentiation and muscle IGF-1 receptor signaling pathway activation after N-glycosylation inhibition.

The role of N-glycosylation in the myogenic process remains poorly understood. Here, we evaluated the impact of N-glycosylation inhibition by Tunicamycin (TUN) or by phosphomannomutase 2 (PMM2) gene knockdown, which encodes an enzyme essential for catalyzing an early step of the N-glycosylation pathway, on C2C12 myoblast differentiation. The effect of chronic treatment with TUN on tibialis anterior (TA) and extensor digitorum longus (EDL) muscles of WT and MLC/mIgf-1 transgenic mice, which overexpress muscle Igf-1Ea mRNA isoform, was also investigated. TUN-treated and PMM2 knockdown C2C12 cells showed reduced ConA, PHA-L, and AAL lectin binding and increased ER-stress-related gene expression (Chop and Hspa5 mRNAs and s/uXbp1 ratio) compared to controls. Myogenic markers (MyoD, myogenin, and Mrf4 mRNAs and MF20 protein) and myotube formation were reduced in both TUN-treated and PMM2 knockdown C2C12 cells. Body and TA weight of WT and MLC/mIgf-1 mice were not modified by TUN treatment, while lectin binding slightly decreased in the TA muscle of WT (ConA and AAL) and MLC/mIgf-1 (ConA) mice. The ER-stress-related gene expression did not change in the TA muscle of WT and MLC/mIgf-1 mice after TUN treatment. TUN treatment decreased myogenin mRNA and increased atrogen-1 mRNA, particularly in the TA muscle of WT mice. Finally, the IGF-1 production and IGF1R signaling pathways activation were reduced due to N-glycosylation inhibition in TA and EDL muscles. Decreased IGF1R expression was found in TUN-treated C2C12 myoblasts which was associated with lower IGF-1-induced IGF1R, AKT, and ERK1/2 phosphorylation compared to CTR cells. Chronic TUN-challenge models can help to elucidate the molecular mechanisms through which diseases associated with aberrant N-glycosylation, such as Congenital Disorders of Glycosylation (CDG), affect muscle and other tissue functions.

Neuromodulatory Contribution to Muscle Force Production after Short-Term Unloading and Active Recovery.

PURPOSE: Prior evidence has shown that neural factors contribute to the loss of muscle force after skeletal-muscle disuse. However, little is known about the specific neural mechanisms altered by disuse. Persistent inward current (PIC) is an intrinsic property of motoneurons responsible for prolonging and amplifying the synaptic input, proportionally to the level of neuromodulation, thus influencing motoneuron discharge rate and force production. Here, we hypothesized that short-term unilateral lower-limb suspension (ULLS) would reduce the neuromodulatory input associated with PICs, contributing to the reduction of force generation capacity. Additionally, we tested whether physical exercise would restore the force generation capacity by re-establishing the initial level of neuromodulatory input. METHODS: In 12 young adults, we assessed maximal voluntary contraction (MVC) pre- and post- 10 days of ULLS and following 21 days of active recovery (AR) based on resistance exercise. PIC was estimated from high-density surface electromyograms of the vastus lateralis muscle as the delta frequency (∆F) of paired motor units calculated during isometric ramped contractions. RESULTS: The values of ∆F were reduced after 10 days of ULLS (-33%, p < 0.001), but were fully re-established after the AR (+29.4%, p < 0.001). The changes in estimated PIC values were correlated (r = 0.63, p = 0.004) with the reduction in MVC after ULLS (-29%, p = 0.002) and its recovery after the AR (+28.5%, p = 0.003). CONCLUSIONS: Our findings suggest that PIC estimates are reduced by muscle disuse and may contribute to the loss of force production and its recovery with exercise. Overall, this is the first study demonstrating that, in addition to peripheral neuromuscular changes, central neuromodulation is a major contributor to the loss of force generation capacity after disuse, and can be recovered after resistance exercise.

Lower limb suspension induces threshold-specific alterations of motor units properties that are reversed by active recovery.

PURPOSE: This study aimed to non-invasively test the hypothesis that (a) short-term lower limb unloading would induce changes in the neural control of force production (based on motor units (MUs) properties) in the vastus lateralis muscle and (b) possible changes are reversed by active recovery (AR). METHODS: Ten young males underwent 10 days of unilateral lower limb suspension (ULLS) followed by 21 days of AR. During ULLS, participants walked exclusively on crutches with the dominant leg suspended in a slightly flexed position (15°-20°) and with the contralateral foot raised by an elevated shoe. The AR was based on resistance exercise (leg press and leg extension) and executed at 70% of each participant's 1 repetition maximum, 3 times/week. Maximal voluntary isometric contraction (MVC) of knee extensors and MUs properties of the vastus lateralis muscle were measured at baseline, after ULLS, and after AR. MUs were identified using high-density electromyography during trapezoidal isometric contractions at 10%, 25%, and 50% of the current MVC, and individual MUs were tracked across the 3 data collection points. RESULTS: We identified 1428 unique MUs, and 270 of them (18.9%) were accurately tracked. After ULLS, MVC decreased by 29.77%, MUs absolute recruitment/derecruitment thresholds were reduced at all contraction intensities (with changes between the 2 variables strongly correlated), while discharge rate was reduced at 10% and 25% but not at 50% MVC. Impaired MVC and MUs properties fully recovered to baseline levels after AR. Similar changes were observed in the pool of total as well as tracked MUs. CONCLUSION: Our novel results demonstrate, non-invasively, that 10 days of ULLS affected neural control predominantly by altering the discharge rate of lower-threshold but not of higher-threshold MUs, suggesting a preferential impact of disuse on motoneurons with a lower depolarization threshold. However, after 21 days of AR, the impaired MUs properties were fully restored to baseline levels, highlighting the plasticity of the components involved in neural control.

Tutorial: Analysis of central and peripheral motor unit properties from decomposed High-Density surface EMG signals with openhdemg.

High-Density surface Electromyography (HD-sEMG) is the most established technique for the non-invasive analysis of single motor unit (MU) activity in humans. It provides the possibility to study the central properties (e.g., discharge rate) of large populations of MUs by analysis of their firing pattern. Additionally, by spike-triggered averaging, peripheral properties such as MUs conduction velocity can be estimated over adjacent regions of the muscles and single MUs can be tracked across different recording sessions. In this tutorial, we guide the reader through the investigation of MUs properties from decomposed HD-sEMG recordings by providing both the theoretical knowledge and practical tools necessary to perform the analyses. The practical application of this tutorial is based on openhdemg, a free and open-source community-based framework for the automated analysis of MUs properties built on Python 3 and composed of different modules for HD-sEMG data handling, visualisation, editing, and analysis. openhdemg is interfaceable with most of the available recording software, equipment or decomposition techniques, and all the built-in functions are easily adaptable to different experimental needs. The framework also includes a graphical user interface which enables users with limited coding skills to perform a robust and reliable analysis of MUs properties without coding.

Effects of short-term unloading and active recovery on human motor unit properties, neuromuscular junction transmission and transcriptomic profile.

Electrophysiological alterations of the neuromuscular junction (NMJ) and motor unit potential (MUP) with unloading are poorly studied. We aimed to investigate these aspects and the underlying molecular mechanisms with short-term unloading and active recovery (AR). Eleven healthy males underwent a 10-day unilateral lower limb suspension (ULLS) period, followed by 21-day AR based on resistance exercise. Quadriceps femoris (QF) cross-sectional area (CSA) and isometric maximum voluntary contraction (MVC) were evaluated. Intramuscular electromyographic recordings were obtained during 10% and 25% MVC isometric contractions from the vastus lateralis (VL). Biomarkers of NMJ molecular instability (serum c-terminal agrin fragment, CAF), axonal damage (neurofilament light chain) and denervation status were assessed from blood samples and VL biopsies. NMJ and ion channel transcriptomic profiles were investigated by RNA-sequencing. QF CSA and MVC decreased with ULLS. Increased CAF and altered NMJ transcriptome with unloading suggested the emergence of NMJ molecular instability, which was not associated with impaired NMJ transmission stability. Instead, increased MUP complexity and decreased motor unit firing rates were found after ULLS. Downregulation of ion channel gene expression was found together with increased neurofilament light chain concentration and partial denervation. The AR period restored most of these neuromuscular alterations. In conclusion, the human NMJ is destabilized at the molecular level but shows functional resilience to a 10-day unloading period at least at relatively low contraction intensities. However, MUP properties are altered by ULLS, possibly due to alterations in ion channel dynamics and initial axonal damage and denervation. These changes are fully reversed by 21 days of AR. KEY POINTS: We used integrative electrophysiological and molecular approaches to comprehensively investigate changes in neuromuscular integrity and function after a 10-day unilateral lower limb suspension (ULLS), followed by 21 days of active recovery in young healthy men, with a particular focus on neuromuscular junction (NMJ) and motor unit potential (MUP) properties alterations. After 10-day ULLS, we found significant NMJ molecular alterations in the absence of NMJ transmission stability impairment. These findings suggest that the human NMJ is functionally resilient against insults and stresses induced by short-term disuse at least at relatively low contraction intensities, at which low-threshold, slow-type motor units are recruited. Intramuscular electromyography analysis revealed that unloading caused increased MUP complexity and decreased motor unit firing rates, and these alterations could be related to the observed changes in skeletal muscle ion channel pool and initial and partial signs of fibre denervation and axonal damage. The active recovery period restored these neuromuscular changes.

Altered muscle mitochondrial, inflammatory and trophic markers, and reduced exercise training adaptations in type 1 diabetes.

Growing evidence of impaired skeletal muscle health in people with type 1 diabetes points toward the presence of a mild myopathy in this population. However, this myopathic condition is not yet well characterised and often overlooked, even though it might affect the whole-body glucose homeostasis and the development of comorbidities. This study aimed to compare skeletal muscle adaptations and changes in glycaemic control after 12 weeks of combined resistance and aerobic (COMB) training between people with type 1 diabetes and healthy controls, and to determine whether the impaired muscle health in type 1 diabetes can affect the exercise-induced adaptations. The COMB training intervention increased aerobic capacity and muscle strength in both healthy and type 1 diabetes sedentary participants, although these improvements were higher in the control group. Better glucose control, reduced glycaemic fluctuations and fewer hypoglycaemic events were recorded at post- compared to pre-intervention in type 1 diabetes. Analysis of muscle biopsies showed an alteration of muscle markers of mitochondrial functions, inflammation, ageing and growth/atrophy compared to the control group. These muscular molecular differences were only partially modified by the COMB training and might explain the reduced exercise adaptation observed in type 1 diabetes. In brief, type 1 diabetes impairs many aspects of skeletal muscle health and might affect the exercise-induced adaptations. Defining the magnitude of diabetic myopathy and the effect of exercise, including longer duration of the intervention, will drive the development of strategies to maximise muscle health in the type 1 diabetes population. KEY POINTS: Type 1 diabetes negatively affects skeletal muscle health; however, the effect of structured exercise training on markers of mitochondrial function, inflammation and regeneration is not known. Even though participants with type 1 diabetes and healthy control were comparable for cardiorespiratory fitness ( V̇O2max${\dot{V}_{{{\rm{O}}_{\rm{2}}}{\rm{max}}}}$ ) and muscle strength at baseline, molecular markers related to muscle health were decreased in type 1 diabetes. After training, both groups increased V̇O2max${\dot{V}_{{{\rm{O}}_{\rm{2}}}{\rm{max}}}}$ and muscle strength; however, a larger improvement was achieved by the control group. The training intervention decreased glucose fluctuations and occurrence of hypoglycaemic events in type 1 diabetes, while signs of mild myopathy found in the muscle of participants with type 1 diabetes only partially improved after training Improving muscle health by specific exercise protocols is of considerable clinical interest in therapeutic strategies for improving type 1 diabetes management and preventing or delaying long-term complications.

Peripheral Amino Acid Appearance Is Lower Following Plant Protein Fibre Products, Compared to Whey Protein and Fibre Ingestion, in Healthy Older Adults despite Optimised Amino Acid Profile.

Plant-based proteins are generally characterised by lower Indispensable Amino Acid (IAA) content, digestibility, and anabolic properties, compared to animal-based proteins. However, they are environmentally friendlier, and wider consumption is advocated. Older adults have higher dietary protein needs to prevent sarcopenia, a disease marked by an accelerated loss of muscle mass and function. Given the lower environmental footprint of plant-based proteins and the importance of optimising dietary protein quality among older adults, this paper aims to assess the net peripheral Amino Acid (AA) appearance after ingestion of three different plant protein and fibre (PPF) products, compared to whey protein with added fibre (WPF), in healthy older adults. In a randomised, single-blind, crossover design, nine healthy men and women aged ≥65 years consumed four test meals balanced in AA according to the FAO reference protein for humans, matched for leucine, to optimally stimulate muscle protein synthesis in older adults. A fasted blood sample was drawn at each visit before consuming the test meal, followed by postprandial arterialise blood sampling every 30 min for 3 h. The test meal was composed of a soup containing either WPF or PPF 1-3. The PPF blends comprised pea proteins with varying additional rice, pumpkin, soy, oat, and/or almond protein. PPF product ingestion resulted in a lower maximal increase of postprandial leucine concentration and the sum of branched-chain AA (BCAA) and IAA concentrations, compared to WPF, with no effect on their incremental area under the curve. Plasma methionine and cysteine, and to a lesser extent threonine, appearance were limited after consuming the PPF products, but not WPF. Despite equal leucine doses, the WPF induced greater postprandial insulin concentrations than the PPF products. In conclusion, the postprandial appearance of AA is highly dependent on the protein source in older adults, despite providing equivalent IAA levels and dietary fibre. Coupled with lower insulin concentrations, this could imply less anabolic potential. Further investigation is required to understand the applicability of plant-based proteins in healthy older adults.

Treatment of Achilles Tendinopathy in Recreational Runners with Peritendinous Hyaluronic Acid Injections: A Viscoelastometric, Functional, and Biochemical Pilot Study.

BACKGROUND: Achilles tendinopathy (AT) affects ca. 10 million recreational runners in Europe; the practice of hyaluronic acid (HA) infiltration is being increasingly adopted. The aim of this pilot study was to monitor the effects of a three-local time-spaced injections regimen of HA in the treatment of AT in middle-aged runners combining for the first time viscoelastometric, biochemical, and functional methodologies with routine clinical examinations. METHODS: Eight male runners (Age 49.3 ± 3.9), diagnosed for unilateral AT, were given three ultrasound (US) guided peritendinous HA injections at the baseline (T0) and every fifteenth day with a follow-up on the forty-fifth day (T1, T2, and T3). At all-time points patients were assessed for viscoelastic tone and stiffness, maximal voluntary isometric contraction (MVIC), and pain level (Likert scale 0-5). The peritendinous effusions of the injured tendon were collected at T0 and T2 to quantify the volume variations and the IL-1ß and MMP-3 levels. RESULTS: At T0 MVIC and pain score were significantly lower and higher, respectively, in injured tendons. The volume, IL-1ß and MMP-3 levels decreased in the course of treatment and the clinical endpoints ameliorated over time. Tone, stiffness, and functional performance also varied significantly at T2 and T3, as compared to T0. CONCLUSIONS: The sequential peritendinous injections of HA were effective in the amelioration of the clinical symptoms, as well as of the functional and viscoelastic state associated with AT. The determination of the viscoelastometric state may help to precisely evaluate the healing process in AT patients.

Cancer Related Anemia: An Integrated Multitarget Approach and Lifestyle Interventions.

Cancer is often accompanied by worsening of the patient's iron profile, and the resulting anemia could be a factor that negatively impacts antineoplastic treatment efficacy and patient survival. The first line of therapy is usually based on oral or intravenous iron supplementation; however, many patients remain anemic and do not respond. The key might lie in the pathogenesis of the anemia itself. Cancer-related anemia (CRA) is characterized by a decreased circulating serum iron concentration and transferrin saturation despite ample iron stores, pointing to a more complex problem related to iron homeostatic regulation and additional factors such as chronic inflammatory status. This review explores our current understanding of iron homeostasis in cancer, shedding light on the modulatory role of hepcidin in intestinal iron absorption, iron recycling, mobilization from liver deposits, and inducible regulators by infections and inflammation. The underlying relationship between CRA and systemic low-grade inflammation will be discussed, and an integrated multitarget approach based on nutrition and exercise to improve iron utilization by reducing low-grade inflammation, modulating the immune response, and supporting antioxidant mechanisms will also be proposed. Indeed, a Mediterranean-based diet, nutritional supplements and exercise are suggested as potential individualized strategies and as a complementary approach to conventional CRA therapy.

Delayed effect of different exercise modalities on glycaemic control in type 1 diabetes mellitus: A systematic review and meta-analysis.

BACKGROUND AND AIMS: Despite the crucial role of exercise in the prevention of comorbidities and complications in type 1 diabetes mellitus (T1DM), people living with the disease are often insufficiently physically active, mainly due to the fear of hypoglycaemia. Research using continuous glucose monitoring (CGM) devices has shown that exercise affects glycaemic control in T1DM for over 24 h. The aim of this systematic review and meta-analysis is, therefore, to investigate the delayed effects of different exercise modalities on glycaemic control in adults with T1DM. METHODS AND RESULTS: The literature search of experimental studies was conducted on PubMed, SPORTDiscus and EMBASE from January 2000 to September 2019. Twelve studies using CGM devices were included. Compared to endurance, intermittent exercise increased the time spent in hypoglycaemia (0.62, 0.07 to 1.18; standardised effect size, 95% CI) and reduced the mean interstitial glucose concentration (-0.88, -1.45 to -0.33). No differences emerged in the time spent in hyperglycaemia (-0.07, -0.58 to 0.45) or in the proportion of exercisers experiencing hypoglycaemic events (0.82, 0.45 to 1.49; proportion ratio, 95% CI) between conditions. The systematic review also found a reduced risk of hypoglycaemia if exercise is performed in the morning rather than in the afternoon, and with a 50% rapid-acting insulin reduction. It was not possible to determine the benefits of resistance exercise. CONCLUSIONS: For the first time, we systematically investigated the delayed effect of exercise in adults with T1DM, highlighted undetected effects, shortcomings in the existing literature, and provided suggestions to design future comparable studies.

Effects of acute aerobic, resistance and combined exercises on 24-h glucose variability and skeletal muscle signalling responses in type 1 diabetics.

PURPOSE: To compare the effect of high-intensity aerobic (AER), resistance (RES), and combined (COMB: RES + AER) exercise, on interstitial glucose (IG) variability and skeletal muscle signalling pathways in type 1 diabetes (T1D). METHODS: T1D participants (6 M/6F) wore a flash glucose monitoring system in four randomized sessions: one control (CONT), and one AER, RES and COMB (40 min each). Mean amplitude of glycemic excursions (MAGE), standard deviation (SD) and coefficient variation (CV) of IG were used to compare the 24 h post-exercise IG variability. Blood and muscle samples were collected to compare exercise-induced systemic and muscle signalling responses related to metabolic, growth and inflammatory adaptations. RESULTS: Both RES and COMB decreased the 24 h MAGE compared to CONT; additionally, COMB decreased the 24 h SD and CV. In the 6-12 h post-exercise, all exercise modalities reduced the IG CV while SD decreased only after COMB. Both AER and COMB stimulated the PGC-1α mRNA expression and promoted the splicing of IGF-1Ea variant, while Akt and p38MAPK phosphorylation increased only after RES and COMB. Additionally, COMB enhanced eEF2 activation and RES increased myogenin and MRF4 mRNA expression. Blood lactate and glycerol levels and muscle IL-6, TNF-α, and MCP-1 mRNAs increased after all exercise sessions, while serum CK and LDH level did not change. CONCLUSION: COMB is more effective in reducing IG fluctuations compared to single-mode AER or RES exercise. Moreover, COMB simultaneously activates muscle signalling pathways involved in substrate metabolism and anabolic adaptations, which can help to improve glycaemic control and maintain muscle health in T1D.