Exercise increases phosphorylation of the putative mTORC2 activity readout NDRG1 in human skeletal muscle

In mice, exercise is suggested to activate the mechanistic target of rapamycin complex 2 (mTORC2) in skeletal muscle, and mTORC2 is required for normal muscle glucose uptake during exercise. Whether this translates to human skeletal muscle and what signaling pathways facilitate the exercise-induced mTORC2 activation is unknown but important to determine given the important role of mTORC2 in metabolism. We herein tested the hypothesis that exercise increases mTORC2 activity in human skeletal muscle and investigated if β2-adrenergic receptor (AR) activation mediates exercise-induced mTORC2 activation. We examined several mTORC2 activity readouts (p-NDRG1 Thr346, p-Akt Ser473, p-mTOR S2481, and p-Akt Thr450) in human skeletal muscle biopsies after uphill walking or cycling exercise. In mouse muscles, we assessed mTORC2 activity readouts following acute activation of muscle β2-adrenergic or Gs signaling and during in vivo and ex vivo muscle contractions. Exercise increased phosphorylation of NDRG1 Thr346 in human soleus, gastrocnemius, and vastus lateralis muscle, without changing p-Akt Ser473, p-Akt Thr450, and p-mTOR Ser2481. In mouse muscle, stimulation of β2-adrenergic or Gs signaling and ex vivo contractions failed to increase p-NDRG1 Thr346, while in vivo contractions were sufficient to induce p-NDRG1 Thr346. In conclusion, the mTORC2 activity readout p-NDRG1 Thr346 is a novel exercise-responsive signaling protein in human skeletal muscle. Notably, contraction-induced p-NDRG1 Thr346 appears to require a systemic factor. Unlike exercise, and in contrast to published data obtained in cultured muscles cells, stimulation of β2-adrenergic signaling is not sufficient to trigger NDRG1 phosphorylation in mature mouse skeletal muscle.

RPS6 phosphorylation occurs to a greater extent in the periphery of human skeletal muscle fibers, near focal adhesions, after anabolic stimuli

Following anabolic stimuli (mechanical loading and/or amino acid provision) the mechanistic target of rapamycin complex 1 (mTORC1), a master regulator of protein synthesis, translocates toward the cell periphery. However, it is unknown if mTORC1-mediated phosphorylation events occur in these peripheral regions or prior to translocation (i.e. in central regions). We therefore aimed to determine the cellular location of a mTORC1-mediated phosphorylation event, RPS6Ser240/244, in human skeletal muscle following anabolic stimuli. Fourteen young, healthy males either ingested a protein-carbohydrate beverage (0.25g/kg protein, 0.75g/kg carbohydrate) alone (n=7;23±5yrs;76.8±3.6kg;13.6±3.8%BF, FED) or following a whole-body resistance exercise bout (n=7;22±2yrs;78.1±3.6kg;12.2±4.9%BF, EXFED). Vastus lateralis muscle biopsies were obtained at rest (PRE) and 120 and 300min following anabolic stimuli. RPS6Ser240/244 phosphorylation measured by immunofluorescent staining or immunoblot was positively correlated (r=0.76, p<0.001). Peripheral staining intensity of p-RPS6Ser240/244 increased above PRE in both FED and EXFED at 120min (~54% and ~138% respectively, p<0.05) but was greater in EXFED at both post-stimuli time points (p<0.05). The peripheral-central ratio of p-RPS6240/244 staining displayed a similar pattern, even when corrected for total RPS6 distribution, suggesting RPS6 phosphorylation occurs to a greater extent in the periphery of fibers. Moreover, p-RPS6Ser240/244 intensity within paxillin-positive regions, a marker of focal adhesion complexes, was elevated at 120min irrespective of stimulus (p=0.006) before returning to PRE at 300min. These data confirm that RPS6Ser240/244 phosphorylation occurs in the region of human muscle fibers to which mTOR translocates following anabolic stimuli and identifies focal adhesion complexes as a potential site of mTORC1 regulation in vivo.

The Study of Muscle, Mobility, and Aging (SOMMA): An Overview

SOMMA is an NIA-funded cohort study to identify biological determinants of mobility and fitness. The overall aim of SOMMA is to use biopsies, novel biomarkers, advanced imaging, and intensive physical and cognitive assessments to elucidate the biological processes that contribute to changes in mobility and physical fitness with aging. SOMMA will recruit 875 people age 70+ (of whom about 200 have been enrolled.) We take biopsies of the vastus lateralis muscle to quantify mitochondrial content and function of the electron transport chain. We use 31PMR spectroscopy to quantify mitochondrial capacity to generate ATP in quadriceps muscle (ATPmax). We will quantify other biological properties in biopsies including denervation, autophagy and accumulated biochemical damage and use gene expression to discover pathways that contribute to mobility and fitness. SOMMA uses MR for quadriceps volume and D3Cr dilution for total skeletal muscle mass, cardiopulmonary exercise testing to measure fitness (VO2 peak). We are also making many other intensive assessments of physical and cognitive function. Mobility endpoints include baseline and three year change in 400 m and 4 meter gait speed and fitness. SOMMA is building a large biobank of muscle, adipose blood, and urine specimens that will be available for ancillary studies. In this Symposium, we will present results from analyses of associations between muscle mitochondrial function and strength, muscle mass, cognitive performance, gait speed, and fitness. The symposium will also preview opportunities for collaborations and ancillary studies with SOMMA.

Changes in muscle architecture on ultrasound in patients early after stroke

BACKGROUND: Early muscle changes are believed to occur in patients with stroke. However, there are insufficient data on the changes in muscle mass and architecture of these patients. OBJECTIVES: This study investigates differences in ultrasound-derived muscle architecture parameters of the hemiplegic upper and lower limbs in patients with subacute stroke. METHODS: This is a prospective observational study, which recruited 40 adult patients who had experienced a first ever unilateral stroke (ischemic or hemorrhagic), with a duration of < 1 month post stroke. The brachialis, vastus lateralis and medial gastrocnemius on both the hemiplegic and normal side were evaluated via ultrasound. We recorded clinical variables including Motricity Index, Modified Ashworth Scale (MAS) and Functional Independence Measure (FIM)-walk. RESULTS: We found reduced mean muscle thickness (p < 0.001) and increased echo intensity (p < 0.001) in the brachialis muscle, increased echo intensity (p = 0.002) in the vastus lateralis muscle, and reduced muscle thickness (p < 0.001) with increased echo intensity (p < 0.001) in the medial gastrocnemius muscle compared to the normal side. There were no significant correlations between ultrasound findings and Motricity Index. CONCLUSIONS: We report changes in ultrasound-derived muscle architecture in the hemiplegic limbs of patients with subacute stroke, with consistent findings of decreased muscle mass and increased echo intensity.

Temporal disruption of neuromuscular communication and muscle atrophy following non-invasive ACL injury in rats

Many patients with anterior cruciate ligament (ACL) injuries have persistent quadriceps muscle atrophy, even after considerable time in rehabilitation. Understanding the factors that regulate muscle mass, and the time course of atrophic events, is important for identifying therapeutic interventions. Using a non-invasive animal model of ACL injury, a longitudinal study was performed to elucidate key parameters underlying quadriceps muscle atrophy. Male Long-Evans rats were euthanized at 6, 12, 24, 48-hrs and 1, 2, 4-wks after ACL injury that was induced via tibial compression overload; controls were not injured. Vastus Lateralis muscle size was determined by wet weight and fiber CSA. Evidence of disrupted neuromuscular communication was assessed via the expression of NCAM and genes associated with denervation and neuromuscular junction instability. Abundance of MuRF-1, MAFbx, and 45s pre-rRNA along with 20S proteasome activity were determined to investigate mechanisms related to muscle atrophy. Lastly, muscle damage-related parameters were assessed by measuring IgG permeability, centronucleation, CD68 mRNA and satellite cell abundance. Compared to controls, we observed a greater percentage of NCAM positive fibers at 6-hrs post-injury, followed by higher MAFbx abundance 48-hrs post-injury, and higher 20S proteasome activity at 1-wk post-injury. A loss of muscle wet weight, smaller fiber CSA and the elevated expression of Runx1 were also observed at the 1-wk post-injury time point relative to controls. There also were no differences observed in any damage markers. These results indicate that alterations in neuromuscular communication precede the upregulation of atrophic factors that regulate quadriceps muscle mass early after non-invasive ACL injury.

Calcium and strontium contractile activation properties of single skinned skeletal muscle fibres from elderly women 66-90 years of age

The single skinned muscle fibre technique was used to investigate Ca2+- and Sr2+- activation properties of skeletal muscle fibres from elderly women (66-90 years). Muscle biopsies were obtained from the vastus lateralis muscle. Three populations of muscle fibres were identified according to their specific Sr2+- activation properties: slow-twitch (type I) fast-twitch (type II) and hybrid (type I/II) fibres. All three fibre types were sampled from the biopsies of 66 to 72 years old women, but the muscle biopsies of women older than 80 years yielded only slow-twitch (type I) fibres. The proportion of hybrid fibres in the vastus lateralis muscle of women of circa 70 years of age (24%) was several-fold greater than in the same muscle of adults (<10%), suggesting that muscle remodelling occurs around this age. There were no differences between the Ca2+- and Sr2+- activation properties of slow-twitch fibres from the two groups of elderly women, but there were differences compared with muscle fibres from adults with respect to sensitivity to Ca2+, steepness of the activation curves, and characteristics of the fibre-type dependent phenomenon of spontaneous force oscillations (SOMO) occurring at sub-maximal levels of activation. The maximal Ca2+ activated specific force from all the fibres collected from the seven old women use in the present study was significantly lower by 20% than in the same muscle of adults. Taken together these results show there are qualitative and quantitative changes in the activation properties of the contractile apparatus of muscle fibres from the vastus lateralis muscle of women with advancing age, and that these changes need to be considered when explaining observed changes in womens mobility with aging.

Early Deconditioning of Human Skeletal Muscles and the Effects of a Thigh Cuff Countermeasure

Muscle deconditioning is a major consequence of a wide range of conditions from spaceflight to a sedentary lifestyle, and occurs as a result of muscle inactivity, leading to a rapid decrease in muscle strength, mass, and oxidative capacity. The early changes that appear in the first days of inactivity must be studied to determine effective methods for the prevention of muscle deconditioning. To evaluate the mechanisms of muscle early changes and the vascular effect of a thigh cuff, a five-day dry immersion (DI) experiment was conducted by the French Space Agency at the MEDES Space Clinic (Rangueil, Toulouse). Eighteen healthy males were recruited and divided into a control group and a thigh cuff group, who wore a thigh cuff at 30 mmHg. All participants underwent five days of DI. Prior to and at the end of the DI, the lower limb maximal strength was measured and muscle biopsies were collected from the vastus lateralis muscle. Five days of DI resulted in muscle deconditioning in both groups. The maximal voluntary isometric contraction of knee extension decreased significantly. The muscle fiber cross-sectional area decreased significantly by 21.8%, and the protein balance seems to be impaired, as shown by the reduced activation of the mTOR pathway. Measurements of skinned muscle fibers supported these results and potential changes in oxidative capacity were highlighted by a decrease in PGC1-α levels. The use of the thigh cuff did not prevent muscle deconditioning or impact muscle function. These results suggest that the major effects of muscle deconditioning occur during the first few days of inactivity, and countermeasures against muscle deconditioning should target this time period. These results are also relevant for the understanding of muscle weakness induced by muscle diseases, aging, and patients in intensive care.