Characterization of the mechanical properties of the mouse Achilles tendon enthesis by microindentation. Effects of unloading and subsequent reloading.

The fibrocartilaginous tendon enthesis, i.e. the site where a tendon is attached to bone through a fibrocartilaginous tissue, is considered as a functionally graded interface. However, at local scale, a very limited number of studies have characterized micromechanical properties of this transitional tissue. The first goal of this work was to characterize the micromechanical properties of the mineralized part of the healthy Achilles tendon enthesis (ATE) through microindentation testing and to assess the degree of mineralization and of carbonation of mineral crystals by Raman spectroscopy. Since little is known about enthesis biological plasticity, our second objective was to examine the effects of unloading and reloading, using a mouse hindlimb-unloading model, on both the micromechanical properties and the mineral phase of the ATE. Elastic modulus, hardness, degree of mineralization, and degree of carbonation were assessed after 14 days of hindlimb suspension and again after a subsequent 6 days of reloading. The elastic modulus gradually increased along the mineralized part of the ATE from the tidemark to the subchondral bone, with the same trend being found for hardness. Whereas the degree of carbonation did not differ according to zone of measurement, the degree of mineralization increased by >70 % from tidemark to subchondral bone. Thus, the gradient in micromechanical properties is in part explained by a mineralization gradient. A 14-day unloading period did not appear to affect the gradient of micromechanical properties of the ATE, nor the degree of mineralization or carbonation. However, contrary to a short period of unloading, early return to normal mechanical load reduced the micromechanical properties gradient, regardless of carbonate-to-phosphate ratios, likely due to the more homogeneous degree of mineralization. These findings provide valuable data not only for tissue bioengineering, but also for musculoskeletal clinical studies and microgravity studies focusing on long-term space travel by astronauts.

Effects of hindlimb unloading and subsequent reloading on the structure and mechanical properties of Achilles tendon-to-bone attachment.

While muscle and bone adaptations to deconditioning have been widely described, few studies have focused on the tendon enthesis. Our study examined the effects of mechanical loading on the structure and mechanical properties of the Achilles tendon enthesis. We assessed the fibrocartilage surface area, the organization of collagen, the expression of collagen II, the presence of osteoclasts, and the tensile properties of the mouse enthesis both after 14 days of hindlimb suspension (HU) and after a subsequent 6 days of reloading. Although soleus atrophy was severe after HU, calcified fibrocartilage (CFc) was a little affected. In contrast, we observed a decrease in non-calcified fibrocartilage (UFc) surface area, collagen fiber disorganization, modification of morphological characteristics of the fibrocartilage cells, and altered collagen II distribution. Compared to the control group, restoring normal loads increased both UFc surface area and expression of collagen II, and led to a crimp pattern in collagen. Reloading induced an increase in CFc surface area, probably due to the mineralization front advancing toward the tendon. Functionally, unloading resulted in decreased enthesis stiffness and a shift in site of failure from the osteochondral interface to the bone, whereas 6 days of reloading restored the original elastic properties and site of failure. In the context of spaceflight, our results suggest that care must be taken when performing countermeasure exercises both during missions and during the return to Earth.

Severe Muscle Deconditioning Triggers Early Extracellular Matrix Remodeling and Resident Stem Cell Differentiation into Adipocytes in Healthy Men.

Besides the loss of muscle mass and strength, increased intermuscular adipose tissue (IMAT) is now a well-recognized consequence of muscle deconditioning as experienced in prolonged microgravity. IMAT content may alter the muscle stem cell microenvironment. We hypothesized that extracellular matrix structure alterations and microenvironment remodeling induced by fast and severe muscle disuse could modulate fibro-adipogenic progenitor fate and behavior. We used the dry immersion (DI) model that rapidly leads to severe muscle deconditioning due to drastic hypoactivity. We randomly assigned healthy volunteers (n = 18 men) to the control group (only DI, n = 9; age = 33.8 ± 4) or to the DI + thigh cuff group (n = 9; age = 33.4 ± 7). Participants remained immersed in the supine position in a thermo-neutral water bath for 5 days. We collected vastus lateralis biopsies before (baseline) and after DI. 5 days of DI are sufficient to reduce muscle mass significantly, as indicated by the decreased myofiber cross-sectional area in vastus lateralis samples (−18% vs. baseline, p < 0.05). Early and late adipogenic differentiation transcription factors protein levels were upregulated. Platelet-derived growth Factors alpha (PDGFR⍺) protein level and PDGFR⍺-positive cells were increased after 5 days of DI. Extracellular matrix structure was prone to remodeling with an altered ECM composition with 4 major collagens, fibronectin, and Connective Tissue Growth Factor mRNA decreases (p < 0.001 vs. baseline). Wearing thigh cuffs did not have any preventive effect on the measured variable. Our results show that altered extracellular matrix structure and signaling pathways occur early during DI, a severe muscle wasting model, favoring fibro-adipogenic progenitor differentiation into adipocytes.

Ergothioneine Improves Aerobic Performance Without Any Negative Effect on Early Muscle Recovery Signaling in Response to Acute Exercise.

Physical activity is now recognized as an essential element of healthy lifestyles. However, intensive and repeated exercise practice produces a high level of stress that must be managed, particularly oxidative damage and inflammation. Many studies investigated the effect of antioxidants, but reported only few positive effects, or even muscle recovery impairment. Secondary antioxidants are frequently highlighted as a way to optimize these interactions. Ergothioneine is a potential nutritional supplement and a secondary antioxidant that activates the cellular NRF2 pathway, leading to antioxidant response gene activation. Here, we hypothesized that ergothioneine could improve performance during aerobic exercise up to exhaustion and reduce exercise-related stress without impairing early muscle recovery signaling. To test this hypothesis, 5-month-old C56B6J female mice were divided in two groups matched for maximal aerobic speed (MAS): control group (Ctrl; n = 9) and group supplemented with 70 mg ergothioneine/kg/day (ET; n = 9). After 1 week of supplementation (or not), mice performed a maximum time-to-exhaustion test by running on a treadmill at 70% of their MAS, and gastrocnemius and soleus muscles were collected 2 h after exercise. Time to exhaustion was longer in the ET than Ctrl group (+41.22%, p < 0.01). Two hours after exercise, the ET group showed higher activation of protein synthesis and satellite cells, despite their longer effort. Conversely, expression in muscles of metabolic stress and inflammation markers was decreased, as well as oxidative damage markers in the ET group. Moreover, ergothioneine did not seem to impair mitochondrial recovery. These results suggest an important effect of ergothioneine on time-to-exhaustion performance and improved muscle recovery after exercise.

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.

Evaluation of an Antioxidant and Anti-inflammatory Cocktail Against Human Hypoactivity-Induced Skeletal Muscle Deconditioning.

Understanding the molecular pathways involved in the loss of skeletal muscle mass and function induced by muscle disuse is a crucial issue in the context of spaceflight as well as in the clinical field, and development of efficient countermeasures is needed. Recent studies have reported the importance of redox balance dysregulation as a major mechanism leading to muscle wasting. Our study aimed to evaluate the effects of an antioxidant/anti-inflammatory cocktail (741 mg of polyphenols, 138 mg of vitamin E, 80 µg of selenium, and 2.1 g of omega-3) in the prevention of muscle deconditioning induced by long-term inactivity. The study consisted of 60 days of hypoactivity using the head-down bed rest (HDBR) model. Twenty healthy men were recruited; half of them received a daily antioxidant/anti-inflammatory supplementation, whereas the other half received a placebo. Muscle biopsies were collected from the vastus lateralis muscles before and after bedrest and 10 days after remobilization. After 2 months of HDBR, all subjects presented muscle deconditioning characterized by a loss of muscle strength and an atrophy of muscle fibers, which was not prevented by cocktail supplementation. Our results regarding muscle oxidative damage, mitochondrial content, and protein balance actors refuted the potential protection of the cocktail during long-term inactivity and showed a disturbance of essential signaling pathways (protein balance and mitochondriogenesis) during the remobilization period. This study demonstrated the ineffectiveness of our cocktail supplementation and underlines the complexity of redox balance mechanisms. It raises interrogations regarding the appropriate nutritional intervention to fight against muscle deconditioning.