Role of Myostatin in Muscle Degeneration by Random Positioning Machine Exposure: An in vitro Study for the Treatment of Sarcopenia

Abstract

Several scientific evidence have shown that exposure to microgravity has a significant impact on the health of the musculoskeletal system by altering the expression of proteins and molecules involved in bone-muscle crosstalk, which is also observed in the research of microgravity effect simulation. Among these, the expression pattern of myostatin appears to play a key role in both load-free muscle damage and the progression of age-related musculoskeletal disorders, such as osteoporosis and sarcopenia. Based on this evidence, we here investigated the efficacy of treatment with anti-myostatin (anti-MSTN) antibodies on primary cultures of human satellite cells exposed to 72 h of random positioning machine (RPM). Cell cultures were obtained from muscle biopsies taken from a total of 30 patients (controls, osteoarthritic, and osteoporotic) during hip arthroplasty. The Pax7 expression by immunofluorescence was carried out for the characterization of satellite cells. We then performed morphological evaluation by light microscopy and immunocytochemical analysis to assess myostatin expression. Our results showed that prolonged RPM exposure not only caused satellite cell death, but also induced changes in myostatin expression levels with group-dependent variations. Surprisingly, we observed that the use of anti-MSTN antibodies induced a significant increase in cell survival after RPM exposure under all experimental conditions. Noteworthy, we found that the negative effect of RPM exposure was counteracted by treatment with anti-MSTN antibodies, which allowed the formation of numerous myotubes. Our results highlight the role of myostatin as a major effector of the cellular degeneration observed with RPM exposure, suggesting it as a potential therapeutic target to slow the muscle mass loss that occurs in the absence of loading.

Keywords: muscle degeneration; myostatin; random positioning machine; sarcopenia; satellite cells.

Figure 1

Morphological analysis by light microscopy of primary cultures of human satellite cells subjected to normogravity regimen and RPM exposure. In normogravity conditions, primary cultures from CTRL (A) and OA (B) patients were characterized by a heterogeneous population of numerous myotubes (arrows) and rare single satellite cells (asterisks), whereas primary cultures from OP patients (C) consisted of rare myotubes (arrows) and numerous single satellite cells (asterisks). RPM exposure caused an increase in the number of myotubes (arrows) in primary cultures from CTRL (D) and OA (E) patients. Samples derived from OP patients (F) showed not only single satellite cells (asterisks), but also several myotubes (arrows). Satellite cells (asterisks) were characterized by immunofluorescence for Pax7 (see Supplementary Figure S1). 4× images, scale bar represents 100 μm; 20× images, scale bar represents 50 μm.

Figure 2

Evaluation of myostatin expression by immunocytochemistry. In cell cultures exposed to normogravity regimen, a significant increase in myostatin expression was observed in OA (B) and OP (C) patients respect to the CTRL group (A). Note that a significant statistical difference was reported between the following groups (D): CTRL vs. OA, ^**p < 0.01; CTRL vs. OP, ^****p < 0.0001; OA vs. OP, ^****p < 0.0001. After RPM exposure, primary cultures derived from OP patients (G) showed a significant reduction in myostatin expression, whereas higher levels of myostatin were found in CTRL (E) and OA (F) patients. Note that a significant statistical difference was found only between the OA and OP groups (OA vs. OP, ^**p < 0.01; H). 4x images, scale bar represents 100 μm.

Figure 3

Effects of anti-MSTN antibodies on primary cultures of human satellite cells subjected to RPM exposure. (A) In CTRL patients, myotubes subjected to RPM exposure showed slowed growth; whereas treatment with anti-MSTN antibodies (D) allowed to counteract this negative effect, promoting in some places the formation of a cellular multilayer. (B) In OA patients, myotubes subjected to RPM exposure exhibited slowed growth; treatment with anti-MSTN antibodies (E) induced growth and formation of new myotubes. (C) In OP patients, RPM exposure dramatically reduced the ability of cells to form myotubes; while treatment with anti-MSTN antibodies (F) promoted cell aggregation and the tendency to form new myotubes even after RPM exposure. 10x images, scale bar represents 100 μm.

Figure 4

Role of myostatin in the pathophysiology of muscle tissue. In normogravity, myostatin is produced by skeletal muscle on which it acts as a negative regulator, reducing satellite cell function and their ability to repair damaged muscle fibers, inducing degeneration and muscle mass loss, and so promoting the progression of sarcopenia. After RPM exposure, the increases myostatin expression caused cell degeneration and reduction in muscle size, volume, and strength. These conditions amplified atrophy and muscle weakening and accelerated the sarcopenia progression. The anti-MSTN antibodies treatment blocks the action of myostatin and promote an increase in cell survival, the formation of new myotubes and muscle regeneration.