Fourteen days of bed rest induces a decline in satellite cell content and robust atrophy of skeletal muscle fibers in middle-aged adults

Abstract

Bed rest, a ground-based spaceflight analog, induces robust atrophy of skeletal muscle, an effect that is exacerbated with increasing age. We examined the effect of 14 days of bed rest on skeletal muscle satellite cell content and fiber type atrophy in middle-aged adults, an understudied age demographic with few overt signs of muscle aging that is representative of astronauts who perform long-duration spaceflight. Muscle biopsies were obtained from the vastus lateralis of healthy middle-aged adults [n= 7 (4 male, 3 female); age: 51 ± 1 yr] before (Pre-BR) and after (Post-BR) 14 days of bed rest. Immunohistochemical analyses were used to quantify myosin heavy chain (MyHC) isoform expression, cross-sectional area (CSA), satellite cell and myonuclear content, and capillary density. Peak oxygen consumption, knee extensor strength, and body composition were also measured Pre-BR and Post-BR. Post-BR MyHC type 2a fiber percentage was reduced, and mean CSA decreased in all fiber types (-24 ± 5%;P< 0.05). Satellite cell content was also reduced Post-BR (-39 ± 9%;P< 0.05), and the change in satellite cell content was significantly correlated with the change in mean fiber CSA (r(2)= 0.60;P< 0.05). A decline in capillary density was observed Post-BR (-23 ± 6%;P< 0.05), and Post-BR capillary content was significantly associated with Post-BR peak aerobic capacity (r(2)= 0.59;P< 0.05). A subtle decline in myonuclear content occurred during bed rest (-5 ± 1%;P< 0.05). The rapid maladaptation of skeletal muscle to 14 days of mechanical unloading in middle-aged adults emphasizes the need for robust countermeasures to preserve muscle function in astronauts.

Keywords: Pax7; capillary; mechanical unloading; myonuclei; spaceflight.

Fig. 1.

Myosin heavy chain (MyHC) type 2a fiber frequency is reduced after 14 days of bed rest. A: representative immunohistochemical image of MyHC type 1 (pink), type 2a (green), and type 2x (orange) fiber identification. Scale bar = 100 μm. B: quantification of MyHC fiber type from immunohistochemical analysis, presented as mean fiber type frequency ± SE. *Significantly different from pre-bed rest (Pre-BR) value (P < 0.05).

Fig. 2.

Mean fiber cross-sectional area (CSA) is decreased after 14 days of bed rest. A: fiber CSA (μm²) of MyHC type 1, 1/2a, 2a, and 2x fibers presented as means ± SE. B: pooled fiber CSA presented as means ± SE. C: histogram distribution of pooled fiber CSA. *Significantly different from Pre-BR value (P < 0.05).

Fig. 3.

14 days of bed rest decreases muscle capillary content, and capillary content is associated with peak oxygen consumption following bed rest. A: representative immunohistochemical image of capillaries labeled with rhodamine-conjugated Ulex Europaeus agglutinin I (agglutinin, red) and arterioles labeled with α-smooth muscle actin (α-SMA, green). Arterioles are denoted as α-SMA positive and agglutinin positive (white arrowhead) and capillaries as α-SMA negative and agglutinin positive (yellow arrow). Scale bar = 100 μm. B: quantification of capillary content, presented as mean capillaries per fiber ± SE. C: quantification of capillary content, presented as mean capillaries per mm² of muscle ± SE. D: quantification of arteriole content, presented as mean arterioles per fiber ± SE. E: quantification of arteriole content, presented as mean arterioles per mm² of muscle ± SE. F: correlation of Pre-BR capillary content with Pre-BR peak oxygen consumption [V̇o_{2 peak}; ml/(kg·min)]. G: correlation of Post-BR capillary content with Post-BR peak oxygen consumption. *Significantly different from Pre-BR value (P < 0.05).

Fig. 4.

Satellite cells associated with MyHC type 1 and 2 fibers decrease following 14 days of bed rest. A: representative immunohistochemical image demonstrating laminin (green), MyHC type 1 (pink), Pax7 (yellow), and DAPI (blue). Pax7-positive satellite cells associated with MyHC type 1 fiber (white arrowhead) and MyHC type 2 fiber (yellow arrow) are identified. Scale bar = 25 μm. B: quantification of fiber type-specific Pax7-positive satellite cell content, expressed as mean satellite cells per fiber ± SE. C: quantification of Pax7-positive satellite cell content, expressed as mean satellite cells per fiber ± SE. D: representative immunohistochemical image demonstrating laminin (green), MyHC type 1 (pink), CD56 (white), and DAPI (blue). CD56-positive satellite cells associated with MyHC type 1 fiber (white arrowhead) and MyHC type 2 fiber (yellow arrow) are identified. Scale bar = 50 μm. E: quantification of fiber type-specific CD56-positive satellite cell content, expressed as mean satellite cells per fiber ± SE. F: quantification of CD56-positive satellite cell content, expressed as mean satellite cells per fiber ± SE. G: correlation of Pre-BR CD56-positive satellite cell content with Pre-BR Pax7-positive satellite cell content. H: correlation of Post-BR CD56-positive satellite cell content with Post-BR Pax7-positive satellite cell content. *Significantly different from Pre-BR value (P < 0.05). †Main effect for fiber type (P < 0.05).

Fig. 5.

The change in satellite cell content is associated with the change in mean fiber CSA following 14 days of bed rest. A: correlation of Pre-BR Pax7-positive satellite cell content with Pre-BR mean fiber CSA. B: correlation of Post-BR Pax7-positive satellite cell content with Post-BR mean fiber CSA. C: correlation of the change in Pax7-positive satellite cell content with the change in mean fiber CSA.

Fig. 6.

14 days of bed rest induces a nonsignificant reduction in MyoD-positive satellite cells. A: representative immunohistochemical image demonstrating laminin (green), MyHC type 1 (pink), MyoD (white), and DAPI (blue). A MyoD-positive satellite cell associated with a MyHC type 1 fiber (white arrowhead) is identified. Scale bar = 50 μm. B: quantification of fiber type-specific MyoD-positive satellite cell content, expressed as mean satellite cells per fiber ± SE. C: quantification of MyoD-positive satellite cell content, expressed as mean satellite cells per fiber ± SE.

Fig. 7.

Myonuclear content is decreased after 14 days of bed rest. A: representative image demonstrating dystrophin (green), MyHC type 1 (pink), MyHC type 2 (orange), and DAPI (blue). Myonuclei associated with type 1 fibers are denoted with white arrowheads, and myonuclei associated with type 2 fibers are denoted with yellow arrows. Scale bar = 50 μm. B: quantification of fiber type-specific myonuclear content, expressed as mean myonuclei per fiber ± SE. C: quantification of myonuclear content, expressed as mean myonuclei per fiber ± SE. D: quantification of myonuclear domain, calculated as the mean fiber CSA (μm²) expressed relative to the number of myonuclei per fiber ± SE. *Significantly different from Pre-BR value (P < 0.05). †Main effect for bed rest (P < 0.05).

Fig. 8.

14 days of bed rest increases the frequency of NCAM-positive muscle fibers. A: representative image demonstrating laminin (green), MyHC type 1 (pink), NCAM/CD56 (white), and DAPI (blue). An NCAM/CD56-positive muscle fiber is denoted with a yellow arrow, and a NCAM/CD56-positive satellite cell is denoted with a white arrowhead. Scale bar = 50 μm. B: quantification of NCAM-positive fibers, expressed as mean percentage of fibers positive for NCAM ± SE. *Significantly different from Pre-BR value (P < 0.05).

Fig. 9.

Individual data plots for fiber type-specific satellite cells, myonuclear content, mean fiber CSA, and capillary content. A: individual data plot for MyHC type 1 Pax7-positive satellite cells. B: individual data plot for MyHC type 2 Pax7-positive satellite cells. C: individual data plot for all Pax7-positive satellite cells. D: individual data plot for MyHC type 1 myonuclei. E: individual data plot for MyHC type 2 myonuclei. F: individual data plot for all myonuclei. G: individual data plot for mean fiber CSA. H: individual data plot for mean capillary content.