Female mice may have exacerbated catabolic signalling response compared to male mice during development and progression of disuse atrophy

Abstract

Background: Muscle atrophy is a common pathology associated with disuse, such as prolonged bed rest or spaceflight, and is associated with detrimental health outcomes. There is emerging evidence that disuse atrophy may differentially affect males and females. Cellular mechanisms contributing to the development and progression of disuse remain elusive, particularly protein turnover cascades. The purpose of this study was to investigate the initial development and progression of disuse muscle atrophy in male and female mice using the well-established model of hindlimb unloading (HU).

Methods: One hundred C57BL/6J mice (50 male and 50 female) were hindlimb suspended for 0 (control), 24, 48, 72, or 168 h to induce disuse atrophy (10 animals per group). At designated time points, animals were euthanized, and tissues (extensor digitorum longus, gastrocnemius, and soleus for mRNA analysis, gastrocnemius and extensor digitorum longus for protein synthesis rates, and tibialis anterior for histology) were collected for analysis of protein turnover mechanisms (protein anabolism and catabolism).

Results: Both males and females lost ~30% of tibialis anterior cross-sectional area after 168 h of disuse. Males had no statistical difference in MHCIIB fibre area, whereas unloaded females had ~33% lower MHCIIB cross-sectional area by 168 h of unloading. Both males and females had lower fractional protein synthesis rates (FSRs) within 24-48 h of HU, and females appeared to have a greater reduction compared with males within 24 h of HU (~23% lower FSRs in males vs. 40% lower FSRs in females). Males and females exhibited differential patterns and responses in multiple markers of protein anabolism, catabolism, and myogenic capacity during the development and progression of disuse atrophy. Specifically, females had greater mRNA inductions of catabolic factors Ubc and Gadd45a (~4-fold greater content in females compared with ~2-fold greater content in males) and greater inductions of anabolic inhibitors Redd1 and Deptor with disuse across multiple muscle tissues exhibiting different fibre phenotypes.

Conclusions: These results suggest that the aetiology of disuse muscle atrophy is more complicated and nuanced than previously thought, with different responses based on muscle phenotypes and between males and females, with females having greater inductions of atrophic markers early in the development of disuse atrophy.

Keywords: Females; Males; Muscle loss; Protein anabolism; Protein catabolism; Sex differences.

Figure 1

Mean fibre cross‐sectional area (CSA) data in males and females across different fibre types. (A) Per cent mass loss in the tibialis anterior across different durations of unloading in males and females. (B) Mean CSA of all fibre types combined in the tibialis anterior across different durations of unloading in males and females. (C) Mean CSA of MHCIIB fibres across different durations of unloading in males and females. (D) Mean CSA of MHCIIX/D fibres across different durations of unloading in males and females. (E) Mean CSA of MHCIIA fibres across different durations of unloading in males and females. (F) Representative images of muscle CSA data. All images were acquired at ×10 magnification. Different letters represent statistical differences at Tukey‐adjusted P ≤ 0.05. *Linear trend within a sex. ^ΩQuadratic trend within a sex. ^#Cubic trend within a sex. Female data are italicized and underlined.

Figure 2

Muscle fractional protein synthesis rates (FSRs) in males and females across different durations of unloading. (A) Mixed FSR in the tibialis anterior of males and females across different durations of unloading. (B) Mixed FSR in the gastrocnemius of males and females across different durations of unloading. Different letters represent statistical differences at Tukey‐adjusted P ≤ 0.05. *Linear trend within a sex. ^ΩQuadratic trend within a sex. ^#Cubic trend within a sex. Female data are italicized and underlined.

Figure 3

Immunoblot data for marks of protein synthesis in males and females across different durations of unloading. (A) Akt protein content in the extensor digitorum longus (EDL) of males and females across different durations of unloading. (B) Akt protein content in the gastrocnemius of males and females across different durations of unloading. (C) pAkt^Ser473 protein content in the EDL of males and females across different durations of unloading. (D) pAkt^Ser473 protein content in the gastrocnemius of males and females across different durations of unloading. (E) pAkt^Ser473:Akt protein ratio in the EDL of males and females across different durations of unloading. (F) pAkt^Ser473:Akt protein ratio in the gastrocnemius of males and females across different durations of unloading. (G) 4EBP1 protein content in the EDL of males and females across different durations of unloading. (H) 4EBP1 protein content in the gastrocnemius of males and females across different durations of unloading. (I) p4EBP1^Thr37/46 protein content in the EDL of males and females across different durations of unloading. (J) p4EBP1^Thr37/46 protein content in the gastrocnemius of males and females across different durations of unloading. (K) p4EBP1^Thr37/46:4EBP1 protein ratio content in the EDL of males and females across different durations of unloading. (L) p4EBP1^Thr37/46:4EBP1 protein ratio content in the gastrocnemius of males and females across different durations of unloading. (M) Representative images for EDL immunoblot data in males. (N) Representative images for EDL immunoblot data in females. (O) Representative images for gastrocnemius immunoblot data in males. (P) Representative images for gastrocnemius immunoblot data in females. Different letters represent statistical differences at Tukey‐adjusted P ≤ 0.05. *Linear trend within a sex. ^ΩQuadratic trend within a sex. ^#Cubic trend within a sex. Female data are italicized and underlined.

Figure 4

mRNA data for positive and negative moderators of protein anabolism in the extensor digitorum longus (EDL), gastrocnemius, and soleus of males and females across different durations of unloading. (A) Igf1 mRNA data in males and females in the EDL. (B) Igf1 mRNA data in males and females in the gastrocnemius. (C) Igf1 mRNA data in males and females in the soleus. (D) Pgc1α4 mRNA data in males and females in the EDL. (E) Pgc1α4 mRNA data in males and females in the gastrocnemius. (F) Pgc1α4 mRNA data in males and females in the soleus. (G) Deptor mRNA data in males and females in the EDL. (H) Deptor mRNA data in males and females in the gastrocnemius. (I) Deptor mRNA data in males and females in the soleus. (J) Redd1 mRNA data in males and females in the EDL. (K) Redd1 mRNA data in males and females in the gastrocnemius. (L) Redd1 mRNA data in males and females in the soleus. Different letters represent statistical differences at Tukey‐adjusted P ≤ 0.05. *Linear trend within a sex. ^ΩQuadratic trend within a sex. ^#Cubic trend within a sex. Female data are italicized and underlined.

Figure 5

mRNA data for moderators of protein catabolism in the extensor digitorum longus (EDL), gastrocnemius, and soleus of males and females across different durations of unloading. (A) Atrogin mRNA content in males and females in the EDL. (B) Atrogin mRNA content in males and females in the gastrocnemius. (C) Atrogin mRNA content in males and females in the soleus. (D) MuRF1 mRNA content in males and females in the EDL. (E) MuRF1 mRNA content in males and females in the gastrocnemius. (F) MuRF1 mRNA content in males and females in the soleus. (G) Ubc mRNA content in males and females in the EDL. (H) Ubc mRNA content in males and females in the gastrocnemius. (I) Ubc mRNA content in males and females in the soleus. (J) Gadd45a mRNA content in males and females in the EDL. (K) Gadd45a mRNA content in males and females in the gastrocnemius. (L) Gadd45a mRNA content in males and females in the soleus. Different letters represent statistical differences at Tukey‐adjusted P ≤ 0.05. *Linear trend within a sex. ^ΩQuadratic trend within a sex. ^#Cubic trend within a sex. Female data are italicized and underlined.

Figure 6

mRNA for moderators of muscle regeneration and cell cycle in the extensor digitorum longus (EDL), gastrocnemius, and soleus of males and females across different durations of unloading. (A) Pax7 mRNA content in males and females in the EDL. (B) Pax7 mRNA content in males and females in the gastrocnemius. (C) Pax7 mRNA content in males and females in the soleus. (D) MyoD mRNA content in males and females in the EDL. (E) MyoD mRNA content in males and females in the gastrocnemius. (F) MyoD mRNA content in males and females in the soleus. (G) MyoG mRNA content in males and females in the EDL. (H) MyoG mRNA content in males and females in the gastrocnemius. (I) MyoG mRNA content in males and females in the soleus. (J) mik67 mRNA content in males and females in the EDL. (K) mik67 mRNA content in males and females in the gastrocnemius. (L) mik67 mRNA content in males and females in the soleus. (M) Ccnd1 mRNA content in males and females in the EDL. (N) Ccnd1 mRNA content in males and females in the gastrocnemius. (O) Ccnd1 mRNA content in males and females in the soleus. Different letters represent statistical differences at Tukey‐adjusted P ≤ 0.05. *Linear trend within a sex. ^ΩQuadratic trend within a sex. ^#Cubic trend within a sex. Female data are italicized and underlined.