Acute daily psychological stress causes increased atrophic gene expression and myostatin-dependent muscle atrophy

Abstract

Psychological stress is known to attenuate body size and lean body mass. We tested the effects of 1, 3, or 7 days of two different models of psychological stress, 1 h of daily restraint stress (RS) or daily cage-switching stress (CS), on skeletal muscle size and atrophy-associated gene expression in mice. Thymus weights decreased in both RS and CS mice compared with unstressed controls, suggesting that both models activated the hypothalamic-pituitary-adrenal axis. Body mass was significantly decreased at all time points for both models of stress but was greater for RS than CS. Mass of the tibialis anterior (TA) and soleus (SOL) muscles was significantly decreased after 3 and 7 days of RS, but CS only significantly decreased SOL mass after 7 days. TA mRNA levels of the atrophy-associated genes myostatin (MSTN), atrogin-1, and the phosphatidylinositol 3-kinase inhibitory subunit p85alpha were all significantly increased relative to unstressed mice after 1 and 3 days of RS, and expression of MSTN and p85alpha mRNA remained elevated after 7 days of RS. Expression of muscle ring finger 1 was increased after 1 day of RS but returned to baseline at 3 and 7 days of RS. MSTN, atrogin-1, and p85alpha mRNA levels also significantly increased after 1 and 3 days of CS but atrogen-1 mRNA levels had resolved back to normal levels by 3 days and p85alpha with 7 days of CS. p21CIP mRNA levels were significantly decreased by 3 days of CS or RS. Finally, body mass was minimally affected, and muscle mass was completely unaffected by 3 days of RS in mice null for the MSTN gene, and MSTN inactivation attenuated the increase in atrogin-1 mRNA levels with 4 days of RS compared with wild-type mice. Together these data suggest that acute daily psychological stress induces atrophic gene expression and loss of muscle mass that appears to be MSTN dependent.

Fig. 1.

Body mass loss in response to acute daily stress. Body mass loss in grams for restrained (RS), cage-switched (CS), and handled and weighed (HC) mice after 1, 3, and 7 days. Body mass decreased for all 3 groups across the time line of the study, but RS resulted in the greatest decrease in body mass at each time point. Each bar represents the mean ± SE for n = 4–5 mice. P < 0.05, significantly different from 1-day time point (*), significantly different from 3-day time point (†), and significantly different from HC at the same time point (‡).

Fig. 2.

Tibialis anterior (TA) and soleus (SOL) mass are decreased by acute daily stress. Mean TA (A, C, and E) and SOL (B, D, and F) mass after 1 (A and B), 3 (C and D), or 7 (E and F) days. As described in methods, the conditions are baseline control (BC); eating control (EC), which was fed the same amount eaten as the RS mice; HC, which was handled and weighed each day like the RS and CS mice; RS; and CS. RS resulted in a significant decrease in TA at 1, 3, and 7 days and in SOL mass at 3 and 7 days, and CS resulted in a significant decrease in SOL mass at 7 days. Bars represent means ± SE for n = 4–5 mice/group. P < 0.05, significantly different from BC (*), significantly different from EC at the same time point (†), significantly different from HC at the same time point (‡), and significantly different from CS at the same time point (¥).

Fig. 3.

Myostatin (MSTN) mRNA levels are increased by acute daily stress. MSTN mRNA levels relative to β-actin in the TA muscle of the various groups after 1 (A), 3 (B), or 7 (C) days. MSTN mRNA levels significantly increased in the HC, CS, and RS groups after 1 day and in the CS and RS groups after 3 and 7 days. Bars in each panel represent means ± SE for n = 4–5 animals/group. P < 0.05, statistically significantly different from BC at the same time point (*), significantly different from EC at the same time point (†), and significantly different from HC at the same time point (‡).

Fig. 4.

p85α mRNA levels are increased by acute daily stress. p85α mRNA levels relative to β-actin in the TA muscle of the various groups after 1 (A), 3 (B), or 7 (C) days. p85α mRNA levels significantly increased in the EC, HC, CS, and RS groups after 1 day, in the CS and RS groups after 3 days, and in the RS group after 7 days. Bars in each panel represent means ± SE for n = 4–5 animals/group. P < 0.05, statistically significantly different from BC at the same time point (*), significantly different from EC at the same time point (†), significantly different from HC at the same time point (‡), and significantly different from CS at the same time point (¥).

Fig. 5.

Atrogin-1 mRNA levels are increased by acute daily stress. Atrogin-1 mRNA levels relative to β-actin in the TA muscle of the various groups after 1 (A), 3 (B), or 7 (C) days. Atrogin-1 mRNA levels significantly increased in the HC, CS, and RS groups after 1 day and in the RS groups after 3 days but were not significantly different for any group after 7 days. Bars in each panel represent means ± SE for n = 4–5 animals/group. P < 0.05, statistically significantly different from BC at the same time point (*), significantly different from EC at the same time point (†), significantly different from HC at the same time point (‡), and significantly different from CS at the same time point (¥).

Fig. 6.

Muscle ring finger 1 (MuRF1) mRNA levels are increased by acute daily stress. MuRF1 mRNA levels relative to β-actin in the TA muscle of the various groups after 1 (A), 3 (B), or 7 (C) days. MuRF1 mRNA levels significantly were increased in the HC, CS, and RS groups after 1 day but were not significantly different for any group after 3 or 7 days. Bars in each panel represent means ± SE for n = 4–5 animals/group. P < 0.05, statistically significantly different from BC at the same time point (*) and significantly different from EC at the same time point (†).

Fig. 7.

p21CIP1 mRNA levels are increased by acute daily stress. p21CIP1 mRNA levels relative to β-actin in the TA muscle of the various groups after 1 (A), 3 (B), or 7 (C) days. p21CIP1 mRNA levels were not significantly different for any group after 1 or 7 days but significantly decreased in the CS and RS groups after 3 days relative to the BC. Bars in each panel represent means ± SE for n = 4–5 animals/group. *P < 0.05, statistically significantly different from BC at the same time point.

Fig. 8.

MSTN inactivation attenuates the changes with stress. A: body mass loss in grams of wild-type (WT) and MSTN null (MSTN KO) unstressed (HC) and stressed (RS) mice. Stress again resulted in a significant decrease in body mass in WT mice, whereas MSTN null mice loss significantly less mass than WT mice with stress. B: fraction of TA mass for WT and MSTN KO unstressed (HC) and stressed (RS) mice. The mean mass of unstressed WT or MSTN null mice was used to normalize the masses of stressed and unstressed mice so as to express them as a fraction. Stress significantly decreased TA mass relative to unstressed in the WT mice, but this decrease was abolished in the MSTN null mice. C: fraction of SOL mass for WT and MSTN KO unstressed (HC) and stressed (RS) mice. The mean mass of unstressed WT or MSTN null mice was used to normalize the masses of stressed and unstressed mice so as to express them as a fraction. Stress significantly decreased SOL mass relative to unstressed in the WT mice, but this decrease was abolished in the MSTN null mice. D: atrogin-1 mRNA expression relative to β-actin in the TA muscle of WT and MSTN KO unstressed (HC) and stressed (RS) mice. Stress significantly increased atrogin-1 mRNA relative to unstressed in the WT mice, but this increase was attenuated in the MSTN null mice. Bars in each panel represent means ± SE for n = 4–5 animals/group. P < 0.05, statistically significantly different from unstressed HC controls for that genotype (*) and significantly different from WT stressed mice values (†).

Fig. 9.

Corticosterone and blood glucose changes with stress are blunted in MSTN null mice. A: serum corticosterone levels of WT and MSTN KO unstressed (HC) and stressed (RS) mice. Serum corticosterone levels significantly increased 2- to 3-fold 1 h after the fourth of four daily RS bouts in WT mice relative to unstressed mice, but were not significantly different in stressed MSTN null mice compared with unstressed MSTN null mice. B: plasma glucose levels of WT and MSTN null (MSTN KO) unstressed (HS) and stressed (RS) mice. Plasma glucose levels significantly increased 1 h after the final four daily RS bouts in WT mice relative to unstressed mice, but were not significantly different in stressed MSTN null mice compared with unstressed MSTN null mice. Bars in each panel represent means ± SE for n = 4–5 animals/group. *P < 0.05, statistically significantly different from unstressed HC controls for that genotype.