Heat shock transcription factor 1-deficiency attenuates overloading-associated hypertrophy of mouse soleus muscle

Abstract

Hypertrophic stimuli, such as mechanical stress and overloading, induce stress response, which is mediated by heat shock transcription factor 1 (HSF1), and up-regulate heat shock proteins (HSPs) in mammalian skeletal muscles. Therefore, HSF1-associated stress response may play a key role in loading-associated skeletal muscle hypertrophy. The purpose of this study was to investigate the effects of HSF1-deficiency on skeletal muscle hypertrophy caused by overloading. Functional overloading on the left soleus was performed by cutting the distal tendons of gastrocnemius and plantaris muscles for 4 weeks. The right muscle served as the control. Soleus muscles from both hindlimbs were dissected 2 and 4 weeks after the operation. Hypertrophy of soleus muscle in HSF1-null mice was partially inhibited, compared with that in wild-type (C57BL/6J) mice. Absence of HSF1 partially attenuated the increase of muscle wet weight and fiber cross-sectional area of overloaded soleus muscle. Population of Pax7-positive muscle satellite cells in HSF1-null mice was significantly less than that in wild-type mice following 2 weeks of overloading (p<0.05). Significant up-regulations of interleukin (IL)-1β and tumor necrosis factor mRNAs were observed in HSF1-null, but not in wild-type, mice following 2 weeks of overloading. Overloading-related increases of IL-6 and AFT3 mRNA expressions seen after 2 weeks of overloading tended to decrease after 4 weeks in both types of mice. In HSF1-null mice, however, the significant overloading-related increase in the expression of IL-6, not ATF3, mRNA was noted even at 4th week. Inhibition of muscle hypertrophy might be attributed to the greater and prolonged enhancement of IL-6 expression. HSF1 and/or HSF1-mediated stress response may, in part, play a key role in loading-induced skeletal muscle hypertrophy.

Figure 1. Changes in the body weight, and absolute and relative muscle weight of heat shock transcription factor 1 (HSF1)-null and wild-type mice following 2 and 4 weeks of functional overloading.

Relative muscle weight: the muscle weight relative to body weight (BW); +/+, wild-type mice; −/−, HSF1-null mice; Control, control muscle; Overloaded, overloaded muscle; 2 weeks, 2 weeks of overloading; 4 weeks, 4 weeks of overloading. Values are means ± SEM. n = 6/group at each time point. *: significant difference at p<0.05.

Figure 2. The rates of increase in the relative muscle weight and protein content to body weight.

Each bar represents the relative value of overloaded muscle vs. the contralateral control muscle, which is 1.0. See figure 1 for other abbreviations. Values are means ± SEM. n  =  6 /group at each time point. *: significant difference at p<0.05.

Figure 3. A: Transverse cryosections of the midbelly region of mouse soleus muscle stained with haematoxylin and eosin (H&E).

B: Responses of the mean fiber cross-sectional area. See figure 1 for other abbreviations. Values are means ± SEM. n  =  6 /group at each time point. *: significant difference at p<0.05.

Figure 4. Changes in the population of Pax7-positive nuclei relative to the total myonuclei in response to functional overloading.

Values are means ± SEM. n  =  6 /group at each time point. *: significant difference at p<0.05.

Figure 5. Changes in mean mRNA expression levels of heat shock transcription factor (HSF) 1, HSF2, and HSF4 in soleus muscle during the experimental period.

hsf1, HSF1 mRNA; hsf2, HSF2 mRNA; hsf4, HSF4 mRNA. See figure 1 for other abbreviations. Values are means ± SEM. n  =  6 /group at each time point. *: significant difference at p<0.05.

Figure 6. Changes in mean mRNA expressions of heat shock protein (HSP) 25, HSP47, HSC70, HSP72, and HSP90α.

hsp25, HSP25 mRNA; hsp47, HSP47 mRNA; hsc70, HSC70 mRNA; hsp72, HSP72 mRNA; hsp90α, HSP90α mRNA. See figure 1 for other abbreviations. Values are means ± SEM. n  =  6 /group at each time point. *: significant difference at p<0.05.

Figure 7. Expressions of heat shock proteins (HSPs) and heat shock cognate protein (HSC) in soleus muscle.

A: Representative protein expression patterns of HSP25, HSP47, HSC70, HSP72, HSP90α and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). C: control muscle, O: overloaded muscle. B: Changes in the mean expression levels of HSP25, HSP47, HSP70, HSP72, and HSP90α proteins. See figure 1 and 6 for other abbreviations. Values are means ± SEM. n  =  6 /group at each time point. *: significant difference at p<0.05.

Figure 8. Changes in the expression level of Akt protein in soleus muscle.

p-Akt: phosphorylated form of Akt (protein kinase B), t-Akt: total protein of Akt. See figure 1 and 7 for other abbreviations. Values are means ± SEM. n  =  6 /group at each time point. *: significant difference at p<0.05.

Figure 9. Changes in mean mRNA expression levels of pro-inflammatory cytokines in soleus muscle.

IL-6, interleukin-6; ATF3, activating transcription factor 3; IL-1β, interleukin1β; TNF, tumor necrosis factor. See figure 1 for other abbreviations. Values are means ± SEM. n = 6/group at each time point. *: significant difference at p<0.05.