1. Effect of Unloading on Myofiber Profiles in Male and Female Rats Hailey C. Ramsey, Shuhan Li, Anna Seo, Matthew A. Adan, Michael R. Deschenes Department of Kinesiology & Health Sciences, and Program in Neuroscience The College of William & Mary, Williamsburg, VA
Conclusions
In the Soleus muscle, there is a significant difference in the cross-sectional area of combined myofibers between the male and female experimental groups (unloaded)
There is also a significant difference in the cross-sectional area of type IIA myofibers between the male and female control groups
In the Plantaris, there is a significant difference in the cross-sectional area of all combined myofibers between the male and female experimental groups (unloaded)
There is also a significant difference in the cross-sectional area of type IIA and IIX myofibers between the male and female control groups
There are no significant results from the myofiber type composition or the percentage of area occupied by a fiber type in either the Soleus or the Plantaris
Atrophy occurred in both males and females, and more so in females than males
Introduction
Many injuries and surgeries result in necessary muscle unloading to aid in recovery. It is known that muscle unloading can lead to muscle atrophy1. A previous study conducted in this lab found that females suffer greater decline in strength than males, as a result of unloading1. Another experiment confirmed that females are more susceptible to adaptations due to unloading and suggested that this is caused by the neural drive from the central nervous system2. This study aims to explain what causes the gender-related discrepancy in strength after unloading by looking at the effect of unloading on the myofiber profiles of male and female rats. The knowledge generated from this experiment can be used to aid in the recovery process from injuries and surgeries that require unloading for recovery.
Type I
Type IIA
Type IIB
Overlay
Methods and Materials
Subjects
Forty Wistar rats (6 months old), twenty male and twenty female, were purchased from Charles River Laboratories.
Experimental Design
The forty rats were randomly assigned to 4 treatment groups: 1) male control, 2) male muscle unloading, 3) female control, 4) female muscle unloading. Rats in both of the unloading groups experienced two weeks of hindlimb unloading.
After the end of two weeks, all rats were euthanized and Soleus and Plantaris muscles were immediately removed from rats, cleared of fat and connective tissue, frozen at resting length and stored at -85º C until analysis. The Soleus is a weight-bearing muscle, and the Plantaris is a non-weight-bearing muscle. This disparity makes these two leg muscles ideal to determine the effects of the treatment on myofiber profiles. Myofiber profiles include average fiber size and fiber type composition. An immunofluorescent procedure was used to assess both myofiber profiles.
Statistical Analysis
Once analysis of all 40 specimens was complete, each variable of interest was analyzed with a 2-way ANOVA with main effects for sex and treatment. In the event of a significant F-ratio, post-hoc procedures were used to identify pair wise differences. Statistical significance was established when p<0.05.
Myofiber at 10X
Figure 1.
Results
Myofiber Profile Data: Soleus
Cross-sectional area (µm2)
Male Control
Male Unloaded
Female Control
Female Unloaded
Types combined
3300 ± 151*
2616 ± 191
2892 ± 135†
2179 ± 189
Type I
3320 ± 151‡
2631 ± 197
2674 ± 300
2205 ± 187
Type IIA
2368 ± 266
1531 ± 421
2218 ± 230
1189 ± 236#
Myofiber type composition (%)
96.4 ± 1.1
97.4 ± 0.6
95.4 ± 1.0
92.7 ± 2.4
3.6 ± 1.3
2.6 ± 1.1
4.6 ± 0.8
7.3 ± 4.0
Percentage area occupied by fiber type
97.8 ± 1.0
99.1 ± 0.4
98.5 ± 0.7
96.9 ± 1.8
2.2 ± 0.2
0.9 ± 0.9
1.5 ± 0.7
2.1 ± 1.3
Future Research
In this study, unloading resulted in significant myofiber atrophy in both genders and in gender differences in response to unloading. A study conducted simultaneously, using the same subjects, found no significant difference between genders or due to unloading on the morphology of the neuromuscular junction. Therefore, it is known that differences in strength after unloading and atrophy are due to myofiber adaptations rather than morphological changes to the NMJ. Going forward, it would be of interest to examine additional presynaptic and postsynaptic variables of the NMJ to continue searching for morphological changes in response to unloading, including the location and quantity of calcium channels, the proximity of calcium channels to vesicle tethering sites, and the amount of tethering proteins such as bassoon present in the NMJ.
Values are means ± SE, N=10/group.
*indicates significant (P ≤ 0.05) difference from Male Unloaded and Female Unloaded.
† indicates significant (P ≤ 0.05) difference from Female Unloaded.
‡ indicates significant (P ≤ 0.05) difference from all other groups.
# indicates significant (P ≤ 0.05) difference from Male Control and Female Control.
Myofiber P{rofile Data: Plantaris
Cross-sectional area (µm2)
Male Control
Male Unloaded
Female Control
Female Unloaded
Types combined
3194 ± 284*
2730 ± 180
2138 ± 284
2281 ± 166
Type I
2144 ± 249*
1858 ± 166
1527 ± 75
1516 ± 83
Type IIA
2351 ± 252*
2052 ± 154*
1476 ± 82
1572 ± 90
Type IIX
3485 ± 367*
3059 ± 200*
2215 ± 146
2335 ± 132
Type IIB
4623 ± 436*
3841 ± 328
2983 ± 284
3109 ± 193
Myofiber Type Composition (%)
12.8 ± 1.8
15.5 ± 4.2
15.2 ± 2.6
15.6 ± 2.1
31.1 ± 1.7
33.4 ± 2.0
30.5 ± 3.2
27.4 ± 2.6
36.3 ± 2.2
32.2 ± 2.3
31.2 ± 2.6
31.1 ± 2.6
19.8 ± 4.5
18.9 ± 3.1
24.1 ± 5.3
25.9 ± 5.6
Percentage Area Occupied by Fiber Type
10.6 ± 2.0
12.1 ± 4.5
12.8 ± 3.5
7.9 ± 1.6
24.2 ± 2.0
25.1 ± 1.9
21.3 ± 3.3
24.5 ± 2.6
39.2 ± 3.2
36.3 ± 2.8
37.4 ± 3.2
33.2 ± 3.4
26.0 ± 6.4
26.5 ± 4.8
28.5 ± 7.1
34.4 ± 6.3
References
Deschenes, M. R., Mccoy, R. W., Holdren, A. N., Eason, M. K. Gender influences neuromuscular adaptations to muscle unloading. European Journal of Applied Physiology, 105(6): , 2009.
Deschenes, M. R., Mccoy, R. W., & Mangis, K. A. Factors relating to gender specificity of unloading-induced declines in strength. Muscle & Nerve, 46(2), : 2012.
Acknowledgments
This study was supported by an Undergraduate STEM Research Scholarship from the Virginia Space Grant Consortium
Values are means ± SE, N=9/group.
*indicates significant (P ≤ 0.05) difference from Female Control and Female Unloaded.