Upregulation of Muscle Genes in Estrogen Treated MiceIan Macato, UNLV Summer Science Camp

A.M.S.A.T. Magnet Program, Ed W. Clark High School

Background

Conclusions

Methods

Results

This material is based on research conducted by Dr. Barbara St. Pierre Schneider, DNSc, RN, CNE on crush muscle injury in estrogen treated mouse groups sponsored by the US Dept. of Defense, Air Force. This study was sponsored by the 2014 UNLV Summer Science Program organized by Dr. Joanna Jezierska and Dr. Schneider.

Estrogen is the principal type of sex hormone in females, affecting a wide range of metabolic pathways. One of estrogen’s active forms, 17β-estradiol, regulates the first part of skeletal muscle differentiation by inducing “the rapid Akt phosphorylation which is necessary for the rapid translocation of Glut-4 at membranes1”. In addition, estradiol “can regulate skeletal muscle mass” as several studies show that “estrogen replacement therapy in postmenopausal women enhances both muscle mass and strength2”. The purpose of the current study is to investigate the effect of 17 β-estradiol on the regulation of six genes related to myogenesis or muscle contraction:

Data were obtained from three independent milestones of a larger project: M15, M9, and M11. In M15, one group of ovariectomized mice were treated with 17β-estradiol and another was placebo-treated for one week. Next the right gastrocnemius muscle of these mice underwent crush injury. Twenty-four hours after the crush injury, all mice were exposed to 8 hours of hypobaria. After hypobaria, muscle was harvested. In M11, ovarian-intact mice underwent similar gastrocnemius crush injury and hypobaria protocols as in M15. In addition, a separate group of mice underwent normobaria instead of hypobaria. Muscle was harvested. In M9, ovarian-intact female mice underwent crush injury of the right gastrocnemius muscle only. No mice were exposed to hypobaria. The left gastrocnemius served as the uninjured control. Muscle was harvested.

Microarray analysis was performed using mRNA from muscle samples from M9, M11, and M15, and the results of gene upregulation and downregulation were entered into a Microsoft Excel spreadsheet. Upregulation was defined as a ≥1.5 fold change.

17 β-estradiol has a significant effect on the upregulation of four muscle related genes: Mstn, Mybph, Myh1, and Myh3. Future studies may be done to compare Myh8 and Myl6b with controlled data points since there were no comparable data points in this study. While several studies have explained the use of ER-a and ER-β receptors, as well as IGF-I receptors, the whole metabolic pathway is not understood for Mstn. In addition, Mybph, Myh1, and Myh3 have no concrete studies where the pathway is investigated. The implications for future research of this pathway is essential to understand and improve skeletal muscle recovery as well as maintaining/gaining muscle mass during menopause for females.

Figure1. A comparison of milestone groups and analysis of significant upregulation in genes of interest. IF: intact female. OE: estrogen treated. OC: placebo treated. CI: crush injury. UI: uninjured. NB: normobaric. HB: hypobaric. NS: not significant as the fold change did not pass the 1.5 threshold. ND: not done. S: Signifcant

Gene Symbol

Protein Function

Mstn Myostatin/Growth Differentiation Factor 8

Inhibits muscle differentiation and Akt-induced protein synthesis4.

Mybph Myosin-binding protein H Able to bind to and organize myosin into thick filaments5.

Myh1 Adult skeletal muscle myosin heavy chain 1

Not widely studied, associated with muscle contraction6.

Myh3 Embryonic skeletal muscle myosin heavy chain 3

Involved in cell movement and transport of materials within and between cells7.

Myh8 Perinatal skeletal muscle myosin heavy chain 8

Involved in skeletal muscle contraction8.

Myl6b Smooth muscle and non-muscle myosin light chain alkali 6B

A regulatory light chain of myosin9.

Downregulation was defined as a -1.5 or lower fold change. This study focused on upregulated genes only.

To investigate the 17β-estradiol regulation of muscle-related genes, the root word “myo,” meaning “muscle,” was entered into the search command in the M15 Excel spreadsheet. To further validate whether this effect was specific for 17β-estradiol, microarray gene data from M9 and M11 were examined to determine whether these genes’ fold change surpassed +/- 1.5 to count as a significant change.

Mstn Mybph Myh1 Myh3-1.5

-1-0.5

00.5

11.5

22.5

Up-Regulation of M15 (Estrogen Treated Mice) Genes vs.

M9 (Non-estrogen Treated Mice) Genes

M15 M9Gene of Interest

Fold

Cha

nge

Estrogen Type

Injury Type Pressure Type

32 hours

48 hours

96 hours

192 hours

IF CI v. UI NB ND NS ND NDOE v. IF CI v. CI HB NS ND NS NS

OC v. IF CI v. CI HB NS ND NS NSOE v. OC CI v. CI HB S ND NS S

Figure 2. A bar graph comparison of the log fold change in specific genes of interest. The four genes’ fold changes for M15 was measured 32 hours post crush injury. For M9, the fold changes were measured 24 hours post crush injury. The fold change for M9 is negative, therefore, these genes were downregulated. Fold change difference is measured by subtracting the fold change of M9 from M15. All M15 fold change values are significant.

In M15, ovariectomized, estrogen treated mice were compared with intact female mice and there was no significant fold change during 96 or 192 hours. In addition, placebo treated mice were compared with intact female mice, and no significant change was noted either. However, when ovariectomized, estrogen treated mice who underwent hypobaria and a crush injury were compared withovariectomized, placebo treated mice who underwent hypobaria and a crush injury, they were significantly different. Estrogen treated mice had genes of interest upregulated, however, placebo treated mice had genes of interest downregulated. Therefore, this supports the idea that 17β-estradiol has an enhancing effect on these particular genes of interest based on isolated comparison.

However, there were some genes left out of the comparison because no sufficient standard was completed to compare Myh8 and Myl6b with. Clinical Significance

This study’s purpose is to determine the effect of 17 β-estradiol on the regulation of these muscle-related genes of interest. Another goal of the experiment was to determine whether there is an existing and understood pathway that describes the relationship of 17 β-estradiol on the specific genes. In addition, this study has the potential to provide rationale for estrogen based treatments for diseases or symptoms of decreased muscle mass or force.

Acknowledgements

Methods

Figure1. Crystal structure of the myostatin: follistatin-like 3 Complex in Mus musculus.

Results

Table 1. Muscle genes of interest and transcribed protein’s functions.

Citations1. Galluzzo, P. (2009). 17β-estradiol regulates the first steps of skeletal muscle cell different via ER-α-mediated signals. American Journal of Physiology. Cell Physiology., 297, 1,12.2. Jacobsen, D. (2008). Efficacy of tibolone and raloxifene for the maintenance of skeletal muscle strength, bone mineral density, balance,… : st. Trials, 9, 1.3. Myogenesis. (n.d.). Myogenesis. Retrieved August 5, 2014, from http://neuromuscular.wustl.edu/mother/myogenesis.html4. Trendelenburg, A. U. (2009). Myostatin reduces Akt/TORC1/p70S6K signaling, inhibiting myoblast differentiation and myotube size. American Journal of Physiology. Cell Physiology., 296, 1, 10, 11.

5. MYBP gene family. (n.d.). Genetics Home Reference. Retrieved August 5, 2014, from http://ghr.nlm.nih.gov/geneFamily/mybp6. MYH1 Gene. (n.d.). GeneCards. Retrieved August 5, 2014, from http://www.genecards.org/cgi-bin/carddisp.pl?gene=MYH17. MYH3 gene. (n.d.). Genetics Home Reference. Retrieved August 5, 2014, from http://ghr.nlm.nih.gov/gene/MYH38. MYH8 gene. (n.d.). Genetics Home Reference. Retrieved August 5, 2014, from http://ghr.nlm.nih.gov/gene/MYH89. MYL6B Gene. (n.d.). GeneCards. Retrieved August 5, 2014, from http://www.genecards.org/cgi-bin/carddisp.pl?gene=MYL6B