Carol A. Morales, Elena Kudryavtseva, and Gordon Huggins Department of MCRI and Tufts University, Boston, MA.

Outline Alcoholism Background  Focus of Project Muscle Fiber Development Background Rapamycin  Project Design  Results!!!  Summary  Conclusion  Acknowledgements

Alcoholism Alcoholism is a very common disease in the United States. Alcohol has many toxic affects on the body which include: − Central Nervous System Damage − Cardiovascular Disease − Etc….. There are two major arguments: 1) Alcoholics tend to have weakened muscles and are more susceptible to muscle damage, due to alcohols direct and toxic affect on muscle cell morphology. 2) Alcoholics poor sense of nutrition is the main reason for weakened muscles and susceptibility to muscle damage.

Focus of Project The focus of this project is to test the argument that alcohol damages the ability of muscle precursor cells, satellite cells or Myoblasts, to differentiate into mature Myotubes. Myotubes are formed from Myoblasts. Myotubes are important in muscle fiber development. Lets take one step back and look at the importance of the parts that make up a muscle.

Myoblasts  Satellite cells are tissue committed stem cells.  Myoblasts are slightly further differentiated than satellite cells. When satellite cells become activated for cell repair or “Growth,” this is the point where they are labeled Myoblasts.  For this project the mouse myoblast cell line, C2C12, will be grown in culture and subjected to treatments.  The mouse precursor muscle cell line grows and differentiates almost exactly the same as the human precursor muscle cells do.

Myotubes  Myotubes form to make Myofibers.  Myofibers assemble to make muscle fiber. Picture by: Wikipedia

Myotubes  Myotubes are surrounded by Myoblasts, which are available to the muscle for further differentiation.  These Myoblasts or satellite cells are stem cells that are tissue committed and can be further differentiated to Myoblasts and then to Myotubes if the muscle fiber needs repair.

Rapamycin  mTOR, a protein kinase, is genetically linked to the predetermination of cell size and shape.  Rapamycin, an inhibitor of mTOR, changes the cells morphology and will be used as the experiments positive control.

Project Design  Hypothesis: Myotubes formed by cells treated with alcohol will be thinner, more elongated and contain fewer nuclei when compared to cells that have not been subjected to alcohol.  Methods: The mouse C2C12 cell line was grown in cell culture in 20% Fetal Bovine Serum until cells became confluent. The media was then switched to 2% Horse Serum, the change in serum concentration caused the cells to differentiate. Once the cells switched to the 2% Horse Serum they began their treatment.

Project Design Continued  Methods Cont’d: The cells were treated according to the following: − 1 st group treated with only 2% Horse Serum. − 2 nd group treated with 0.5% alcohol in 2% Horse Serum. − 3 rd group treated with 100nM rapamycin in 2% Horse Serum. − 4 th group treated with both 0.5% alcohol and 100nM rapamycin in 2% Horse Serum. ― The cells were grown under these conditions for approximately 7 to 10 days.

Project Design Continued  Methods Cont’d: ― Immunohistochemistry was used to stain both the cytoplasm and nucleus of the cells: − myoD, mostly found in the nuclei of cells, is responsible for the regulation of cells transitioning to myotubes from myoblast. (rabbit antibody) − α sarcomeric actin is specific for proteins that are found in the cytoplasm of the cell. (mouse antibody) Control Cell msm7 5d-G 6d-D Alcohol Cell msm7 5d-G 6d-D

Project Design Continued Methods Cont’d: Measurements of Cells after staining: − The program used for measurement was Image Pro Plus: − Approximately 10 measurements for number of nuclei, length and width were obtained for each cell. Measurements were compiled in Excel and then a Statistical program was used to obtain a p value for results. ○ A p value of less than 0.05 simply states that the compared data differ significantly, more so than would be left to chance. Control Cell msm7 5d-G 6d-D with measurement lines.

Results!!! Control C2C12 Cells 7 Day Differentiation Alcohol C2C12 Cells 7 Day Differentiation These are examples of some pictures, taken with a microscope, of a control plate and an alcohol treated plate. Visually these results show there is a drastic affect on the width of the Myotubes caused by alcohol. Control Cells: Experiment 3 C2C12 7 Day Treatment Alcohol Cells: Experiment 3 C2C12 7 Day Treatment

Results Continued  Since the cell line showed such a drastic difference in the width of the cell, live mice were then euthanized for extraction of Myoblasts. The width of the Myotubes formed proved to be statistically significant, while the length and number of nuclei did not.  These cells, called primary cells, were treated in the exact same manner as the cell line. Control msm8 Cells 2 Day Differentiation Alcohol msm8 Cells 2 Day Differentiation

Results Continued Experiment 3: 7day- Differentiation Control Cell 1 Measurement Value (um) 1 Width15 2 Width15 3 Width16 4 Width18 5 Width14 6 Width12 7 Width16 8 Width8 9 Width10 10 Width12 Average13.6 Standard Deviation3.06 This is an example of how the measured widths for one control cell and one alcohol cell were tabulated. About 240 cells were measured over the course of the project. Control Cell Alcohol Cell Experiment 3: 7day- Differentiation Alcohol Cell 1 Measurement Value (um) 1 Width5 2 Width6 3 Width6 4 Width6 5 Width7 6 Width5 7 Width6 8 Width7 9 Width3 10 Width3 Average5.33 Standard Deviation 1.37

Results Continued  The average width was then compiled and put into the statistical program. Cell LineTreatmentSample SizeAvgStdevP-value C2C12 (Exp 2)Control20 Cells <0.001 C2C12 (Exp 2)Alcohol20 Cells Primary (msm8)Control20 Cells Primary (msm8)Alcohol20 Cells

Summary The difference between control and alcohol, in both C2C12 and primary cells, proved to be statistically significant. C2C12 cells illustrated more drastically the effect of alcohol, on myotube formation, than primary cells. The difference in the number of nuclei and length of cells treated with alcohol proved to be insignificant.

Conclusion  Alcohol directly impairs muscle precursor cell differentiation.

Acknowledgements Dr. Gordon Huggins Dr. Elena Kudryavtseva Dr. Huggins Lab The Sackler Summer Research Program NIH Grant GMO