Carol A. Morales, Elena Kudryavtseva, and Gordon Huggins
Department of MCRI and Tufts University, Boston, MA.
Outline
• Alcoholism
 Background
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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:
−1st group treated with only 2% Horse Serum.
−2nd group treated with 0.5% alcohol in 2% Horse Serum.
−3rd group treated with 100nM rapamycin in 2% Horse Serum.
−4th 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:
Control Cell msm7 5d-G 6d-D with measurement lines.
−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.
Results!!!
Control Cells:
Experiment 3
C2C12
7 Day Treatment
Alcohol Cells:
Experiment 3
C2C12
7 Day Treatment
Control C2C12 Cells 7 Day Alcohol C2C12 Cells 7 Day
Differentiation
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.
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
Alcohol Cell 1
Experiment 3:
7day- Differentiation
Control Cell 1
Measurement
1 Width
2 Width
3 Width
4 Width
5 Width
6 Width
7 Width
8 Width
9 Width
10 Width
Average
Standard
Deviation
Value (um)
15
15
16
18
14
12
16
8
10
12
13.6
3.06
Control
Cell
Alcohol
Cell
Measurement
1 Width
2 Width
3 Width
4 Width
5 Width
6 Width
7 Width
8 Width
9 Width
10 Width
Average
Standard
Deviation
Value (um)
5
6
6
6
7
5
6
7
3
3
5.33
1.37
•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.
Results Continued

The average width was then compiled and put
into the statistical program.
Cell Line
Treatment
Sample Size
Avg
Stdev
C2C12 (Exp 2)
Control
20 Cells
14.64
3.73
C2C12 (Exp 2)
Alcohol
20 Cells
7.27
1.91
Primary (msm8)
Control
20 Cells
10.33
3.01
Primary (msm8)
Alcohol
20 Cells
9.30
2.66
P-value
<0.001
0.031
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 GMO7667-30