1. L-Arginine Remediation of Stressed C2C12 Cells Warren Austin Pittsburgh Central Catholic High School Grade 12 3 rd Year in PJAS

2. Tissue Engineering Developing and manipulating Artificial implants, laboratory grown tissues, and genetically engineered cells and/or molecules Replace or support the function of defective or injured body parts as a result of hereditary conditions, disease, trauma, and aging The potential for supplementing muscle tissue

3. Stem Cells Unspecialized cells capable of self-renewing through cell division Can be induced to become tissue- or organ specific cells with special functions. Offer new potentials for treating diseases such as diabetes and heart disease

4. C2C12 Cells In 1977, discovered through experimentation of murine thigh muscle growth Subclone of post-natal mouse myoblastic stem cell line Immortalized and a frequent model used in T.E. experiments Rapid differentiation Produces extending myotubes and characteristic muscle proteins Allows a study of differentiation of non-muscle to skeletal muscle cells

5. L-Arginine One of the 20 most common natural amino acids Produced by the body Commonly sold at supplement stores in liquid and powder forms Precursor for the synthesis of nitric oxide Can be used to heal wounds, build muscle, and increase vascular circulation

6. Past Study with L-Arginine L-arginine: proposed as a pharmacological treatment to attenuate the dystrophic pattern of DMD Despite little understanding about the mechanism of signaling events Could decrease inflammation and able to enhance muscle regeneration in mdx mouse Inhibits the activity of MMP-9, which is activated by NFκB Promotes muscle membrane integrity in dystrophic muscle NFκB-related signaling cascades suggest therapeutic approaches Inhibition of MMP-9 activity drastically reduced deterioration, inflammation, cell death, and fibrosis (scarring) Improvement of skeletal muscle regeneration and contractile functions in mdx mice

7. Stress Disrupts regular cellular processes and reduces survivorship mechanical (violent shaking) chemical (toxins) oxidative (UV, X-ray) ischemic (lack of oxygen)

8. Oxidative Stress H2O2 stress Two electron reduction state Formed by dismutation of O 2 - or direct reduction of O 2 Lipid soluble Can be caused through overproduction of oxygen during physical exercise Increases oxidant production in cells Free radical accumulation can lead to compounding stress Results in cellular degeneration May cause direct cell death or induce cancer High glucose levels induce an increase in enzymatic activity leading to the synthesis of amino acids

9. Purpose To determine the remediation effects of L-arginine on the proliferation, differentiation, and survivorship of stressed C2C12 cells

10. Hypotheses Null Hypothesis: L-arginine will not significantly effect proliferation and differentiation of H2O2 stressed C2C12 stem cells Alternative Hypothesis: L-arginine will significantly increase the proliferation and differentiation of H2O2 stressed C2C12 stem cells

11. Materials Cryotank 75mm2 tissue culture treated flask 25 mm2 tissue culture treated flasks Fetal bovine serum (FBS) C2C12 myoblastic cell line Trypsin-EDTA Pen/strep Macropipette + sterile macropipette tips (1 mL, 5 mL, 10, mL, 20 mL) Micropipettes + sterile tips DMEM Media - 1% and Complete Media (4 mM L-glutamine, 4500 mg/L glucose, 1 mM sodium pyruvate, and 1500 mg/L sodium bicarbonate + [ 10% fetal bovine serum for complete]) 75 mL culture flask L-arginine

12. Materials (Continued) H2O2 Incubator Nikon Inverted Microscope with imaging technology Laminar Flow Hood Laminar Flow Hood UV Sterilizing Lamp Sterilizing Lamp Sharpie pen Hemacytometer Sterile PBS Ethanol (70%) Sterile Water Purple Nitrile gloves

13. Procedure (Cell Line Culturing) A 1 mL aliquot of C2C12 cells from a Cryotank was used to inoculate 30 mL of 10% serum DMEM media in two 75mm2 culture flasks yielding a cell density of approximately 10^6 to 2x10^6 cells/mL. The media was replaced with 15 mL of fresh media to remove cryo-freezing fluid and incubated (37° C, 5% CO2) for 2 days until a cell density of approximately 4x10^6 to 5x10^6 cells/mL was reached. The culture was passed into 7 flasks in preparation for the experiment and incubated for 2 days at 37°C, 5% CO2

14. Procedure: Proliferation-Addition of the Variable After trypsinization, cells from all of the flasks were pooled into 1 common 75mm2 flask (cell density of approximately 1 million cells/mL). 27 T25 were prepared with 5 mL of 10% DMEM media 0.1 mL of cell suspension was then transferred to the 27 T25 flasks, thus, a cell density of 10^5 cells/flasks was created Flasks were placed back into incubator and cells were allowed to attach for several hours 1 mM stock concentration of hydrogen peroxide created by adding 0.1 ml of peroxide to 8.7 ml of sterile water. T25 flasks were removed from incubator and L-Arginine and H2O2 was added to reach the following desired concentrations (3 T25 flasks for each of the concentrations, 2 for proliferation and 1 for differentiation) The cells were placed back into the incubation state for the remainder of the study

15. Concentrations of Variables-Proliferation Experiment H 2 O 2 Zero (0 µM)H 2 O 2 Low (1 µm)H 2 O 2 High (10 µM) L-Arginine Zero (0%=0X) Media= 5 mL L-Arginine= 0 mL H 2 O 2 = 0 mL Total Volume= 5mL Media= 4.995 mL L-Arginine= 0 mL H 2 O 2 = 0.005 mL Total Volume= 5 mL Media= 4.95 mL L-Arginine= 0 mL H 2 O 2 = 0.05 mL Total Volume= 5 mL L-Arginine Low (0.06%=0.1X) Media= 4.997 mL L-Arginine= 0.003 mL H 2 O 2 = 0 mL Total Volume= 5 mL Media= 4.992 mL L-Arginine=0.003 mL H 2 O 2 =0.005 mL Total Volume= 5 mL Media= 4.947 mL L-Arginine= 0.003 mL H 2 O 2 = 0.05 mL Total Volume= 5 mL L-Arginine High (0.6%=X) Media= 4.97 mL L-Arginine=0.03 mL H 2 O 2 = 0 mL Total Volume= 5 mL Media= 4.965 mL L-Arginine= 0.03 mL H 2 O 2 = 0.005 mL Total Volume= 5 mL Media= 4.92 mL L-Arginine= 0.03 mL H 2 O 2 = 0.05 mL Total Volume= 5 mL A normal dosage of L-Arginine was modeled by assuming 30 mL dissolved in 5 L of blood. That working concentration is X=0.6%

16. Procedure: Day 1 and 3 of Proliferation Experiment Using one flask from each group, cell densities were determined as follows: The cells were trypsinized and collected into cell suspension. 20 µl aliquots were transferred to a Hemacytometer for quantification (four counts per flask). Day 1 and Day 3 The previous procedure for determining densities was used again, and a Nikon Inverted Microscope was used to take images of representative areas of each flask.

17. Procedure: Differentiation Day 1 of imaging The original media was removed and replaced with 1% DMEM media (serum starvation) to induce myotube differentiation. Day 1, 4, 6 of imaging Using the Nikon Inverted Microscope, images of representative areas of each of the groups were taken.

18. Statistical Analyses Used for Interpretation of the Proliferation Experiment ANOVA Compares variation within groups to variation between groups Using the ANOVA, if a p-value less than the alpha of 0.05 is generated (significant variation), it suggests that the null hypothesis can be rejected Dunnett’s Test Compares each experimental group to control individually 0.05 alpha was used, and each generated T-value was compared to the T- critical value of 2.67

19. The Effects of L-Arginine on Stressed C2C12 Cells Day 1 Synergistic P-Value=2.21E-03 P-value=6.07E-04 P-value=4.59E-02

20. The Effects of L-Arginine on Stressed C2C12 Cells Day 3 Synergistic P-Value=1.10E-03 P-value=1.07E-03 P-value=5.09E-08

21. Results of the ANOVA Statistical Test for the Proliferation Experiment Day 1 QuestionP-Value Does hydrogen peroxide at the highest concentration affect C2C12 cells? 3.75E-02 Significant Did L-Arginine affect unstressed C2C12 cells?4.59E-02 Significant Did L-Arginine at the highest concentration affect stressed C2C12 cells? 6.60E-04 Significant

22. Results of the ANOVA Statistical Test for the Proliferation Experiment Day 3 QuestionP-Value Does hydrogen peroxide at the highest concentration affect C2C12 cells? 3.91E-02 Significant Did L-Arginine affect unstressed C2C12 cells? 5.09E-08 Significant Did L-Arginine at the highest concentration affect stressed C2C12 cells? 4.77E-06 Significant

23. Results from Differentiation Experiment: Day 1 1.L-Arginine 0%, H 2 O 2 0 µm 2.L-Arginine 0%, H 2 O 2 10 µm 3.L-Arginine 0.6%, H 2 O 2 0 µm 4.L-Arginine 0.6%, H 2 O 2 10 µm 5.L-Arginine 0.6%, H 2 O 2 1 µm

24. Results from Differentiation Experiment: Day 4 1.L-Arginine 0%, H 2 O 2 0 µm 2.L-Arginine 0%, H 2 O 2 10 µm 3.L-Arginine 0.6%, H 2 O 2 0 µm 4.L-Arginine 0.6%, H 2 O 2 10 µm 5.L-Arginine 0.6%, H 2 O 2 1 µm

25. Results from Differentiation Experiment: Day 6 1.L-Arginine 0%, H 2 O 2 0 µm 2.L-Arginine 0%, H 2 O 2 10 µm 3.L-Arginine 0.6%, H 2 O 2 0 µm 4.L-Arginine 0.6%, H 2 O 2 10 µm 5.L-Arginine 0.6%, H 2 O 2 1 µm

26. Conclusions L-arginine does significantly affect C2C12 myoblastic stem cell proliferation H2O2 does significantly affect C2C12 myoblastic cell proliferation L-arginine does significantly affect the survival of H2O2 stressed C2C12 cells Reject the null hypothesis

27. Limitations Variation in counts using hemacytometer Cell Clumping Technique Errors Number of flask Lag time More varying concentrations LD50 for hydrogen peroxide Use of other stem cell lines, such as 3T3 or MG63 CyQUANT™ Cell Proliferation Assay, which gives better quantitative data and uses a fluorescent dye that bonds to nucleic acid in cell Future Extensions to the Experiment

28. Sources of Information http://www.netdoctor.co.uk/focus/nutrition/facts/oxidative_stress/oxidativestress.htm www.PTEI.org www.tissue-engineering.net http://www.webmd.com/vitaminssupplements/ingredientmono-875- LARGININE.aspx?activeIngredientId=875&activeIngredientName=L-ARGININE http://www.webmd.com/vitaminssupplements/ingredientmono-875- LARGININE.aspx?activeIngredientId=875&activeIngredientName=L-ARGININE https://www.landesbioscience.com/article/9338/full_text_xml/ "Cell Biology Lab- Tissue Culture C2C12 Cells." Cell Biology Lab- Tissue Culture C2C12 Cells. Burattini, S., P. Ferri, M. Battistelli, R. Curci, F. Luchetti, and E. Falcieri. "C2C12 Murine Myoblasts as a Model of Skeletal Muscle Development: Morpho-functional Characterization." Ejh.it. European Journal of Histochemistry

29. Acknowledgements Dr. Phil Campbell Conrad M. Zapanta, Ph.D. Biomedical Engineering Laboratory, Carnegie Mellon University Mr. Mark Krotec, PTEI

30. ANOVA Data: Day 1 Peroxide 0 micromolarPeroxide 1 micromolarPeroxide 10 micromolar Arginine 0.0%554306254 538447294 464453394 401413212 379335288 409510301 341462236 549313327 Arginine 0.06%479334303 440339425 389516398 498584330 463480460 471352365 499387239 457446256 Arginine 0.6%401590388 594542308 588434366 535407327 535476515 557455340 488496410 557373393

31. ANOVA Data: Day 3 Peroxide 0 micromolarPeroxide 1 micromolarPeroxide 10 micromolar Arginine 0.0% 492410330 463547370 464420427 490556439 450473410 409420370 578456330 512413311 Arginine 0.06% 480322374 560437493 450513472 570459421 570640310 500590450 530460467 500480499 Arginine 0.6% 614477370 707508560 716408472 807417510 698533362 650670394 676598500 752420482

32. The Effects of Hydrogen Peroxide on C2C12 Cells Day 1 Synergistic P-Value=2.21E-03 P-value=4.59E-02 P-value=6.07E-04

33. The Effects of Hydrogen Peroxide on C2C12 Cells Day 3 Synergistic P-Value=1.10E-03 P-value=5.09E-08 P-value=1.07E-03

34. Results of the Dunnett’s Test The Effects of L-Arginine (Day 1) Peroxide (0 µm) T-Value Peroxide (1 µm) T-Value Peroxide (10 µm) T-Value L-Arginine (0.0%) to L-Arginine (0.06%) =0.2388 Insignificant L-Arginine (0.0%) to L-Arginine (0.06%) =0.6181 Insignificant L-Arginine (0.0%) to L-Arginine (0.06%) =1.7407 Insignificant L-Arginine (0.0%) to L-Arginine (0.6%) =2.4274 Insignificant L-Arginine (0.0%) to L-Arginine (0.6%) =1.6586 Insignificant L-Arginine (0.0%) to L-Arginine (0.6%) =2.7444 Significant The Effects of L-Arginine (Day 3) Peroxide (0 µm) T-Value Peroxide (1 µm) T-Value Peroxide (10 µm) T-Value L-Arginine (0.0%) to L-Arginine (0.06%) =1.4594 Insignificant L-Arginine (0.0%) to L-Arginine (0.06%) =0.6040 Insignificant L-Arginine (0.0%) to L-Arginine (0.06%) =1.9904 Insignificant L-Arginine (0.0%) to L-Arginine (0.6%) =8.5149 Significant L-Arginine (0.0%) to L-Arginine (0.6%) =0.9852 Insignificant L-Arginine (0.0%) to L-Arginine (0.6%) =2.6446 Significant T-Crit = 2.61

35. Results of the Dunnett’s Test The Effects of Hydrogen Peroxide (Day 1) L-Arginine 0.0% T-Value L-Arginine 0.0% T-Value L-Arginine 0.0% T-Value Peroxide (0 Micromolar) to Peroxide (1 micromolar) =1.3462 Insignificant Peroxide (0 Micromolar) to Peroxide (1 micromolar) =0.8827 Insignificant Peroxide (0 Micromolar) to Peroxide (1 micromolar) =1.8239 Insignificant Peroxide (0 Micromolar) to Peroxide (10 micromolar) =4.5178 Significant Peroxide (0 Micromolar) to Peroxide (10 micromolar) =3.1477 Significant Peroxide (0 Micromolar) to Peroxide (10 micromolar) =4.571 Significant The Effects of Hydrogen Peroxide (Day 3) L-Arginine 0.0% T-Value L-Arginine 0.0% T-Value L-Arginine 0.0% T-Value Peroxide (0 Micromolar) to Peroxide (1 micromolar) =0.7729 Insignificant Peroxide (0 Micromolar) to Peroxide (1 micromolar) =0.8963 Insignificant Peroxide (0 Micromolar) to Peroxide (1 micromolar) =5.1827 Insignificant Peroxide (0 Micromolar) to Peroxide (10 micromolar) =4.1303 Significant Peroxide (0 Micromolar) to (10 micromolar) =2.3325 Insignificant Peroxide (0 Micromolar) to Peroxide (10 micromolar) =6.4235 Significant T-Crit = 2.61