Seth Hampton and Dr. Robert Hyldahl, Exercise Science
Fibroblasts play a key role in repairing injured tissue by secreting collagen and growth factors into the tissue. In states of disease and overuse, fibroblast activity can become overly abundant and can lead to skeletal muscle fibrosis, decreasing strength and muscle elasticity. This fibrotic condition can lead to muscle wasting disorders, such as muscular dystrophies, that severely decrease the length and quality of life. Previous studies have shown that the TGF-beta signaling family directly increases fibroblast proliferation. It has also been shown that inhibition of myostatin, a member of the TGF-beta family, reduces symptoms of muscular dystrophies and decreases fibrosis. Previous research suggested that a novel small molecule, SGI-1252 (SGI), could serve as a pharmacological inhibitor of myostatin. The purpose of this project was to determine how treatment of fibroblasts with SGI affects proliferation. We hypothesized that SGI would decrease fibroblast proliferation, while myostatin would increase fibroblast proliferation.
Fibroblasts were isolated from mice tissue that had been minced and incubated in culture medium for three hours. During incubation fibroblasts adhered to the plate and non-adherent, non-fibroblastic cells were discarded. Immunohistochemical techniques were exercised to verify that only fibroblast cells were present in the plate culture. Fibroblast cells were grown in culture and divided into four separate treatment groups: control cells with solubilization agent, cells treated with SGI only, cells treated with myostatin only, and cells treated with both myostatin and SGI. Cells treated with SGI were incubated at four different concentrations: 100nM, 500nM, 1uM and 10uM.
Fibroblast proliferation was measured using Edu staining procedures. This staining process fluorescently tags newly transcribed DNA, highlighting all new nuclei that proliferate during the incubation time. The Edu staining agents were added to each treatment group of cells at the beginning time point, and the cells were incubated and collected at varying time points: 3 hours, 12 hours, and 24 hours. Cells were then analyzed under microscope, and total nuclei and proliferated nuclei were counted and compared for each treatment group.
As expected, the control group showed no fibroblast proliferation at any time point. Cells incubated with myostatin showed a small increase in proliferation, averaging .07 proliferated nuclei per total nuclei (PN) after the 24-hour treatment. The SGI treatment group experienced greater proliferation than the myostatin group, averaging a PN of .144 for the 12-hour treatment, and ,109 for the 24-hour treatment. The greatest proliferation was observed in the 24-hour combined SGI and myostatin treatment. This group observed an average PN of .208, with the highest proliferation occurring in SGI concentration groups of 100nM and 500nM, recording a PN of .253 and .270 respectively.
The results of our study present data directly opposite to our hypothesis. Cells treated with SGI increased fibroblast proliferation rather than decreasing proliferation as we hypothesized. Cells treated with myostatin did increase fibroblast proliferation from basal levels as predicted, but proliferation was minimal when compared to SGI treated cells. It is of interest to note that proliferation in the SGI group generally decreased from the 12 to 24 hour time points, from .144 PN to .109 PN respectively. Total number of nuclei also declined in a similar fashion from the 12 to 24 hour time point, falling from an average of 1886 nuclei to an average of 1341 nuclei. From these observations it is possible to conclude that in the SGI treatment, the drug begins to exercise negative effects on the cells at a time point after 12 hours which begins to inhibit further fibroblast proliferation and even damage present cells.
It is most interesting that the combined treatment group, SGI and myostatin, produced the greatest proliferation of fibroblasts, especially at the 24-hour time point. Considering the discussion presented above, it may be possible that myostatin counteracts the destructive effects of the SGI, allowing the fibroblast cells to continually benefit from the positive interaction with SGI, increasing proliferation through 24 hours. As opposed to the SGI only group, cells treated with myostatin and SGI experienced no change in total nuclei between the 12-hour and 24-hour treatments (12hr: 1472 nuclei. 24hr: 1455 nuclei). This further suggests that myostatin counteracted possible negative effects produced by the SGI beyond the 12-hour time point, providing a maintenance of fibroblast cells and allowing for increased fibroblast proliferation at 24 hours.
Using the findings of this study, along with the results of other work performed in our lab, we have discovered that SGI-1252 is less impactful as a TGF-beta pathway inhibitor and works more effectively as an inhibitor of Jak/STAT signaling. Further research will need to be conducted to determine the effectiveness of this drug as a Jak/STAT inhibitor and to understand the implications involved with this new finding.