Jacob Parmley and Robert Hyldahl, Department of exercise Science
The repair and regeneration processes of skeletal muscle rely on the activation, proliferation and differentiation of skeletal muscle stem cells (satellite cells), which are necessary sources for muscle increase (hypertrophy) and regeneration (Stewart, 2006). A muscles ability to regenerate diminishes due to age or various diseases, such as muscular dystrophy (Mariol, 2001). Interventions aimed at altering the skeletal muscle environment to optimize muscle stem cell activity and regenerative processes represents a promising approach to enhance muscle regenerative capacity. Recent data from our lab has identified increased levels of the cytokine Interferon Gamma-Inducible Protein 10 (IP-10) during the muscle regenerative period following damage in human muscle (Hyldahl, 2014). IP-10 is primarily considered to function as a chemoattractant for monocytes and T lymphocytes, playing an important role in mediating physiological inflammatory responses. Additionally, it has been suggested that IP-10 may play a distinct role in vascular remodeling as it induced smooth muscle proliferation and migration following vascular injury (Wang, 1996). Currently, no biological functions of IP-10 in the repair and regeneration processes of skeletal muscle have been reported. I collected data showing that introducing IP-10 in vitro increased both proliferation and differentiation of human primary myoblasts. With these data, we hypothesized that IP-10 plays a role in the proliferation and differentiation of muscle satellite cells in vivo. By utilizing a mouse model of IP-10 deficiency, we sought to determine how IP-10 contributes to the skeletal muscle regenerative process at various time points following a controlled injury. We hypothesized that the muscle fiber (myofiber) cross-sectional area would remain significantly lower following muscle injury in IP-10 knockout mice. The cross-sectional area of myofibers exhibiting centralized nuclei provides insight into skeletal muscle structure and the progress of repair (Lee, 2013). We also hypothesized that IP-10 knockout mice will experience significantly lower rates of satellite cell proliferation following injury. Originally, we sought to measure proliferation by injecting the mice with EdU (5-ethynyl-2’-deoxyuridine) during treatment, a compound used to label the DNA of actively proliferating cells. However, in order to maximize the tissue sample for other uses, we decided that measurement of satellite cell proliferation should be done by staining fixed tissue for the satellite cell marker Pax7 (Seale, 2000)
We obtained wild-type C57BL/10ScSnJ (WT) and knockout B6.129S4-Cxcl10tm1Adl/j (IP-10 KO) mice from Jackson Laboratories (Bar Harbor, ME) at six to eight weeks of age (n= 7 and 10 respectively). To induce muscle damage, WT and KO mice were treated with 10 micromolar (twenty-five micro-liter volume) cardiotoxin, which was injected into the tibialis anterior (TA) muscle. Only one TA muscle of each mouse was injected with cardiotoxin. The other TA muscle served as a control and was injected with saline. At 2, 7 and 14 days post injury (DPI), the TA muscles were excised, weighed, frozen, cut by cross section and mounted on glass slides for histological studies. Mounted muscle samples were stained using hematoxylin and eosin to identify central nucleated fibers and quantify cross sectional area as they are an indicator for muscle cell regeneration (Lee et al., 2013). Mounted muscle samples were also immunohistochemically stained using an antibody for Pax7 to measure satellite cell quantity.
Compared to the control TA, the CTX-injected TA muscle mass was significantly reduced (p<0.05) at 7 DPI (WT=863%, KO=919%) compared to 14 DPI (WT=1058.3%, KO= 10511%). No significant differences in muscle mass were found between genotypes. Cross sectional area and central nucleation of regenerating myofibers was greater at 14 DPI compared to 7 (p<0.05). However, there were no significant differences found between genotypes. I am currently still analyzing the tissue for number of Pax7 positive cells per cross section at each time point. I anticipate completing the analysis by February at the latest.
From the data we collected, we conclude that IP-10 is not necessary for normal muscle regeneration from a toxin-induced muscle injury in mice. However, this does not rule out the possibility that IP-10 could be used as an intervention to increase muscle cell regeneration. I am still optimistic that this could be the case given the data we collected previously in an in vitro study. To test this hypothesis, we would need to develop a way to introduce excess IP-10 in vivo. Furthermore, our gathering of data was hindered due to the need to modify protocols, re-stock our available antibodies, and completing another project alongside this one.
Though we did not see the results we had expected, I am still optimistic about the potential for IP-10 as an intervention for muscle cell regeneration because of the findings from our previous in vitro experiment. Regardless of the outcome, this experiment helped me to gain various skills and insight into how muscles regenerate and into the world of research. While concurrently working on this project, I was asked to contribute to another study because of my acquired skill at cell culture and analysis. This was a major contributing factor to the reason why we have not finished the Pax7 analysis yet. That study is currently in review with the Journal of Clinical & Experimental Pharmacology. It has received good initial reviews and will likely be accepted in the next month (Sorensen et al., 2017).
Hyldahl et al. (2014). Satellite cell activity is differentially affected by contraction mode in human muscle following a work-matched bout of exercise. Frontiers in Physiology, 5:1-11.
Lee et al. (2013). Aged skeletal muscle retains the ability to fully regenerate functional architecture. Bioacrchitecture, 3(2):25-37
Seale et al. (2000). Pax7 Is Required for the Specification of Myogenic Satellite Cells. Cell, 102(6):777-786
Sorensen et al. (2017). Preclinical characterization of the JAK/STAT inhibitor SGI-1252 on skeletal muscle function, morphology, and satellite cell content. Journal of Clinical &; Experimental Pharmacology, in review.
Stewart et al. (2006). Adaptive processes in skeletal muscle: molecular regulators and genetic influences. Journal of Musculoskeletal &; Neuronal Interactions, 6(1):72-86
Wang et al. (1996). Interferon-inducible protein 10 involves vascular smooth muscle cell migration, proliferation, and inflammatory response. The Journal of Biological Chemistry, 271(39):24286-24293