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NOTCH INHIBITION AFFECTS WNT AND PROTEIN SYNTHESIS IN EXERCISED SKELETAL MUSCLE

Abstract

Carl Williams, FACSM. University of North Carolina at Charlotte, Charlotte, NC.

PURPOSE. Notch, Wnt, and mTOR affect the repair of injured skeletal muscle. Notch is important for the early stages of muscle repair while mechanistic target of rapamycin (mTOR) and Wnt affect muscle differentiation. Notch’s role in protein synthesis during muscle repair is not well studied. In addition, little is known if Notch, mTOR, and Wnt signaling interact during myogenesis and protein synthesis. The purpose of this study is to determine the effect of Notch inhibition on protein synthesis, and markers of mTOR and Wnt signaling following exercise in mice. METHODS. Young (2-4 months) male C57BL/6 mice were injected with 100,000 TUs of shRNA Notch1 inhibitor into the left gastrocnemius (Control vector in right) for five consecutive days. Mice were exposed to an injurious bout of downhill running. Mice were euthanized at 1D, 2D, 3D, and 4D post-exercise and gastrocnemius were collected. For immunofluorescence, tissues were sectioned, and labelled for Green Fluorescent Protein (GFP). For western blots, tissues were homogenized and markers of protein synthesis, mTOR and Wnt signaling were measured (mTOR (pmTORSer 2448, Total mTOR, phospho- and total-4eBP1, phospho- and total-p70S6k1) and Wnt signaling (phopho- and total-Gsk3BetaSer9). The SUnSET method and puromycin incorporation were used to measure protein synthesis. One-way analysis of variance (ANOVA) tests were performed to determine differences between experimental groups and post-hoc comparisons were accomplished via a Tukey’s test, with statistical significance set apriori at p < 0.05. RESULTS. There was increased muscle protein synthesis in the vector leg relative to the control leg (p<0.05). Markers of mTOR were also increased in the shRNA leg relative to control (p < 0.05), and Wnt signaling increased in shRNA leg at 4 days post exercise relative to empty vector. Relative to control (p = CONCLUSION. Notch inhibits protein synthesis, mTOR, and Wnt signaling suggesting that Notch needs to be turned off in order for muscle repair to complete. Supported by UNC Charlotte’s Faculty Research Grant to STA.

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