Skip to main content
2024 BMES Annual Meeting Main banner
Icon Legend
This session is not in your schedule.
This session is in your schedule. Click again to remove it.
Back

Drug Delivery

Drug Delivery - Poster Session E

Poster J20 - The Impact of Localized Retinoic Acid Drug Delivery on Markers of Protein Synthesis in Muscle Recovering From Atrophy

Saturday, October 26, 2024
10:00 AM – 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Muscle atrophy is a phenomenon that occurs from disuse, aging, or illness and leads to lower strength and increased mortality and is characterized by a decrease in fiber cross-sectional area (CSA). The decrease in myofiber CSA is closely correlated with a decrease in protein synthesis. Current treatment involves dietary changes and exercise, often requiring months to years for recovery. A pharmacotherapy has yet to be developed for muscle atrophy, though an effective therapy is needed to aid in the muscle recovery process. All-trans retinoic acid (ATRA) is an active form of vitamin A that has been researched as an agent for regenerative medicine and is established to influence muscle regeneration. However, ATRA is sensitive to light, heat, and oxygen leading to degradation and encapsulating ATRA in poly(lactide-co-glycolide) (PLG) microparticles has been shown to maintain its stability and potency.
It has been previously shown that muscle atrophy in the mouse hindlimb casting model closely resembles atrophy due to disuse seen in humans. It is hypothesized that the injection of ATRA microparticles following hindlimb casting will increase the cross-sectional area of myofibers via activation of the AKT-4eBP1-mTOR pathway. The AKT-4eBP1-mTOR pathway is a crucial signaling pathway that promotes protein synthesis and myofiber growth by regulating the activity of mechanistic target of rapamycin (mTOR).

Materials and

Methods:  Male C57BL6 mice (16-weeks of age) were casted with both ankles fixed in plantar flexion for 10 days to induce atrophy of the gastrocnemius and soleus muscles. Following casting, mice were randomly assigned to one of four treatment groups: 90 ng, 30 ng, or 9 ng of ATRA or saline. Mice recovered for 3 or 7 days before euthanasia. Immediately following euthanasia, the left hindlimb muscles were embedded in OCT for histology and the right hindlimb muscles were flash frozen for other measurements.
Immunofluorescence microscopy for laminin was used to visualize and quantify fiber cross sectional area. The total protein concentration within mouse gastrocnemius homogenate was determined using a Bicinchoninic (BCA) assay. Expression of indexes of protein synthesis was examined by Western blot analysis. Equal amounts of total protein were run on SDS-PAGE gels and transferred onto nitrocellulose membrane. Antibodies derived from rabbits were used to detect p-AKT, AKT, p-4eBP1, 4eBP1, p-mTOR, and mTOR proteins. Goat anti-rabbit secondary antibodies were used with chemiluminescence to visualize the protein bands. The resulting bands on the membrane represent the presence and relative abundance of the target protein within the original sample.

Results, Conclusions, and Discussions: The casting model was successful in inducing atrophy indicated by the cross-sectional area (Figure 1). Additionally, the CSA of myofibers increased after treatment with 90 ng of ATRA particles. Western blot analysis showed no difference in the activation of indexes of protein synthesis (p-AKT/AKT, p-4eBP1/4eBP1, and p-mTOR/mTOR) at both the 3-day and 7-day timepoints (Figure 2). Future studies will focus on determining if protein synthesis is occurring at an earlier time point (e.g., 1 day recovered) and assess levels of growth factors (and atrophy factors) over the course of recovery (i.e., 1, 3, and 7 days).

Acknowledgements (Optional):