(B-58) Impact of Senescence Signaling During Aging on Musculoskeletal Function and Morphology

Introduction:: Aging is an inevitable process that increases susceptibility to certain diseases including cardiovascular and musculoskeletal disorders. Aging also causes an increase in cellular senescence, mediated by cyclin dependent kinases CDKN1a/p21 and CDKN2a/p16, and causing cells to exit from the cell cycle, lose the capability to proliferate, and secrete multiple inflammatory cytokines. Tissue repair processes also decrease during both aging and disuse potentially due to stem cell exhaustion and activation of the p53/p21 pathway thereby accelerating aging. Interestingly, several studies have shown that the elimination of CDKN1a/p21 gives rise to limited regenerative abilities to mammals during aging.

The musculoskeletal system can adapt to changes in the mechanical environment and depends on external cues to determine what is required for the body to be able to survive in a particular situation. Skeletal muscles are regenerated by stem cells, known as satellite cells, which decline with aging. The regenerative function of satellite stem cells is negatively affected by oxidative stress and mitochondrial dysfunction, likely contributing to the decline in muscle force, velocity, and power generating ability with age. Fiber types also change with disuse and age. This research will provide insight into new molecular mechanisms that may be targets for potential therapeutics to assist multiple populations, including those suffering from degenerative diseases, chronic inflammatory conditions, and the aging population.


Materials and Methods:: In this project, CDKN1a/p21 knockout (KO) and B6129S2 wildtype (WT) mice were euthanized at 18-weeks, 12-months, 18-months, and 24-months old. Soleus, gastrocnemius, and extensor digitorum longus (EDL) muscles were isolated and weighed post-euthanasia to determine the extent of muscle loss during aging. Muscles were flash frozen for RNA sequencing or PFA fixed for histology. The expression of p21 and related signaling pathways were analyzed using RNAseq. H&E staining was also performed to determine tissue morphology while immunohistochemistry was used for fiber typing. The right side of the 24-month-old KO and WT mice was also used for mechanical testing. Individual fibers isolated from soleus and EDL muscles were tested for their ability to generate force and power using sinusoidal analysis within a month of isolation. Membranes were removed from the fibers, they were t-clipped on either side, mounted on a mechanical testing apparatus, and activated with calcium. After mechanical testing, the muscle fiber myosin heavy chain isoforms were identified using SDS-page gel and silver staining. Statistical analysis will be calculated between WT and CDKN1a/p21 KO mice to determine significance. Two-ANOVA was used to calculate relative muscle weights between age groups.


Results, Conclusions, and Discussions:: It is expected that muscles from CDKN1a/p21 KO mice will repair the accumulated tissue damage that occurs during aging. Thus, muscle from the CDKN1a/p21 KO mice will show less loss of tension and power producing ability than control mice. Preliminary muscle fiber mechanic data on 24-month-old p21 knockout mouse demonstrates that the EDL generates positive power after activation. Soleus has a higher viscosity and elasticity when compared to the EDL. These studies are the first to analyze the influence of age and unloading on single muscle fiber biomechanics in CDKN1a/p21 KO mice.

18-week-old mice KO mice had an increase in body weight when compared to WT mice, but there was a significant decrease in KO mice muscle mass in the gastrocnemius and the right EDL. There was a decrease in soleus weight in the 18-week-old mice but was not significant. The 12-month-old KO mice only had a significant decrease in relative muscle weight in the EDL. The 18-month-old KO mice had significant decreases in relative muscle mass in the soleus, gastrocnemius, and EDL. The body weights of the 12-month-old and 18-month-old did not change significantly when comparing genotypes. The body weight of the 24-month-old KO mice was significantly less than the WT counterpart. This aligned with the comparison between the relative weight of the soleus and EDL, however, the decrease was not significant. When comparing age groups, the EDL experienced the most changes. For example, 18-month-old KO mice had a significant decrease in relative weight compared to 12-month-old WT mice while 12-month-old KO had a significant decrease compared to 18-month-old WT. This means that KO mice did not necessarily aid in the regeneration of muscle mass with age.

Future experiments include performing immunohistochemistry to determine fiber types, and individual muscle fiber mechanics of 24-month WT mice to determine the effects of aging and regeneration on the functionality of the muscle. It expected that there should be an increase in atrophy-related genes in aged mice and the transition to slow twitch fibers from fast twitch. This will aid in the understanding of p21 in muscle mechanics and functionality of muscle regeneration.


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