Skeletal Muscle LINE‐1 Gene Regulation and Inflammatory Response to Chronic, Voluntary Endurance Training in Rodents

Abstract

ObjectiveWe sought to determine the effects of chronic, voluntary wheel running on Long Interspersed Nuclear Element‐1 (LINE‐1) mRNA expression and promoter methylation, as well as markers of the cGAS‐STING inflammatory pathway, in skeletal muscle.HypothesesLINE‐1 mRNA expression will increase with age, and exercise will mitigate this increase. This difference in expression will be driven through changes in promoter methylation. The cGAS‐STING inflammatory pathway will be upregulated with age and downregulated with rats that exercise‐trained.MethodsPlantaris and soleus skeletal muscles were collected from female Lewis rats that belonged to one of three groups: a young control group (CTL; n=10; 6 mos old), an aged sedentary group (SED; n=12; 15 mos old), and an aged exercise group with access to a running wheel for 9 months (EX; n=12; 15 mos old). RNA, DNA, and protein were isolated from the tissues for analysis. Analysis included RT‐qPCR for LINE‐1 mRNA expression, methylated DNA Immunoprecipitation (MeDIP) to assess the methylation status of the LINE‐1 promoter via RT‐qPCR, and Western blotting for markers of the cGAS‐STING pathway and 4Hydroxynonenal (4HNE). Two primer sets were used for RT‐qPCR: L1‐3 probed for the most active form of LINE‐1 and L1‐Tot probed for all full‐length LINE‐1 elements.ResultsThere was no significant difference in plantaris LINE‐1 mRNA expression or for L1‐3 or L1‐Tot. Plantaris LINE‐1 promoter methylation was undetectable in most samples, so the results are not presented herein. Soleus L1‐3 mRNA was significantly higher in EX compared to CTL (p=0.036) and there was a trend between SED and EX (p=0.053). L1‐Tot also was trending (p=0.059), where EX was higher than CTL (p=0.025). Methylation of the LINE‐1 promoter was significantly different for both L1‐3 and L1‐Tot (p=0.021 and p=0.028, respectively). In both instances, LINE‐1 DNA in SED and EX were significantly more methylated compared to CTL. There were no differences in cGAS protein expression, p‐/pan STING protein expression, or 4HNE expression.ConclusionsContrary to the original hypothesis, LINE‐1 mRNA expression and DNA methylation were higher in the exercise group. Additionally, chronic exercise did not affect the cGAS‐STING inflammatory pathway, highlighting this pathway may be more relevant in extreme age and disease states. Previous skeletal muscle research has focused on using mixed muscle types to look at LINE‐1 expression and regulation, so the differences seen between type I (soleus) and type II (plantaris) fibers is a novel finding that warrants further investigation to understand the underlying mechanisms.