Line-1 Retrotransposition and the Impact on Aging Rodent Skeletal Muscle Tissue

Abstract

Purpose: Long INterspersed Element 1 (LINE-1) is a class 1 transposable element known as a retrotransposon. LINE-1 is thought of as a genomic parasite due to its reverse transcription machinery, and ability to randomly copy and paste itself back into the genome. Studies have shown that there are an estimated 500,000 copies of LINE-1 accounting for roughly 17-18% of the total human genome. However, only around 100 of the 500,000 copies are functionally-active. Additional studies have shown that markers of tissue LINE-1 activity increase with age, and due to the ability of LINE-1 to randomly insert itself into the genome, this may negatively impact overall health. Currently, there is no research regarding the effects of aging on LINE-1 activity in rat skeletal muscle tissue. Therefore, the purpose of this study was to identify the effects of aging on LINE-1 activity markers in rat skeletal muscle tissue. Methods: In the current study mixed gastrocnemius muscle from male Fischer 344 rats that were 3, 12, and 24 months (mo) of age (n=9 per age group) were analyzed for LINE-1 mRNA expression, DNA expression, promoter methylation and euchromatin content. Primer sets for qPCR were designed for the youngest and most active form of LINE-1 (L1.3), older LINE-1 elements (L1.Tot), and ORF1. Results: L1.3, L1.Tot and ORF1 mRNA expression was higher in 12 and 24 mo versus 3 mo rats (p<0.05). L1.3 and ORF1 DNA expression was higher in the 24 mo versus other groups (p<0.05). ORF1 protein expression was higher in 12 and 24 mo versus 3 mo rats (p<0.05). L1.3 promoter methylation was numerically lower (but was not significantly different) in 24 mo versus 3 mo rats. Nuclear DNA methyltransferase (DNMT) activity was lower in the 12 and 24 mo versus 3 mo rats (p<0.05). Due to the lower nuclear DMNT activity within skeletal muscle of older rats we aimed to inhibit DNMT activity in L6-derived myotubes with 5-Azacytidine (5-AC) to determine its effects on LINE-1 mRNA expression. However, 5-AC treatments at 3 h and 24 h did not alter L1.3 or L1.Tot mRNA levels relative to vehicle-treated myotubes. Conclusions: Markers of LINE-1 activity increase in rat skeletal muscle across the age spectrum, and this may be related to age-related alterations in LINE-1 methylation and chromatin changes. However, more research is needed to determine factors that alter LINE-1 promoter methylation and chromatin content with aging.