Dietary Butyrate, an HDAC Inhibitor, Primes Muscle Satellite Cells for Differentiation by Altering the Epigenetic Landscape

Abstract

Satellite cells (SC) are a heterogeneous population of skeletal muscle resident stem cells that are responsible for postnatal muscle growth and repair. Satellite cells lie in a state of quiescence along the muscle fiber where they will activate, proliferate, and either self‐renew or differentiate. With the investigation into the genomic regulation of satellite cell fate, we now have an idea as to how the satellite cell epigenome governs myogenesis. Specifically, changes in the chromatin structure and the regulators that direct these changes modulate SC activation, proliferation, and differentiation. Interestingly, histone deacetylase (HDAC) inhibitors have proven to be effective at enhancing the myogenic program of SC, but their precise mode of action in altering the epigenetic landscape of SC remains undetermined. Our objective was to determine how a HDAC inhibitor, butyrate, promotes myogenic differentiation. Twenty female neonatal piglets (24±8hrs old) were fed a standard milk replacer or one supplemented with 0.50% (on a dry‐matter basis) spray dried Tributyrin (TB), a butyrate pro‐drug, for 17d. Satellite cells from TB supplemented piglets showed a decrease in the expression of EZH2 (enhancer of zeste homolog 2), the methyltransferase subunit of the PRC2 (polycomb repressive complex 2), just prior to being induced to differentiate. ChIP‐Seq analysis revealed that SC from TB treated piglets resulted in a global reduction of the repressive chromatin mark H3K27me3. Of the 532 differentially enriched regions (DER), TB treatment decreased binding at 358 sites, specifically those sites associated with genes involved in myogenic differentiation, negative regulation of cell proliferation, and micro‐RNAs normally upregulated during differentiation. ChIP‐Seq analysis for global enrichment of H3K27ac did not reveal any numerical differences in DER resulting from TB treatment. Given that EZH2 is responsible for the deposition of the repressive H3K27me3 mark, SC were transfected with siRNA targeting the EZH2 transcript (siEZH2), treated with 0.5 mM sodium butyrate (NaBu), or both (siEZH2 + NaBu) to determine if this was the primary mechanism through which butyrate alters SC myogenesis. Treatment with NaBu and siEZH2 reduced EZH2 expression in an additive manner. Treatment with NaBu alone and with siEZH2 significantly reduced Pax7 and MyoD expression after 24h of treatment while siEZH2 treatment had no effect on the expression of these myogenic transcription factors. Immunostaining for the contractile protein myosin heavy chain (MyHC) revealed an increase in MyHC+ cells from the siEZH2 treatment group after 72h of differentiation; however, these cells did not form multinucleated myotubes. The epigenetic changes caused by dietary TB supplementation resulted in reduced EZH2 and EZH2 trimethylation of H3K27. Taken together, these results indicate that while EZH2 ablation may force myogenic differentiation, butyrate may operate through a parallel mechanism to enhance differentiation. These findings help support the notion that butyrate may be used to enhance muscle growth and could prove useful at treating myopathic conditions.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.