Endurance training increases a ubiquitylated form of histone H3 in the skeletal muscle, supporting Notch1 upregulation in an MDM2-dependent manner

Abstract

At the onset of training, each exercise session transiently shifts the distribution of histone post-transcriptional modifications (HPTMs) to activate genes that drive muscle adaptations. The resulting cyclic changes in gene expression promote the acquisition of high oxidative capacities and gains in capillaries. If training stops or remains at the same intensity, adaptation ceases. Whether silencing HPTMs helps to halt adaptation remains understudied. The E3 ubiquitin ligase murine double minute (MDM2) and enhancer of zester homolog 2 (EZH2) interact and tri-methylate histone H3 on lysine 27 (H3K27^me3), silencing genes. C57Bl6 mice ran for 9 weeks (5 days a week) maintaining a constant running speed for the last 5 weeks of training. Muscles were collected 72 h after the last run. Training increased MDM2 and EZH2 proteins and led to an H3K27^me3 enrichment in Kdr and Notch1 regulatory sequences. Kdr mRNA levels decreased, following the canonical model that H3K27^me3 silences genes. Notch1 mRNA increased. Trained muscles had greater levels of H3K27^me3 detected at 25 kDa and no change at the expected molecular weight of 17 kDa. The 25 kDa band was identified as a ubiquitylated form of H3 (H3^Ub). C2C12 myotubes exposed to four consecutive days of 90 min electrostimulation had higher levels of H3^Ub. EZH2 inhibition counteracted the electrostimulation-driven accumulation of H3^Ub and increased Notch1 mRNA. Serdemetan, an MDM2 ring domain inhibitor, reduced Notch1 mRNA and H3^Ub level in myotubes. MDM2-dependent ubiquitylation of H3 might upregulate Notch1 when endurance training ceases. The role H3^Ub plays in establishing a new muscle homeostasis remains unclear.

Key points

• Whether epigenetic silencing histone marks play a role once skeletal muscle adaptations have occurred following endurance training remains unclear.
• The E3 ubiquitin ligase MDM2 and the epigenetic writer EZH2 interact to establish H3K27^me3 marks that silence genes, and endurance training increased the expression of both proteins.
• After weeks of training new capillaries were established, and lower levels of Kdr mRNA and increased H3K27^me3 marking on Kdr regulatory sequences question whether silencing of this positive regulator of angiogenesis is required to halt microvascular remodelling.
• Training increases skeletal muscle abundance of a ubiquitylated form of H3 (H3^Ub); in myotubes EZH2 inhibition limits H3^Ub accumulation after contractile activity repeated over 4 days and MDM2 inhibition reduces H3^Ub levels and upregulates Notch1 expression.
• MDM2-dependent ubiquitylation of H3 might explain why H3K27^me3 enrichment fails to silence Notch1 after training; whether H3^Ub is crucial to halt adaptation and establish a new muscle homeostasis requires further investigation.