Nucleus Type-Specific DNA Methylomics Reveals Epigenetic "Memory" of Prior Adaptation in Skeletal Muscle

Abstract

Using a mouse model of conditional and inducible in vivo fluorescent myonuclear labeling (HSA-GFP), sorting purification of nuclei, low-input reduced representation bisulfite sequencing (RRBS), and a translatable and reversible model of exercise (progressive weighted wheel running, PoWeR), we provide the first nucleus type-specific epigenetic information on skeletal muscle adaptation and detraining. Adult (>4 mo) HSA-GFP mice performed PoWeR for 8 wk then detrained for 12 wk; age-matched untrained mice were used to control for the long duration of the study. Myonuclei and interstitial nuclei from plantaris muscles were isolated for RRBS. Relative to untrained, PoWeR caused similar myonuclear CpG hypo- and hyper-methylation of promoter regions and substantial hypomethylation in interstitial nuclear promoters. Over-representation analysis of promoters revealed a larger number of hyper- versus hypo-methylated pathways in both nuclear populations after training and evidence for reciprocal regulation of methylation between nucleus types, with hypomethylation of promoter regions in Wnt signaling-related genes in myonuclei and hypermethylation in interstitial nuclei. After 12 wk of detraining, promoter CpGs in documented muscle remodeling-associated genes and pathways that were differentially methylated immediately after PoWeR were persistently differentially methylated in myonuclei, along with long-term promoter hypomethylation in interstitial nuclei. No enduring gene expression changes in muscle tissue were observed using RNA-sequencing. Upon 4 wk of retraining, mice that trained previously grew more at the whole muscle and fiber type-specific cellular level than training naïve mice, with no difference in myonuclear number. Muscle nuclei have a methylation epi-memory of prior training that may augment muscle adaptability to retraining.

Keywords: epigenetics; exercise training; methylation; muscle memory; myonuclei; skeletal muscle.

© The Author(s) 2021. Published by Oxford University Press on behalf of American Physiological Society.

Figures

Graphical Abstract

Figure 1.

Myonuclear CpG promoter region DNA methylation changes in response to progressive weighted wheel running (PoWeR). (A) Study design schematic showing myonuclear labeling using the HSA-GFP mouse, PoWeR training (6M PoW) and detraining (9M PoW + DT), and age-matched untrained controls (6M UT and 9M UT), doxycycline treatment time points, and fluorescent activated cell sorting (FACS) of myonuclear and interstitial nuclear populations for downstream reduced representation bisulfite sequencing (RRBS) analysis. (B) Myonuclear and interstitial nuclear methylation in promoter regions after 8 wk of PoWeR. (C) Pathway analysis of hypomethylated promoters in myonuclei after PoWeR (relative to 6M UT). (D) Myonuclear genes with a hypomethylated promoter CpG in the NFκβ signaling pathway after PoWeR (FDR 

Figure 2.

Interstitial CpG promoter region DNA methylation changes in response to progressive weighted wheel running (PoWeR). (A) Pathway analysis of hypomethylated promoters in interstitial nuclei after PoWeR (relative to 6M UT). (B) Interstitial nuclear genes with a hypomethylated promoter CpG in the fatty acid (FA) metabolism signaling pathway after PoWeR (FDR 

Figure 3.

Myonuclear promoter region methylation memory of prior PoWeR training may facilitate retraining adaptations. (A) Memory of promoter site-specific CpG methylation in myonuclear Pkd2l1 (FDR < 0.05). (B) Memory of promoter region-specific hypomethylation in myonuclear Gdf10. (C) Memory of promoter region-specific hypermethylation in myonuclear Eif1a, Pitx1, Sun2, Usp43, and Ski (FDR < 0.05). (D) Evidence for promoter region CpG methylation memory of previous PoWeR at the pathway level in myonuclei. (E) Study design schematic showing how mice were subjected to PoWeR for 8 wk, detrained for 3 mo, and retrained for 4 wk (PoW + DT + RT); age-matched mice that only trained for 4 wk served as controls (4 wk PoW). (F) Average nightly running volume during 4 wk of retraining. (G) Body weight at the time of being euthanized. (H) Absolute plantaris muscle weight in milligrams (mg; *P<0.05, directional t-test). (I) Plantaris muscle weight (mg) normalized to body weight in grams (g; *P<0.05, directional t-test). (J) Myosin heavy chain (MyHC) 2A cross sectional area (CSA) of gastrocnemius muscle fibers (*P<0.05, directional t-test). (K) MyHC 2A proportion. (L) MyHC 2A myonuclear number measured using dystrophin and DAPI. (M) Representative image of dystrophin, MyHC 2A fibers, and nuclei in 4 wk PoW and PoW + DT + RT muscles. Scale bar = 50 µm.