Transcriptome and Methylome Profiling in Rat Skeletal Muscle: Impact of Post-Weaning Protein Restriction

Abstract

Skeletal muscle is programmable, and early-life nutritional stimuli may form epigenetic memory in the skeletal muscle, thus impacting adult muscle function, aging, and longevity. In the present study, we designed a one-month protein restriction model using post-weaning rats, followed by a two-month rebound feeding, to investigate how early-life protein restriction affects overall body growth and muscle development and whether these influences could be corrected by rebound feeding. We observed comprehensive alterations immediately after protein restriction, including retarded growth, altered biochemical indices, and disturbed hormone secretion. Transcriptome profiling of the gastrocnemius muscle followed by gene ontology analyses revealed that "myogenic differentiation functions" were upregulated, while "protein catabolism" was downregulated as a compensatory mechanism, with enhanced endoplasmic reticulum stress and undesired apoptosis. Furthermore, methylome profiling of the gastrocnemius muscle showed that protein restriction altered the methylation of apoptotic and hormone secretion-related genes. Although most of the alterations were reversed after rebound feeding, 17 genes, most of which play roles during muscle development, remained altered at the transcriptional level. In summary, early-life protein restriction may undermine muscle function in the long term and affect skeletal muscle development at the both transcriptional and methylation levels, which may hazard future muscle health.

Keywords: methylome; post-weaning; protein restriction; skeletal muscle; transcriptome.

Figure 1

Changes in calorie intake and body weight. (A) Final body weight; (B) Total calories intake; (C) Weight change throughout the experiment; (D) Weekly food intake. Data are shown as means ± standard error of the mean (SEM), n = 6 or 7. CN, control diet (15% casein); LP, low-protein diet (8% casein); CN-R, rebound feeding group fed with control diet after 4 weeks of normal feeding; LP-R, rebound feeding group fed with control diet after 4 weeks of low-protein diet feeding; ns, no significance; *** p < 0.001 vs. CN. ^# p < 0.05, ^## p < 0.01, and ^### p < 0.001 vs. CN-R.

Figure 2

Effects of low protein diet and rebound feeding of control diet on amino acid profiles of LP and CN rats (A–C) during regular feeding in (A) plasma, (B) liver, and (C) gastrocnemius muscle; (D–F) during rebound feeding in (D) plasma, (E) liver, and (F) gastrocnemius muscle. Data are shown as means ± SEM; n = 6 or 7. CN, control diet (15% casein); LP, low-protein diet (8% casein); CN-R, rebound feeding group fed with control diet after 4 weeks of normal feeding; LP-R, re-bound feeding group fed with control diet after 4 weeks of low-protein diet feeding; * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. CN. Val, valine; Leu, leucine; Ile, isoleucine; Thr, threonine; Lys, lysine; Met, methionine; His, histidine; Phe, phenylalanine; Trp, tryptophan; Gly, glycine; Ala, alanine; Ser, serine; Asp, aspartic acid; Glu, glutamic acid; Asn, asparagine; Gln, glutamine; Arg, arginine; Tyr, tyrosine; Pro, proline; Cit, citrulline; Orn, ornithine; β-Ala, β-alanine; Tau, taurine.

Figure 2

Effects of low protein diet and rebound feeding of control diet on amino acid profiles of LP and CN rats (A–C) during regular feeding in (A) plasma, (B) liver, and (C) gastrocnemius muscle; (D–F) during rebound feeding in (D) plasma, (E) liver, and (F) gastrocnemius muscle. Data are shown as means ± SEM; n = 6 or 7. CN, control diet (15% casein); LP, low-protein diet (8% casein); CN-R, rebound feeding group fed with control diet after 4 weeks of normal feeding; LP-R, re-bound feeding group fed with control diet after 4 weeks of low-protein diet feeding; * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. CN. Val, valine; Leu, leucine; Ile, isoleucine; Thr, threonine; Lys, lysine; Met, methionine; His, histidine; Phe, phenylalanine; Trp, tryptophan; Gly, glycine; Ala, alanine; Ser, serine; Asp, aspartic acid; Glu, glutamic acid; Asn, asparagine; Gln, glutamine; Arg, arginine; Tyr, tyrosine; Pro, proline; Cit, citrulline; Orn, ornithine; β-Ala, β-alanine; Tau, taurine.

Figure 3

Study design. CN: control diet; LP: low-protein diet. CN-R, CN followed by rebound feeding. LP-R, LP followed by rebound feeding.