Skeletal Muscle Mitochondria Contain Nuclear-Encoded RNA Species Prior to and Following Adaptation to Exercise Training in Rats

Abstract

Skeletal muscle mitochondria adaptation to exercise training is mediated by molecular factors that are not fully understood. Mitochondria import over 1000 proteins encoded by the nuclear genome, but the RNA population resident within the organelle is generally thought to be exclusively encoded by the mitochondrial genome. However, recent in vitro evidence suggests that specific nuclear-encoded miRNAs and other noncoding RNAs (ncRNAs) can reside within the mitochondrial matrix. Whether these are present in mitochondria of skeletal muscle tissue, and whether this is affected by endurance training-a potent metabolic stimulus for mitochondrial adaptation-remains unknown. Rats underwent 4 weeks of moderate-intensity treadmill exercise training, then were humanely killed and tissues were collected for molecular profiling. Mitochondria from gastrocnemius skeletal muscle were isolated by immunoprecipitation, further purified, and then the resident RNA was sequenced to assess the mitochondrial transcriptome. Exercise training elicited typical transcriptomic responses and functional adaptations in skeletal muscle, including increased mitochondrial respiratory capacity. We identified 24 nuclear-encoded coding or noncoding RNAs in purified mitochondria, in addition to 50 nuclear-encoded miRNAs that met a specified abundance threshold. Although none were differentially expressed in the exercise vs. control group at FDR < 0.05, exploratory analyses suggested that the abundance of 3 miRNAs was altered (p < 0.05) in mitochondria isolated from trained compared with sedentary skeletal muscle. We report the presence of a specific population of nuclear-encoded RNAs in the mitochondria isolated from rat skeletal muscle tissue, which could play a role in regulating exercise adaptations and mitochondrial biology.

Keywords: exercise; mitochondria; skeletal muscle; transcriptome.

FIGURE 1

Effects of 4 weeks of treadmill exercise training (ExT) or sedentary control (Sed) on rat whole‐body characteristics, and heart and skeletal muscle mitochondrial respiratory capacity. Rat body mass (A) at 9 weeks of age after 4‐week treadmill exercise training (ExT) or sedentary control (Sed); data are mean (SD) analyzed by t‐test for n = 16/grp. (B) Heart weight and (C) left ventricle outer wall mass (n = 12 Sed, n = 14 ExT; tissue mass data for 6 animals not recorded due to equipment fault). (D) Left ventricle and (E) red portion of gastrocnemius mitochondrial oxygen consumption per mg tissue during non‐ADP‐stimulated state‐4 (Leak) respiratory state, 2.5 mM ADP‐stimulated oxidative phosphorylation (OXPHOS) or uncoupled electron transport system capacity (ETS) supported by complex I and/or II substrates. Left ventricle respiration analyses were performed on a subset of animals (n = 4 per group); gastrocnemius tissue analysis was performed on n = 15 per group. Data are mean (SD) analyzed by two‐way ANOVA (left ventricle) or Mixed‐effects analysis (Gastrocnemius) with repeated measures, with Bonferroni post hoc test. (F) Citrate synthase and (G) 3‐hydroxyacyl‐CoA dehydrogenase (β‐HAD) maximal enzyme activity from red portion of the gastrocnemius; data are mean (SD) analyzed by t‐test for n = 16 (Sed) and n = 15 (ExT).

FIGURE 2

Transcriptome of rat left ventricle and gastrocnemius skeletal muscle tissue after 4 weeks of treadmill exercise training (ExT) or sedentary control (Sed). Whole tissue lysate from left ventricle and the red portion of the gastrocnemius was subjected to total RNA sequencing. (A) Left ventricle MA plot of all transcripts (≥ 1 CPM in all samples), positive fold‐change (log2FC) indicates upregulation in ExT at unadjusted p < 0.01 (blue) and p > 0.01 (light gray) for n = 7 samples per group. (B) GSEA analysis for enriched Reactome terms in left ventricle with ExT vs. Sed. (C) Gastroc MA plot of all transcripts (≥ 1 CPM in all samples), positive fold‐change (FC) indicates upregulation in ExT at FDR < 0.05 (red) and FDR < 0.2 (blue) for n = 15 samples per group. (D) GSEA analysis for enriched Reactome terms in gastroc with ExT vs. Sed; pathway FDR < 0.1. (E) Volcano plot of nuclear‐encoded genes for subunits of OXPHOS complexes I–V (CI—CV) as well as mtDNA‐encoded genes (MT) in gastroc for ExT vs. Sed.

FIGURE 3

Transcriptome of isolated mitochondria from red portion of rat gastrocnemius skeletal muscle. (A) Experimental overview of mitochondrial isolation from gastrocnemius muscle tissue. Isolated mitochondria were enzymatically purified with RNase‐A to remove transcripts on the outer mitochondrial membrane. RNA extracted from these purified mitochondria and their respective whole tissue lysate then underwent RNA sequencing. (B) Purity of the isolated mitochondria preparation indicated by the relative absence of nuclear‐encoded transcripts for subunits of OXPHOS complexes I to V in isolated mitochondria fraction compared with whole muscle lysate samples. Data are the ratio of RNAseq normalized counts for each gene expressed relative to Mt‐co1 normalized counts (the most abundant mRNA in the mitochondrial fraction), presented as mean and individual data points for n = 30 animals (Sed and ExT combined). (C) MA plot of isolated mitochondria (Mt) relative to whole skeletal muscle (SM); mtDNA‐encoded transcripts are shown in red, all nuclear‐encoded transcripts in blue or gray. (D) Correlation of all genes (> 1 CPM) with Mt‐co1 in isolated mitochondria (n = 30 animals, Sed and ExT combined); red and gray points represent mtDNA‐encoded and nuclear‐encoded transcripts, respectively. Positive correlation indicates mitochondrial localization; negative correlation suggests nonmitochondrial localization. (E) Enlarged plot of panel D, showing transcripts with a correlation greater than r = 0.8 with Mt‐co1. (F) The top 25 most highly expressed miRNAs in rat skeletal muscle mitochondria. Data are the average normalized read counts per miR across all samples (n = 25 animals, Sed and ExT combined).

FIGURE 4

Effects of 4‐week exercise training on the transcriptome of mitochondria isolated from rat gastrocnemius skeletal muscle. (A) MA plot of all transcripts (≥ 2 CPM in all samples) for n = 15 samples per group, no genes were significantly DE between ExT and Sed at FDR < 0.05. mtDNA‐encoded transcripts are shown in red, nuclear‐encoded transcripts in gray, and transcripts identified as being mitochondria‐localized candidates from correlation with Mt‐co1 (from Figure 3E) shown in green. (B) Volcano plot of miRNAs detected in isolated mitochondria (> 100 counts in > 80% samples) for ExT vs. Sed. No miRNAs were differentially expressed at the FDR < 0.05 threshold; data shown are p‐values for n = 12 (Sed) and n = 13 (ExT). (C) Correlation heatmap of significant intersecting TargetScan miRNA‐mRNA target predictions in isolated mitochondria.3.