Increased expression of the mitochondrial derived peptide, MOTS-c, in skeletal muscle of healthy aging men is associated with myofiber composition

Abstract

Mitochondria putatively regulate the aging process, in part, through the small regulatory peptide, mitochondrial open reading frame of the 12S rRNA-c (MOTS-c) that is encoded by the mitochondrial genome. Here we investigated the regulation of MOTS-c in the plasma and skeletal muscle of healthy aging men. Circulating MOTS-c reduced with age, but older (70-81 y) and middle-aged (45-55 y) men had ~1.5-fold higher skeletal muscle MOTS-c expression than young (18-30 y). Plasma MOTS-c levels only correlated with plasma in young men, was associated with markers of slow-type muscle, and associated with improved muscle quality in the older group (maximal leg-press load relative to thigh cross-sectional area). Using small mRNA assays we provide evidence that MOTS-c transcription may be regulated independently of the full length 12S rRNA gene in which it is encoded, and expression is not associated with antioxidant response element (ARE)-related genes as previously seen in culture. Our results suggest that plasma and muscle MOTS-c are differentially regulated with aging, and the increase in muscle MOTS-c expression with age is consistent with fast-to-slow type muscle fiber transition. Further research is required to determine the molecular targets of endogenous MOTS-c in human muscle but they may relate to factors that maintain muscle quality.

Keywords: MOTS-c; aging; mitochondria; mitochondrial derived peptides; muscle.

Figure 1

Plasma MOTS-c levels decrease and skeletal muscle levels increase with aging. Plasma MOTS-c (A), correlated with HOMA-IR (B), fat mass (C), lean mass (D), and muscle MOTS-c expression (E) in young, middle-aged and older males. Representative blots are independent and from different participants. Correlation between plasma MOTS-c and muscle MOTS-c expression in young (F), middle (G), and combined (H). Significance was determined using linear regression or one-way ANOVA. Data is presented as means ± SE, n=26 per group except for body composition measurements where data was not available for a young (n=25) and middle-aged (n=25) participant. *p<0.05, **p<0.01, ***p<0.001.

Figure 2

MOTS-c expression is higher in slow-type muscle. Correlations between muscle MOTS-c expression and MYH7 (A) and MYH2 (B) mRNA levels in young, middle-aged and older men. Mouse extensor digitorum longus (EDL), gastrocnemius, tibialis anterior (TA) and soleus (SOL) muscle MOTS-c expression (C), and mRNA levels of fiber type markers (D). Two independent COXIV representative blots with different participants and quantification of MOTS-c relative to COXIV expression (E) in muscle samples from young, middle-aged and older males. Significance was determined using linear regression or one-way ANOVA. Data is presented as means ± SE for n=26 per group. ***p<0.001; ^#p<0.0001 vs soleus muscle.

Figure 3

The association between MOTS-c and muscle area and function. Thigh cross-sectional area (CSA) (A), maximal leg press load (B) and maximal leg press load relative to CSA (C) was correlated with muscle MOTS-c expression in older men. Significance was determined using linear regression or one-way ANOVA. Due to missing pQCT/leg press data n=24.

Figure 4

Muscle MOTS-c and 12S rRNA transcription with aging. 12S rRNA mRNA (MT-RNR1) and MOTS-c mRNA analysis technique (A). Young, middle-aged and older male muscle MOTS-c mRNA levels in the small RNA fraction were determined (B) and correlated with muscle MOTS-c protein expression (C), MT-RNR1 (D) and CYTB (E) mRNA levels in the small RNA fraction and MT-RNR1 mRNA levels in the total RNA fraction (F). MT-RNR1 mRNA levels in the total (G) and small (H) RNA fraction were correlated with MOTS-c protein expression. Muscle mitochondrial to nuclear DNA (mtDNA/nDNA) (I), and MOTS-c mRNA levels relative to mtDNA (J), and correlation of MOTS-c mRNA and MT-RNR1 mRNA in the total RNA relative to mtDNA (K). Significance was determined using linear regression or one-way ANOVA. Results are shown as means ± SE, due to limited sample availability and assay failure, for small RNA fraction assays n=14-18 per group, and for mtDNA n=26 for young, 24 for middle and 25 for old. *p<0.05, **p<0.01, ***p<0.001.

Figure 5

MOTS-c muscle protein or small mRNA expression does not correlate well with antioxidant-response element (ARE) related genes. Correlation between MOTS-c protein (A–D) or small mRNA (E–H) expression and mRNA levels of NFE2L2, HMOX1, NQO1 and SOD2 in muscle samples from young, middle-aged and older men. Significance was determined using linear regression for n=78 (protein correlations) or n=52 (MOTS-c mRNA correlations).