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

Plasma MOTS-c levels are reduced with aging while muscle levels are increased

Plasma MOTS-c levels were measured in young (18-30 y), middle-aged (45-55 y) and older (70-81 y) males that were free from any overt disease following an overnight fast (Figure 1). Characteristics of participants are shown in Table 1, while all age groups had similar HOMA-IR, plasma LDL and HDL, the older-aged groups had higher fat mass and lower lean mass, and the middle-aged group high plasma triglycerides. Consistent with murine data [14], both the middle and older groups had lower circulating MOTS-c (by 11 and 21%, respectively) than the young group. Since exogenous MOTS-c treatment of aged or high fat diet challenged mice improves insulin sensitivity and alters body composition [14] we correlated plasma MOTS-c levels with HOMA-IR, and relative fat and lean mass (Figure 1B–1D). A weak association was observed with relative lean mass, but not fat mass or HOMA-IR (Figure 1B, 1C). To determine if differences between the groups could be explained by differences in clinical blood parameter or body composition, ANCOVA analysis was undertaken with % fat, % lean mass, HOMA-IR and plasma triglycerides as covariates (both independently and combined; Supplementary Table 1). With these covariates included the main effect for age on plasma MOTS-c remained significant (p<0.001; Supplementary Table 1) indicating that this is likely an age-dependent effect. To determine if skeletal muscle MOTS-c levels were similarly reduced with aging, MOTS-c protein expression was measured in vastus lateralis muscle samples at rest. In contrast to both plasma data, and MOTS-c muscle levels reported in aged mice [14], both the older and middle-aged groups had higher levels of muscle MOTS-c than the young group (Figure 1E and Supplementary Table 2). Plasma and muscle MOTS-c positively correlated in young, but not middle-aged or combined groups (Figure 1F–1H). The muscle and plasma samples for the older group were from different participants and therefore this comparison could not be made for the older men.

Age-related increases in muscle MOTS-c levels are associated with fast-to-slow fiber type shift

In addition to loss of muscle mass, aging is associated with a fast-to-slow fiber type shift [31]. Therefore, we determined whether markers of slow (myosin heavy chain type 7, MYH7) and fast (myosin heavy chain type 2, MYH2) type fibers associate with muscle MOTS-c levels. Consistent with the hypothesis that a change in fiber type may account for the increase in muscle MOTS-c levels observed with aging, MYH7 mRNA showed a positive association with muscle MOTS-c levels while MYH2 mRNA was negatively associated (Figure 2A, 2B). Furthermore, MOTS-c expression was higher in mouse soleus muscle (Figure 2C) which has a higher proportion of slow type fibers than EDL, gastrocnemius, and tibialis anterior muscles (Figure 2C). Higher slow-type fiber content of soleus muscle was confirmed by measuring mRNA levels of Myh7 (type I fiber), Myh2 (type IIa fibers), Myh4 (type IIb fibers) and Myh1 (type IIx fibers) in these muscles (Figure 2D). Slow type fibers normally have a greater mitochondrial density, therefore the mitochondrial protein COXIV was determined in muscle samples and used to correct MOTS-c levels for mitochondrial mass. This did not change the increase in muscle MOTS-c expression observed in the middle-aged and older groups compared to the young group (Figure 2E), suggesting that the increase in muscle MOTS-c levels was independent of mitochondrial protein levels.

To assess how muscle MOTS-c levels relate to muscle function, thigh cross-sectional area (CSA) and maximal leg press weight was measured in the older group. There was no association between MOTS-c levels and leg press weight or CSA. However, when maximal leg press weight was corrected for thigh size (CSA) a positive association was seen (Figure 3). This may suggest that muscle MOTS-c levels are associated with improved muscle quality in the elderly.

Transcriptional regulation of MOTS-c and MT-RNR1 with age

The coding sequence for MOTS-c is found within the 12S rRNA gene (MT-RNR1) of mtDNA (Figure 4A). To determine if MOTS-c transcripts are present in muscle independent of MT-RNR1 transcripts we filtered total RNA to enrich small mRNA fragments (<200 nt) and used a custom TaqMan small RNA assay with a probe that was designed against the 51-nucleotide sequence of MOTS-c to measure MOTS-c mRNAs in the enriched small RNA fraction. Similar to muscle MOTS-c protein expression, both the middle and older aged groups had increased muscle MOTS-c mRNA compared to the young group (Figure 4B), and MOTS-c mRNA correlated with MOTS-c protein levels (Figure 4C). Due to the amount of RNA required for this approach, only 52 samples could be included in this analysis; 18 each from young and middle-aged, and 16 from the older groups.

MOTS-c mRNA correlated with MT-RNR1 and CYTB measured in the small RNA fraction but not in total RNA (Figure 4D–4F; and Supplementary Figure 1). Similar results were seen for a second primer pair designed against different location in the MT-RNR1 gene (Supplementary Figure 1), and as a result of enriching for transcripts <200 nt, the expression of these mRNA’s was substantially lower in the small fraction compared to fractions containing larger transcripts (Supplementary Figure 2). This indicates that small RNA transcript levels are independent of the larger transcript levels, suggesting that they may be independently transcribed. Neither MT-RNR1 mRNA measured in total or small RNA fraction correlated with muscle protein MOTS-c protein levels (Figure 4G, 4H).

To determine whether the increase in MOTS-c mRNA is a function of differences in mtDNA content, mitochondrial-to-nuclear ratio (mtDNA/nDNA) was measured and used to standardize MOTS-c mRNA levels (Figure 4I, 4J). Similar to MOTS-c protein expression, when corrected for mtDNA content, both the middle and older groups had greater MOTS-c mRNA relative to mtDNA than the young group (Figure 4J), and this standardization did not improve the association between MOTS-c mRNA and MT-RNR1 mRNA total RNA fraction (Figure 4K).

MOTS-c has been shown to bind to nuclear DNA in HEK293 cells and interact with Nrf2 (NFE2L2) to regulate ARE-related gene expression [22]. A weak correlation was observed between skeletal muscle NRF2 mRNA and MOTS-c small mRNA levels, and HMOX-1 mRNA and MOTS-c protein expression (Figure 5B and 5E). However, overall there was no association between ARE-responsive genes (HMOX-1, NQO1 and SOD2) and MOTS-c small mRNA, or NQO1 and SOD2 mRNA and MOTS-c protein levels in skeletal muscle (Figure 5A–5H).

Participants

One hundred and four healthy young, middle-aged or older males were recruited (Table 1 and [45]) and gave written informed consent before the commencement of the study. Participants were sedentary to recreationally active, non-smokers, free of chronic illnesses including cardiovascular or metabolic disease and were not taking any medication. Body composition was assessed by dual X-ray absorptiometry (DXA; model iDXA, GE-Lunar, Madison, WI), and thigh muscle cross-sectional area was determined via anthropometric measurement as previously reported [46]. One repetition maximum (1-RM) for leg press was estimated using the Brzycki equation [47]. Two groups of older participants were recruited as blood samples were not available for the group that had muscle samples collected (Older 1). All experimental procedures were approved by the Health and Disability (New Zealand) and Deakin University Human (Melbourne, Australia) Research Ethics Committee’s, and performed in accordance with the Helsinki declaration.

Murine housing

Mice were maintained in a temperature-controlled animal facility with 12 h light-dark cycle and ad libitum access to water, a standard rodent chow diet (Teklad TB 2018; Harlan, Madison, WI). At 10-12 weeks of age in-house bred male C57BL/6J mice were culled by cervical dislocation and muscle excised, snap frozen in liquid nitrogen and stored at -80°C until analysis. All experiments were approved by the University of Auckland Animal Ethics Committee, New Zealand.

Muscle and blood collection

Following an overnight fast, and having abstained from strenuous physical activity for at least 48h prior to arriving at the laboratory in the morning (before 0900 h), venous blood was drawn into a 10 ml EDTA vacutainer tube, and a percutaneous muscle biopsy was obtained from the vastus lateralis muscle using suction-modified Bergstrom biopsy needle under local anaesthesia. Muscle samples were snap-frozen in liquid nitrogen and blood samples were centrifuged immediately upon collection at 4° C, 1900 x g for 15 min. Plasma and muscle samples were subsequently stored at -80°C until analysis.

Plasma analysis

Glucose (C311 autoanalyzer, Roche, Mannheim, Germany) and insulin (cobas e11, Roche, Mannheim, Germany) were measured in plasma and the homeostatic model assessment (HOMA) was used to estimate insulin resistance (HOMA2-IR2; The Oxford Centre for Diabetes, Endocrinology and Metabolism, UK) [48]. As described previously [14], MOTS-c was extracted from plasma in acetonitrile-hydrochloric acid and measured using an in-house sandwich ELISA developed at the University of Southern California by the laboratory that first described MOTS-c in human and murine plasma. This ELISA has been extensively validated, and peptide blocking experiments have been undertaken to show specificity of the antibody [14].

Immunoblotting

~20 mg of muscle was homogenized for 40s at 20 Hz using a TissueLyser (Qiagen, Venlo, Netherlands) in lysis buffer containing 10 μL/mg 25 mM Tris 0.5% v/v Triton X-100 supplemented with Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific, Waltham, MA). The supernatant was collected after centrifugation to determine protein concentration using the BCA protein assay kit (Thermo Scientific, Waltham, MA). Laemmli's buffer was added to protein homogenates and boiled for 5 min at 95 °C. Denatured proteins were separated by SDS-PAGE on 8%–16% TGX pre-cast gels (Bio-Rad, Hercules, USA) and proteins were transferred to PVDF membranes (Bio-Rad, Hong Kong, China) using a TransBlot semidry transfer apparatus (Bio-Rad, Hongkong, China). Membranes were blocked in 5% BSA in TBST for 1h at room temperature, and probed using specific antibodies for MOTS-c (custom produced by YenZym Antibodies, LLC, South San Francisco, CA and described previously [14], 1:500) COXIV (Molecular probes #A6431, 1:10,000) and α-tubulin (Sigma-Aldrich #T9026, 1:10,000). Primary antibodies were incubated overnight at 4 °C with gentle agitation. The following morning, the membranes were probed with either anti-rabbit or anti-mouse linked to horseradish peroxidase secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA) (1:10,000 dilution) for 1h at room temperature. Membranes were exposed using enhanced chemiluminescence reagent (Western Lightning^® Ultra, Perkin Elmer, Waltham, MA, USA) and chemiluminescent signals were captured using ChemiDoc image device (BioRad, HongKong, China). Densitometry analysis of protein bands were quantified using ImageJ software (National Institutes of Health, Bethesda, MD). The intensity of each band was recorded relative to their respective α-tubulin intensity and then normalized to the ratio of target to loading protein in the pooled control sample ran on each gel, and log transformed if required.

Real-time polymerase chain reaction

Total RNA and DNA was extracted from ~20 mg of tissue using the AllPrep® DNA/RNA/miRNA Universal Kit (QIAGEN GmbH, Hilden, Germany) and small RNA’s (<200 nt) enriched using the Zymo RNA Clean and Concentrator Kit (Cat # R1016, Zymo Research, Irvine, CA) per the manufacturer’s instructions, and enrichment confirmed using RNA NANO Chip using a 2100 Bioanalyzer (Agilent Technologies; data not shown). Total RNA was reverse transcribed using a High-Capacity RNA-to-cDNA™ kit (Life Technologies, Carlsbad, CA) and quantitative real-time PCR was performed on a QuantStudio 6 PCR System using the SYBR green select master mix (Applied Biosystems, Foster City, CA). Reactions were performed in duplicate and relative quantification achieved using the ∆∆Ct method with geometric mean of four reference genes (human sample: C1ORF43, CHMP2A, VCPEMC7; mouse sample, Gapdh, Actb B2mb Rplpo) as an internal control. Primer sequences used are listed in Supplementary Table 3, and for mitochondrial (mtDNA) to nuclear (nDNA) ratio primers were used to MTND4 and 18S rRNA. For small RNA assays, 50 ng of fractioned RNA was converted to cDNA using TaqMan® microRNA RT kit #4366596 (Life Technologies, Carlsbad, CA) and a multiplexed approach [49] was utilized with specific TaqMan small RNA assays (Life Technologies, Carlsbad, CA) for MOTS-c (designed using Custom TaqMan® Small RNA Assay Design Tool against MOTS-c mtDNA sequence), RNU44 (assay ID: 001094) and RNU48 (assay ID: 001006) primers as per the manufactures instruction and as described previously [50]. The expression of MOTS-c mRNA was normalized to the geomean RNU44 and RNU48 using the ∆∆Ct method.

Statistical analyses

Statistical analyses were performed using Prism 8 (GraphPad Software Incorporated, California, USA) and SPSS (IBM SPSS, NY, USA, version 25), with statistical significance determined as p ≤ 0.05, and data are presented as individual data points and mean ± standard error of the mean unless stated otherwise. Statistical significance was determined with Pearson linear regression, one-way ANOVA with Holm-Sidak’s post-hoc analysis or ANCOVA.