Human iPS cloning and generation of muscle stem cells

The healthy donor-derived human pluripotent stem cell (hiPSC) clone 414C2 [29] was used to harvest iPSC-derived muscle stem cells (iMuSCs) after myogenic differentiation. Myogenic induction of hiPSCs was performed as previously described to obtain iMuSCs [30]. Briefly, undifferentiated hiPSCs were plated onto a 6-well plate coated with perlecan-binding laminin 421E8 fragments (p421E8) in StemFit AK02N (Ajinomoto) and 10 µM Y-27,632 (Nacalai) at 5 × 103 cells/well. After three days, the medium was replaced with CDMi supplemented with 10 µM CHIR99021 (CHIR, Wako) and 5 µM SB431542 (SB, Wako). CDMi is composed of IMDM (Wako) and Ham’s F-12 medium (Wako) (ratio, 1:1) supplemented with 1% bovine serum albumin (BSA) (Sigma-Aldrich, Burlington, MA, USA), 1% penicillin-streptomycin mixed solution (Nacalai), 1% CD lipid concentrate (Thermo Fisher Scientific, Waltham, MA, USA), 1% insulin-transferrin selenium (Thermo Fisher Scientific), and 450 µM 1-thioglycerol (Sigma-Aldrich).

After seven days of differentiation, the cells were dissociated with Accutase (Nacalai) and plated onto a p421E8-coated 6-well plate (4.5 × 105 cells/well) in CDMi medium supplemented with 10 µM CHIR, 5 µM SB, and 10 µM Y-27,632. On differentiation day 14, the cells were dissociated with Accutase and placed onto an iMatrix-511 (Nippi)-coated 6-well plate (8 × 105 cells/well) in CDMi medium supplemented with 10 µM Y-27,632. The medium was replaced with serum-free culture medium (SF-O3; Sanko Junyaku) supplemented with 0.2% BSA, 200 µM 2-mercaptoethanol (2-ME), 10 ng/mL IGF-1 (PeproTech), 10 ng/mL recombinant human bFGF (Oriental Yeast), and 10 ng/mL recombinant human HGF (PeproTech) after three days. The medium was changed twice a week until day 38 of differentiation. On differentiation day 38, it was replaced with 2% horse serum (HS, Sigma-Aldrich) in DMEM (Nacalai) supplemented with 200 µM 2-ME (Nacalai), 10 ng/mL IGF-1, 5 µM SB, 0.5% penicillin-streptomycin mixed solution (Nacalai), and 2 mM L-glutamine (Nacalai). Thereafter, the medium was changed every 2–3 days, and the cells were cultured in the medium until weeks 11–12 of differentiation.

Isolation of iMuSCs through flow cytometric sorting

The cells were harvested for flow cytometric sorting after 11–12 weeks of differentiation [31]. Briefly, cells were dissociated with a Collagenase mix solution at 37 °C for 7 min. The collagenase mix solution is composed of DMEM supplemented with 0.05% Collagenase H (Meiji Seika Pharma) and 0.5% Collagenase G (Meiji Seika Pharma). Accutase was added to the solution and incubated at 37 °C for 10 min. The solution was subsequently neutralized with DMEM supplemented with 10% HS and centrifuged at 380 × g for 7 min at 4 °C. Next, the supernatant was removed. Cells were then resuspended in DMEM supplemented with 10% HS and filtered through a 40-µm cell strainer (Corning). Density gradient centrifugation was performed using Optiprep (Serumwerk Bernburg) in DMEM supplemented with 10% HS (ρ = 1.11 g/mL) according to the manufacturer’s instructions to remove debris and dead cells. The fraction of living cells was resuspended in HBSS (Thermo Fisher Scientific) and 1% BSA before incubation with an APC-conjugated anti-CDH13 antibody (5 × 106 cells/mL) on ice for 15 min [32]. Flow cytometry was performed using Aria II (BD) in accordance with the manufacturer’s instructions. Gating of the CDH-13-positive fraction was determined using unstained cells as the baseline control. After sorting the CDH-13-positive cells, the suspension was centrifuged at 780 × g for 10 min at 4 °C. The supernatant was removed, and the 2 × 105 sorted cells were cryopreserved in 200 µL Bambanker (NIPPON Genetics).

Re-culture and myogenic differentiation of sorted iMuSCs

Frozen sorted cells were rapidly thawed in a water bath at 37 °C. They were then suspended in a growth medium containing 39% DMEM (Gibco), 39% Ham’s F12, 20% FBS (Gibco), and 1% UltroserG (Pall Life Sciences). Next, the cells were centrifuged at 400 × g for 10 min at 4°C, as described previously [33, 34]. After removal of the supernatant, the cells were resuspended in a growth medium and plated on to an iMatrix-coated (nippi) 12-well plate (2 × 104 cells/well). The medium was replaced with a differentiation medium containing 2% HS in DMEM after three days. Thereafter, the medium was changed every 2 days until analysis. The growth and differentiation media were supplemented with 1% penicillin-streptomycin.

Preparation and culture of primary bovine muscle stem cells

The thoracic longissimus muscle was excised from a 22-month-old Holstein cow. Primary bovine muscle stem cells were isolated as described previously, with some minor modifications [35, 36]. Connective tissue, blood vessels, and fat tissue were removed from isolated muscle tissue under dissection microscopy. Minced muscle pieces were treated with 0.2% collagenase type II (Worthington Biochemical Corporation) in HBSS (Thermo Fisher) for 30–40 min at 37 °C. Muscle slurry was homogenized with an 18G needle passing through 10 times and subsequently incubated at 37 °C for another 10 min. Debris was filtrated through 100-µm and 40-µm cell strainers (Corning Japan KK). The obtained cells were cultured on non-coated dishes in 20% FBS (Biowest) in F10 (Thermo Fisher) supplemented with 2.5 ng/mL bFGF (Nacalai Tesque, Inc.) for 60 min. Next, a non-adherent fraction was collected as primary bovine MuSCs and cultured on collagen-coated dishes (AGC Techno Glass Co., Ltd.) for expansion. Cells with a few passages were cryopreserved and used for the experiments. Enrichment of primary bovine MuSCs and myotubes were confirmed through immunostaining with an antibody against sarcomeric-α-actinin, which is specifically expressed in skeletal muscles. The cells generated multinucleated myotubes after myogenic differentiation. In addition, > 50% of the cultured cells were positive for sarcomeric-α-actinin, which is specifically expressed in skeletal muscle cells.

Frozen bovine cells were rapidly thawed in a water bath at 37 °C and followed the same protocol as that for iMuSCs. Briefly, bovine cells were resuspended in growth medium and plated on to an iMatrix-coated (nippi) 12-well plate (2 × 104 cells/well). The medium was replaced with a differentiation medium containing 2% HS in DMEM after two days. Thereafter, the medium was changed every 2 days until analysis. The growth and differentiation media were supplemented with 1% penicillin-streptomycin.

Overexpression of Adenovirus-mediated large MAFs

The following adenoviral vectors were used to overexpress the large MAF transcription factor family in human iMuSCs and primary bovine MuSCs: pAV[Exp]-mCherry-CMV > hMAFA, pAV[Exp]-mCherry-CMV > hMAFB, and pAV[Exp]-mCherry-CMV > hMAF. pAV[Exp]-mCherry-CMV was used for control. All vectors were constructed and packaged by VectorBuilder Inc. (Chicago, IL, USA), with vector IDs as follows: VB010000-9300kfr (mCherry), VB900139-7528hhe (hMAFA), VB900139-7526uvc (hMAFB), and VB900139-7527axe (hMAF). The myotubes were incubated with adenovirus at a multiplicity of infection of 250 in differentiation medium for 48 h at two days post-differentiation of human iMuSCs or primary bovine MuSCs. The medium was then replaced with fresh differentiation medium without adenovirus. The myotubes were used for subsequent experiments at four days post-infection.

Immunofluorescence

Cultured cells were washed twice with PBS after removing the media and fixed with 4% paraformaldehyde for 15 min. The cells were permeabilized and blocked using 5% goat serum in 0.1% Triton PBS for 1 h at room temperature and subsequently washed thrice with PBS. The cultured cells were incubated with primary antibodies at 4 °C overnight. All immunostained samples were visualized using appropriate Alexa Fluor conjugated secondary antibodies (Thermo Fisher Scientific) for 1 h. Primary antibodies against RFP (600-401-379; Rockland), α-actinin (A7811; Sigma-Aldrich) were used. Moreover, secondary antibodies conjugated with Alexa Fluor-488, Alexa Fluor-555, and Alexa Fluor-647 were used.

Analysis of actively translated mRNA in human myotubes using AHA-mediated ribosomal isolation

The active ribosome was isolated with the AHARIBO RNA system (Immagina) as described previously [37] to analyze MYH4 mRNA translation in human myotubes. Briefly, adenovirus-infected human myotubes were washed once with PBS and treated for 40 min with methionine-free growth medium (Thermo Fisher Scientific) supplemented with 2% FBS and 0.8 mM l-leucine to deplete methionine reserves. After 40 min, 10 µL of AHA reagent was added to the medium. The cells were then incubated at 37 °C for 5 min, following the addition of 2.6 µL sBlock for 5 min at 37 °C. Next, they were placed on ice and washed once with 1 mL of cold PBS. PBS was carefully removed with a pipette, and cells were lysed with 45 µL of cold lysis buffer using a cell scraper. The cell lysate was transferred to a 1.5-mL microcentrifuge tube, and cell debris was pelleted through centrifugation at 20,000 × g for 5 min at 4 °C. The supernatant was transferred to a new tube and kept on ice for 20 min. Absorbance was measured by NanoDrop at 260 nm, with lysis buffer as blank subtraction.

Two absorbance units (AU) of the sample was transferred to a new tube, and the volume was adjusted to 100 µL with freshly prepared SWB buffer to capture active ribosomal complexes. To this, 100 µL of sBeads was added, and the mixture was incubated for 60 min at 4 °C on a rotating wheel. Next, the supernatant was removed, and the beads were washed twice with WSS buffer. The beads were resuspended in 200 µL of SWB buffer. RNA was then extracted from the resuspended SWB buffer containing the beads. The extracted RNA concentration was measured at 260 nm using a NanoDrop spectrophotometer, and cDNA was subsequently synthesized.

Quantitative analysis of transcripts using reverse transcription PCR

Total RNA was extracted from human and bovine myotubes using TRIzol reagent (Life Technologies) according to the manufacturer’s instructions. RNA (500 ng) or 340 ng of active ribosome-associated RNA was reverse transcribed using either the ReverTra Ace Kit with a genomic DNA remover (Toyobo, Osaka, Japan) or the QuantiTect Reverse Transcription Kit (Qiagen) for cDNA synthesis. Quantitative PCR was performed using the THUNDERBIRD SYBR qPCR system (Toyobo, Osaka, Japan) on a TP850 Thermal Cycler Dice Real-Time System (Takara Bio, Kusatsu, Japan). Primer sequences are listed in Supplementary Table 1. Transcription levels were normalized to the TATA-box-binding protein (TBP) transcript levels for each sample.

RNAscope

Adenovirus-infected human myotubes were fixed in 4% paraformaldehyde for 15 min to visualize MYH4 transcripts. Fixed cells were processed using the RNAscope Multiplex Fluorescent V2 Assay (323270; Advanced Cell Diagnostics), with MYH4 transcripts hybridized to Hs-MYH4-C1 probes (1583381-C1, ADC). Briefly, fixed cells were washed twice with PBS before incubation for 10 min in hydrogen peroxide solution. Cells were then treated with protease III and diluted 1:15 in PBS for 10 min. Next, they were washed twice and hybridized with the Hs-MYH4-C1 probe for 2 h at 40 °C. MYH4 transcripts were visualized using the TSA Vivid Fluorophore Kit 520 (R&D Systems) according to the manufacturer’s instructions. Cells were washed twice in PBS, and immunofluorescence analysis was performed using antibodies against α-actinin and RFP, as described above. Nuclei were counterstained with DAPI.

RNA-seq analysis of human iMuSC-derived myotubes

Total RNA was extracted from human myotubes on day 4 of infection using TRIzol reagent (Thermo Fisher Scientific). The RNA sequencing (RNA-seq) library was prepared using the NEBNext Ultra Directional RNA Library Prep Kit (New England Biolabs, Ipswich, MA, USA) after ribosomal RNA (rRNA) depletion (NEBNext rRNA Depletion Kit; New England Biolabs). Paired-end (2 × 36 bases) sequencing was performed using the NextSeq500 platform (Illumina, San Diego, CA, USA). FASTQ files were imported to the CLC Genomics Workbench (Version 10.1.1; Qiagen, Hilden, Germany). Sequence reads were then mapped to the human reference genome (hg19). Gene expression was calculated as total read counts normalized by transcripts per million. Genes with zero counts in any sample were excluded, and differential expression was analyzed using the Empirical Analysis of DGE tool (edgeR test) in the CLC Main Workbench (Version 22.0; Qiagen). Differentially expressed genes (DEGs) were extracted among conditions (pAV-mCherry vs. pAV-MAFA vs. pAV-MAFB vs. pAV-MAF) with a false discovery rate–corrected P < 0.05. The DAVID Bioinformatics Resources 6.8 [38] was used for GO analysis, with P-value < 0.05.

Seahorse XF Glycolysis stress test

iMuSCs were seeded in XFe 24-well plates (Seahorse Bioscience, North Billerica, MA, USA) at a density of 1 × 10⁴ cells/well. Adenoviral infection was performed on iMuSCs after 2 days of differentiation in differentiation medium (DM). The extracellular acidification rate (ECAR) was measured using a Seahorse XFe24 analyzer (Seahorse Bioscience) four days after infection. For the glycolysis stress test, glucose, oligomycin, and 2-deoxyglucose (2-DG) were sequentially added to the wells at final concentrations of 10 mM, 1.0 µM, and 50 mM, respectively. Glucose serves as a substrate for glycolysis, whereas oligomycin inhibits mitochondrial ATP synthase, consequently increasing reliance on glycolysis for energy production. Finally, 2-DG, a competitive inhibitor of glucose, effectively shuts down glycolysis.

Single-nucleus RNA-seq analysis

Single-nucleus RNA-seq data were downloaded from the Human Muscle Ageing Cell Atlas database (https://db.cngb.org/cdcp/hlma/), and the Seurat pipeline was applied to this dataset. The Seurat objects underwent normalization, scaling, and dimensional reduction. Types I and II myonuclei were classified based on previous studies [16]. We used density plots generated through the Nebulosa package in our snRNA-seq analysis to assess the localization of large MAFs within myonuclei [39]. Expression levels of large MAFs and MYH genes were measured in clusters of type II-positive myonuclei. Gene expression in the snRNA-seq data was illustrated using two approaches: first, by plotting the expression levels of the gene across all myonuclei from young (15–46 years old, n = 8) and old (74–99 years old, n = 19) individuals; and second, by comparing the gene expression levels within myonuclei aggregated for each individual, grouped into young and old categories.

Mass spectrometry

Human myotube samples (10 mg) were reduced, alkylated, and digested with trypsin/Lys-C using the iST kit (iST 8x, P.O.00001, PreOmics, Germany) according to the manufacturer’s instructions. The samples were then evaporated to dryness in vacuo to obtain residues. These residues were dissolved in 10 mL of LC-LOAD buffer from the iST kit. Peptides (200 ng) were injected into nanoLC/ESLI-MS/MS systems [LC: ACQUITY UPLC M-Class system (Waters, MA, USA), MS: ZenoTOF7600 (Sciex)]. The peptide samples were subsequently separated using the nanoEase M/Z Peptide CSH C18 column (1.7 mm, 0.075 3 150 mm) (Waters) at a flow rate of 300 nL/min. Mobile phase A consisted of a 0.1% (v/v) solution of formic acid in water, whereas mobile phase B consisted of a 0.1% (v/v) solution of formic acid in acetonitrile. The linear gradient conditions were as follows: 0–17 min: 2% solvent B, 17–125 min: 30% B, 126–127 min: 40% B, 127–132 min: 80% B, 132–133 min: 90% B, 133–153 min: 90% B, 153–154 min: 2% B, 154–180 min: 2% B. The mass spectrometer was operated in a Zeno SWATH acquisition scheme with 100 variable-size windows, and 25 ms accumulation time was used. The Zeno SWATH raw MS data were processed using DIA-NN 1.8.1 [40], available on github (DIA-NN github repository).

The human spectral libraries were generated from the human spectral library and human UniProt database (id UP000005640, reviewed, canonical). The DIA-NN search parameters were as follows: experimental data search enzyme, trypsin; missed cleavage sites, 1; peptide length range, 7–30; precursor mass charge range, 1–4; precursor m/z range, 300–1800; fragment ion m/z range, 200–1800; and static modification, cysteine carbamidomethylation. The protein identification threshold was set at < 1% for both peptide and protein FDR. Each peptide assigned to MYH4 was verified through a BLAST search, and peptides specific to MYH4 were counted. The newly acquired mass spectrometry proteomic data have been deposited to the Proteome Xchange Consortium via jPOSTrepo [41] with the dataset identifier JPST003487.

Comparison of gene expression patterns across multiple animal species

Bgee v15.2.0 was used [42] to compare MYH4 and large MAF expression scores across animal species. Expression scores are based on non-parametric statistics, where conditions are ranked based on the gene expression levels. These ranks are then normalized across conditions, genes, and data types, and transformed into an expression score ranging from 0 (low expression) to 100 (high expression). These non-parametric statistics enable quantitative comparison of gene expression across species and conditions without requiring batch-correction procedures. The anatomical entities included in the analysis were skeletal muscle tissue (opossum, dog, and cow), quadricep muscle (mouse, rat, and human), and general muscle tissue (horse).

Motif enrichment analysis

The presence of MAF recognition element sites in the promoter regions of genes were predicted through the Find Individual Motif Occurrences (FIMO) algorithm of the MEME Suite [43], using the position weight matrix available from JASPAR (https://jaspar.genereg.net/). This analysis was run from 3 kb upstream to 3 kb downstream of the Myh4 transcriptional start in mice, rats, oppossums, humans, dogs, cows, and horses.

Study participants

The Russian part of the study was approved by the Ethics Committee of the Federal Research and Clinical Center of Physical–Chemical Medicine of the Federal Medical and Biological Agency of Russia (protocol no. 2017/04). The Finnish part of the study was approved by the Hospital District of Helsinki and Uusimaa (these data were used with permission; Database of Genotypes and Phenotypes Study Accession: phs001048.v2.p1). Written informed consent was obtained from each participant. The study complied with the Declaration of Helsinki and ethical standards for sport and exercise science research.

The Russian gene expression study (analysis of MAF gene expression in m. vastus lateralis) involved 24 men (mean age ± SD: 32.7 ± 8.9 years; mean height: 180.8 ± 6.8 cm; mean body mass: 80.1 ± 11.6 kg; mean percentage of fast-twitch muscle fibers: 50.3 ± 21.3%; mean percentage of slow-twitch muscle fibers: 52.7 ± 21.6%; cross-sectional area (CSA) of fast-twitch muscle fibers: 5934 ± 1833 µm2; CSA of slow-twitch muscle fibers: 5545 ± 1197 µm2). Outliers were identified using Grubbs’ test (Alpha = 0.05) in GraphPad Prism; three participants with outlying MYH4 expression levels were excluded from the analysis.

The Finnish study involved 291 individuals (166 men, mean age 59.5 ± 8.1 years; mean height: 176.7 ± 6.7 cm; mean body mass: 87.3 ± 15.1 kg; mean percentage of fast-twitch muscle fibers: 58.9 ± 14.7%; mean percentage of slow-twitch muscle fibers: 41.1 ± 14.7%; 125 women, mean age: 60.3 ± 8.1 years; mean height: 162.8 ± 5.6 cm; mean body mass: 71.8 ± 9.8 kg; mean percentage of fast-twitch muscle fibers: 49.9 ± 13.3%; mean percentage of slow-twitch muscle fibers: 50.1 ± 13.3%) from the FUSION study, as previously described [44].

Evaluation of muscle fiber composition in human muscle biopsy samples

Vastus lateralis samples of Russian participants were obtained from the left leg using the modified Bergström needle procedure, with aspiration under local anesthesia using a 2% lidocaine solution. Samples were frozen in liquid nitrogen and stored at -80 °C before analysis. Serial cross-Sect. (7 μm) were obtained from frozen samples using an ultratom (Leica Microsystems, Wetzlar, Germany). Sections were then thaw-mounted on Polysine glass slides, maintained at room temperature (RT) for 15 min and incubated in PBS (3 × 5 min). The sections were then incubated at RT in primary antibodies against slow or fast isoforms of the myosin heavy chains (M8421, 1:5000; M4276; 1:600, respectively; Sigma-Aldrich) for 1 h and incubated in PBS (3 × 5 min). Next, the sections were incubated at RT in secondary antibodies conjugated with FITC (F0257; 1:100; Sigma-Aldrich) for 1 h. Antibodies were removed, and the sections were washed in PBS (3 × 5 min), placed in mounting media, and covered with a cover slip. Images were captured with a fluorescent microscope (Eclipse Ti-U; Nikon, Tokyo, Japan). All analyzed images contained 334 ± 14 fibers. The ratio of the number of stained fibers to the total fiber number was calculated. Fibers stained in serial sections with antibodies against slow and fast isoforms were considered hybrid fibers. The CSAs of fast- and slow-twitch muscle fibers were evaluated using ImageJ software (NIH, USA).

Muscle fiber composition in 287 Finnish individuals was estimated based on the expression of MYH1, MYH2, MYH7, Ca2+ ATPase A1, and Ca2+ ATPase A2 genes, as previously described [44]. Muscle samples were obtained from the vastus lateralis using a conchotome, under local anesthesia with 20 mg∙ml− 1 lidocaine hydrochloride without epinephrine.

RNA-seq analysis of human muscle biopsy samples

Russian participants were asked not to train for one day before biopsy of the vastus lateralis of the left leg to analyze their gene expression profiles at the resting state. The RNeasy Mini Fibrous Tissue Kit (Qiagen) was used to isolate RNA from 24 muscle tissue samples. Frozen tissue samples were placed in a box submerged in liquid nitrogen. Each frozen sample was transferred onto a sterile Petri dish placed on a frozen plastic ice pack. A piece of tissue with a weight of 10 mg was separated with a sterile scalpel and immediately placed in a 2 mL safe-lock microcentrifuge tube containing 300 µL of lysis buffer and one sterile stainless-steel bead with a diameter of 4 mm. Samples were homogenized using the TissueLyser II system (Qiagen) and shaken twice for 2 min at 25 Hz. RNA samples were isolated according to the manufacturer’s guidelines. RNA concentration was measured using the Qubit spectrophotometer (Thermo Fisher Scientific). RNA quality was assessed using the BioAnalyzer electrophoresis system and BioAnalyzer RNA Nano assay (Agilent Technologies, Santa Clara, CA, USA). The RNA integrity number (RIN) was calculated for each RNA sample. Only RNA samples with RIN > 7 were included in the study. Samples were stored at − 80 °C until sequencing libraries were prepared. Total RNA samples were treated with DNase I using the Turbo DNA-free Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Libraries for RNA sequencing were prepared using the Illumina NEBNext Ultra II Directional RNA Library Prep Kit with the NEBNext rRNA Depletion Module (New England Biolabs). RNA libraries were sequenced on the Illumina HiSeq system with 250 cycles. Sequenced reads were pseudoaligned to the hg38 gencode (v37) transcriptome using kallisto v0.48.0 [45] with default settings. Gene-level expression abundances were calculated using the tximport Bioconductor package [46]. Expression of large MAFs and MYH genes is presented in transcripts per million (TPM).

RNA isolation and sequencing in the Finnish samples was performed using strand-specific mRNA-seq, as previously described [44]. Using the basic GENCODE v19 annotations [47], we counted fragments mapping to each gene using htseq-count v0.5.4 [48] and quantified gene expression as TPM.

Statistical analysis

Data are presented as the mean ± SEM, as indicated in the Figure legends. Statistical significance was determined using GraphPad Prism software (v10.4.0) through Student’s t-test or one-way analysis of variance with Tukey’s test. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant.