Mitochondrial types and mitochondria-ER contact sites in aged human muscle precursor cells

hSKMC (human skeletal muscle cells) were purchased from Lonza Walkersville, Inc. (CC-2561) or PromoCell GmbH (C-12530) and initiated from liquid nitrogen at the start of each experiment. The cells were cultured for 24 h to acclimate to the cell culture medium (Promocell, C-23160) and then seeded into the respective culture vessels for each assay. If necessary, the cells were transfected with small interfering RNAs (siRNAs) using Lipofectamine RNAiMAX Reagent (Invitrogen). Silencer Select siRNAs against hMFN1(s31218) and DRP1(s19559) were purchased from Thermo Fisher Scientific. Knockdown efficiency for each siRNA was confirmed by Western blotting.

PLA was performed using the Duolink In Situ Red Starter Kit Mouse/Rabbit (Duolink In Situ Detection Reagents Red (Sigma-Aldrich, DUO92008), Duolink In Situ PLA Probe Anti-Mouse MINUS (Sigma-Aldrich, DUO92004), and Duolink In Situ PLA Probe Anti-Rabbit PLUS (Sigma-Aldrich, DUO92004), according to the manufacturer’s instructions. hSKMC were fixed with 10% formaldehyde in PBS, quenched with an equal volume of 1M Glycine, and washed once with 100 mM glycine. If necessary, they were treated with MTTEMPO (Mito-TEMPO, Final 0.1 µM) for 1 h at 37 °C before fixing. After permeabilization with 0.1% Triton, hSKMC were incubated with Duolink blocking solution for 1 h at 37 °C and then incubated with primary antibodies, VDAC1 (mouse; abcam ab186321) and IP3R1 (rabbit; Invitrogen PA1–901), in Duolink antibody diluent overnight at 4 °C. Secondary antibodies PLUS and MINUS (anti-rabbit and anti-mouse IgG antibodies conjugated with oligonucleotides) were incubated 1:5 in Duolink antibody diluent for 1 h at 37 °C. The ligation solution (ligase 1:40 in ligation buffer) was then incubated for 30 min at 37 °C. The amplification solution (polymerase 1:40 in amplification buffer) was incubated for 1 h 40 min at 37 °C and then incubated with Alexa Fluor 488-phalloidin (Invitrogen, A12379) for 20 min at RT. After incubation, the slides were mounted using the Vectashield Vibrance Antifade Mounting Medium containing DAPI. PLA images were acquired using a Zeiss LSM800 (Zeiss), ZEN acquisition software, at 63 × magnification.

hSKMC were seeded on an 8-well chamber slide (Matsunami) and fixed with 4% Paraformaldehyde in PBS. The cells were then permeabilized for 10 min with 0.2% Triton and blocked by incubation with Blocking One Histo (Nacalai) for 1 h. After blocking, the cells were incubated for 1 h at RT with mouse monoclonal anti-TOM20 antibody (ab56783; 1:40 in PBS-T with 5% Blocking One Histo). Alexa Fluor 555 goat anti-mouse IgG (Invitrogen, A21422) secondary antibodies were incubated at a dilution of 1:500 in PBS-T with 5% Blocking One Hist for 30 min at RT and then incubated with Alexa Fluor 488-phalloidin for 20 min at RT. After incubation, the slides were mounted using the Vectashield Vibrance Antifade Mounting Medium containing DAPI. Images were acquired using Zeiss LSM800 (Zeiss), ZEN acquisition software, at 63 × magnification.

Young and old hSKMC were seeded on a 35 mm petri-dish (Matsunami). The cells were fixed with 2.5% glutaraldehyde (FUJIFILM Techno Products Co., Ltd., 072–02262) in a warm medium (Promocell, C-23160) for 1 h. The cells were scraped and collected using a 0.2% bovine serum albumin/0.1M PBS buffer. After washing the cells three times for five minutes in 0.1M PBS, samples were post-fixed in 1% OsO₄ (Nisshin EM, cat no. 3028) with 1.5% potassium ferrocyanide (ScyTek Laboratories, Inc., PFD125) for 2 h. After rinsing the samples three times for five minutes with H₂O, the cells were incubated in 1% thiocarbohydrazide (Tokyo Chemical Industry Co., Ltd, T1136) for ten minutes. After rinsing the cells with H₂O three times, they were fixed with 1% osmium tetroxide in H₂O for 30 min at room temperature. Samples were washed three times with H₂O and incubated overnight with 2% uranyl acetate at 4 °C. Samples were washed three times with H₂O at room temperature and another three times in H₂O at 50 °C. Following the washing steps, cells were incubated in Walton’s lead at pH 5.2 (0.635% lead nitrate: FUJIFILM WakoPure Chemical Co., 124–00612, and 0.4% aspartic acid: SIGMA-ALDRICH, 01-6810-2) at 50 °C for 20 minutes. The final cell pellet was embedded in 2% low-melting-point agarose (NIPPON GENE Co., LTD., 312–06512) on ice. Agarose blocks containing the cell pellets were cut into 1–2 mm³ and dehydrated in a series of increasing ethanol concentrations (30%, 50%, 70%, and 90%) for 10 min at each concentration. A further dehydration step was performed using 100% ethanol for 10 min, three times, and twice for five minutes in a 1:1 (v/v) ethanol: acetone mixture. Finally, the samples were dehydrated thrice in pure acetone for five minutes three times at room temperature. Following the dehydration steps, resin infiltration was performed by incubating the sample in 1:1 (v/v) acetone: low-viscosity resin (Agar Scientific, AGR 1078) for 30 min with slow rotation using a tube rotator (AS ONE CO., TR-118) at room temperature. After confirming that the agar blocks sank to the bottom of the acetone/resin mixture, the samples were replaced with 100% low-viscosity resin and infiltrated for 1 h under vacuum conditions. The samples were incubated overnight in 100% resin in a vacuum chamber for further infiltration. The samples were replaced with fresh 100% resin and vacuumed for 1 h in a resin silicon mold. After orienting the sample position, resin samples were polymerized at 60 °C for 48 hours in the oven. Low Viscosity Resin mixture was prepared with the formula of LV Resin 48.0 g, VH1 Hardener 16.0 g, VH2 Hardener 36.0 g, and LV Accelerator 2.5 g. Resin blocks were trimmed with glass knives, and ultrathin 60 nm sections were made using a Diamond knife (ultra; DiATOME) in Leica Ultramicrotome (UC7). Ultrathin sections were collected on a 0.5% formvar-coated (SPI Supplies, 02463-MB) one-slot grid (EM, JAPAN) and dried. Observations and imaging were performed using a JEOL JEM-1400 FLASH 100 keV Transmission Electron Microscope.

Quantitation of SA-βGal activity was evaluated using Cellular Senescence Plate Assay Kit – SPiDER-βGal, a fluorogenic substrate for β-galactosidase (Dojindo), following the manufacturer’s instructions.

Cell viability and relative cell growth were assessed using the RealTime-Glo™ MT Cell Viability Assay (Promega) according to the manufacturer’s protocol. Luminescence was measured at the indicated time points, and cell growth ratio was calculated as the fold change in signal intensity relative to the initial time point (0 h), reflecting changes in viable cell number over time.

For TMRM staining, cells were seeded in a 0.17 mm glass-bottom 10-well chamber (Greiner Bio-One, 543978). After being washed once with imaging medium (Skeletal Muscle Cell Basal Medium phenol red free; PromoCell, C-23265), they were incubated with imaging medium containing TMRM (Final 0.2 µM) and MitoBright LT Deep Red (Final 0.8 µM, Dojindo) for 30 min at 37 °C. If necessary, they were treated with FCCP (Final 10 µM) for 10 min at 37 °C after staining. The staining or FCCP medium was replaced with a fresh imaging medium. Images were acquired using Zeiss LSM800 (Zeiss), ZEN acquisition software, at 63 × magnification. For LipiRADICAL [17] (Funakoshi, FDV-0042) staining, cells were seeded in a 0.18 mm glass-bottom 8-well chamber (ibid, 80829). If necessary, they were treated with MTTEMPO (Final 10 µM) for 1 h at 37 °C before staining. After being washed once with imaging medium, they were incubated with imaging medium containing LipiRADICAL (Final 1 µM) and MitoBright LT Deep Red (Final 0.8 µM) for 1 h at 37 °C. After washing once with imaging medium, the medium was replaced with fresh imaging medium. Images were acquired using Zeiss LSM800 (Zeiss), ZEN acquisition software, at 63 × magnification.

hSKMC were plated in a Seahorse XFe24 (Agilent Technologies) Extracellular Flux Analyzer culture plate in culture medium overnight. Prior to the assay, cells were switched to XF DMEM (Seahorse Biosciences) supplemented with 10 mM glucose, 2.0 mM glutamine, and 1.0 mM sodium pyruvate, following the manufacturer’s instructions. The oxygen consumption rate (OCR) during the Mito stress test was measured using a Seahorse XFe24 analyzer (Agilent) and an Extracellular Flux Analyzer (Seahorse Biosciences), following the manufacturer’s instructions. Following the Mito stress test, 1.5 µM oligomycin, 2.0 µM FCCP, and 0.5 µM antimycin A diluted in assay medium were the final concentrations in the wells.

All reactions were performed using QuantStudio 3 (Thermo Fisher Scientific). Each sample was analyzed in triplicate for each PCR. Melting curves were checked for specificity. All mRNA expression levels were normalized to those of Actb. For quantification of mtDNA, total DNA was extracted from hSKMC using a QIAamp DNA Mini Kit (Qiagen). The following primers were used to quantify mtDNA: Actb (Hs01060665_g1), Nd1 (Hs02596873_s1), Nd5 (Hs02596878_g1), and Cytb (Hs02596867_s1), all of which were purchased from Thermo Fisher Scientific.

hSKMC were lysed by RIPA A buffer (Wako) supplemented with Halt protease and phosphatase inhibitor cocktail and EDTA solution for 30 min at 37 °C, and the supernatant was collected after centrifugation (15,000 rpm for 5 min at 4°C). The cell lysates were quantified using the Pierce BCA Protein Assay kit (Thermo Fisher Scientific), separated by 5–20% SDS-PAGE, and blotted onto Immobilon PVDF membranes (IPVH07850, Millipore). Blots were probed with the indicated antibodies, and protein bands were visualized using Amersham^TM ECL^TM Prime (Cytiva). Band images were captured using FUSION SOLOS (Vilber-Lourmat). Relative band intensities were quantified using the Fiji/ImageJ software.

Samples were digested with phase-transfer surfactants according to previously reported methods [18] and subjected to liquid chromatography-tandem mass spectrometry (LC–MS/MS) analysis using an UltiMate 3000 Nano LC system (Thermo Fisher Scientific) coupled with an Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific) for data-independent acquisition. Samples were injected by an autosampler and enriched on a C18 reverse-phase trap column (100 μm inner diameter × 5 mm length; Thermo Fisher Scientific) at a flow rate of 8 μL/min. They were subsequently separated on an IonOpticks 25 cm Aurora column (Bruker Daltonics, Fitzroy, VIC, Australia) at a flow rate of 300 nL/min with a linear gradient from 3% to 35% mobile phase B. Mobile phase B consisted of acetonitrile with 0.1% formic acid, and mobile phase A consisted of distilled water with 0.1% formic acid. The peptides were ionized using nano-electrospray ionization in the positive ion mode. A spray voltage of 2,100 V was applied. MS1 spectra were collected in the m/z range of 497–740 at 15,000 resolution to set an auto-gain control (AGC) target of 3 × 10^6. MS2 spectra were collected in the m/z range of 200–1,800 at 45,000 resolution to set an AGC target of 3 × 10^6, automatic maximum injection time, and stepped normalized collision energies of 22%, 26%, and 30%. Data-independent acquisition (DIA) data were analyzed using DIA-NN (version 1.8.1) against the UniProt human database (https://www.uniprot.org↗; downloaded February 2022 with 20,577 sequences) in the robust liquid chromatography (high precision) mode with retention time–dependent cross-run normalization. The enzyme trypsin was set to be fully specific, allowing for one missed cleavage. Both Mass and MS1 accuracies were set to 10 ppm, a match between runs was enabled, and the precursor false discovery rate was controlled at 1%. Carbamidomethylation of cysteine was set as a fixed modification, and oxidation of methionine was set as a variable modification.

Mitochondrial morphology was analyzed using the plugin, and MiNA morphology was analyzed using StuartLab from the ImageJ/Fiji Software. The PLA images were analyzed manually with ‘Threshold’ and ‘Analyze particles’ commands from ImageJ/Fiji Software, and PLA dot areas were normalized on each cell area. The percentage of MERCS from Electron Microscopy images was measured as the ratio of the mitochondria-ER distance of <25 nm lengths the mitochondrial perimeter. The lengths and perimeters were analyzed manually using ImageJ/Fiji Software. The intensity of LipiRADICAL staining was measured as the integrated density of each cell type. We measured the intensity of TMRM in only the mitochondrial area, analyzed manually with ‘Threshold’ and ‘Analyze particles’ commands from ImageJ/Fiji Software from the MitoBright LT Deep Red images. Statistical significance between two groups was determined using a standard unpaired two-sided t-test with Welch’s t-test. The statistical significance between multiple groups was determined by one-way analysis of variance (ANOVA) to ensure comparable variance, and comparisons for controls were performed using Dunnett’s test. The analysis was done in Prism 10, GraphPad. The asterisk represents a p-values, *p < 0.05, **p < 0.01, ***p < 0.001, and **** p < 0.0001. No statistical methods were used to determine the sample sizes. The investigators were not blinded to the allocation during the experiments or outcome assessment. Because imaging quantification was not performed under blinded conditions, the possibility of measurement bias cannot be excluded.

Primary human skeletal muscle cells used in this study were obtained commercially from PromoCell GmbH and Lonza Group Ltd., which collected donor tissue under informed written consent and with approval from the appropriate ethics committee. All samples were fully anonymized by the supplier prior to distribution. Because the authors used only commercially available, fully anonymized human primary cells, additional ethics approval and participant consent for this research were not required, in accordance with the institutional guidelines and PLOS ONE human subjects research policies.

We compared commercially available human skeletal muscle myoblasts derived from young and elderly donors to determine whether these cells from elderly donors exhibit a senescence phenotype. For all experiments, myoblasts from healthy Caucasian male donors aged 20–30 years and 60–90 years were used as young and old cells, respectively. This study used commercially available human skeletal muscle cells, and therefore ethics approval was not required. To prevent changes in myoblast characteristics due to long-term culture or repeated passages, fresh cell stocks stored in liquid nitrogen were used for each experiment. Passaging was limited to one passage, and the total culture period was maintained at 5 days [19,20]. Examination of SA-βGal activity, Lamin B1, and p16 expression in young and old cells revealed that SA-βGal activity was significantly increased, while Lamin B1 was significantly decreased in old cells (Fig 1a and 1b). In addition, p16 protein levels showed an increasing trend in old myoblasts, although this difference did not reach statistical significance (Fig 1b). Proteomic analysis of whole cell lysates from young and old cells showed enhanced oxidative stress responses in old cells, as indicated by increased expression of SOD2 and ALDH1A3 (retinaldehyde dehydrogenase 3). In contrast, young cells have higher levels of proteins involved in protein synthesis, cell proliferation, and signal transduction, such as DDRGK1(DDRGK domain-containing protein 1), SPRY4 (protein sprouty homolog 4), and RPS20 (small ribosomal subunit protein uS10), indicating their higher proliferative capacity (Fig 1c). Additionally, the PCA plot results showed that young and old cell groups could be distinctly categorized, validating their suitability for aging phenotype analysis (Fig 1d). Old cells exhibited reduced cell growth compared to young cells (Fig 1e), further confirming the senescent phenotype. These findings indicate that myoblasts from the elderly donors used in this study displayed a typical senescence phenotype of proliferative stem cells.

Next, we evaluated the mitochondrial energy metabolism in old cells using a Seahorse Flux Analyzer to measure oxygen consumption. The results demonstrated an increased oxygen consumption in old cells (Fig 2a). Additionally, immunostaining revealed excessive mitochondrial fusion in old cells (Fig 2b), suggesting an increased mitochondrial mass. Importantly, the increased oxygen consumption in old cells was accompanied by a preserved increase in maximal respiration following FCCP treatment (Fig 2a), arguing against a state of pre-existing mitochondrial uncoupling. If mitochondria were already substantially uncoupled, FCCP would not be expected to further elevate oxygen consumption. In addition, FCCP treatment induced marked mitochondrial fragmentation in young cells (Fig 2b), whereas mitochondria in old cells did not display a comparable fragmented morphology, further supporting that elevated OCR in old cells is unlikely to reflect a pre-uncoupled state. Therefore, to determine whether mitochondrial quantity explains the elevated oxygen consumption, we quantified mitochondrial DNA. Mitochondrial DNA content was assessed by quantifying ND1 (NADH-ubiquinone oxidoreductase chain 1), ND5 (NADH-ubiquinone oxidoreductase chain 5), and CYB (cytochrome b). As anticipated, mitochondrial content increased in old cells (Fig 2c), suggesting a correlation with increased oxygen consumption. However, according to proteomics data, there was no change in the expression of respiratory enzymes (S1a in S1 Fig). We further quantified membrane potential per mitochondrial area. Staining with MT Bright Deep Red and TMRM allowed for the evaluation of membrane potential intensity relative to mitochondrial quantity, with no significant difference observed between young and old cells (S1b in S1 Fig). Thus, we did not observe a decline in key aspects of mitochondrial function in myoblasts from elderly donors.

MERCS are associated with various mitochondrial functions, such as bioenergetic regulation via calcium influx [21] and the removal of lipid peroxides through reactive oxygen species [22]. Given that mitochondrial function was maintained in old cells, we examined whether age-associated changes in MERCS were present and therefore quantified MERCS. The results indicated a significant increase in MERCS in old cells (Fig 3a and S2a in S2 Fig). However, proteomic data revealed no changes in the expression levels of tethering proteins in MERCS (S2b in S2 Fig). Considering the proteomics results showing increased SOD2 expression in old cells (Fig 1c) and reports that MERCS increase in oxidatively stressed mitochondria [22], we hypothesized that mitochondrial oxidative stress contributes to increased MERCS in old cells. Lipid peroxide detection using LipiRADICAL Green showed a significant increase in peroxide levels in old cells (Fig 3b). Moreover, suppression of mtROS by MTTEMPO reduced peroxide generation (Fig 3b). Furthermore, reducing mitochondrial oxidative stress in old cells treated with MTTEMPO resulted in a decrease in MERCS (Fig 3c). These findings suggest that mitochondria in old cells are in an oxidative state and that increased MERCS may be involved in cellular responses to mitochondrial oxidative stress.

All relevant data are within the paper and its Supporting Information files. The ImageJ macro used to assist part of the image analysis in this study is publicly available via Zenodo (DOI: https://doi.org/10.5281/zenodo.18251810↗). The macro automates only specific steps of the analysis, and manual operations are required during processing.