High-Intensity Interval Training Mitigates Sarcopenia and Suppresses the Myoblast Senescence Regulator EEF1E1

Abstract

Background: The optimal exercise regimen for alleviating sarcopenia remains uncertain. This study aimed to investigate the efficacy of high-intensity interval training (HIIT) over moderate-intensity continuous training (MICT) in ameliorating sarcopenia.

Methods: We conducted a randomized crossover trial to evaluate plasma proteomic reactions to acute HIIT (four 4-min high-intensity intervals at 70% maximal capacity alternating with 4 min at 30%) versus MICT (constant 50% maximal capacity) in inactive adults. We explored the relationship between a HIIT-specific protein relative to MICT, identified via comparative proteomic analysis, eukaryotic translation elongation factor 1 epsilon 1 (EEF1E1) and sarcopenia in a paired case-control study of elderly individuals (aged over 65). Young (3 months old) and aged (20 months old) mice were randomized to sedentary, HIIT and MICT groups (five sessions/week for 4 weeks; n = 8 for each group). Measurements included skeletal muscle index, hand grip strength, expression of atrophic markers Atrogin1 and MuRF1 and differentiation markers MyoD, myogenin and MyHC-II via western blotting. We examined the impact of EEF1E1 siRNA and recombinant protein on D-galactose-induced myoblast senescence, measuring senescence-associated β-galactosidase and markers like p21 and p53.

Results: The crossover trial, including 10 sedentary adults (32 years old, IQR 31-32) demonstrated significant alterations in the abundance of 21 plasma proteins after HIIT compared with MICT. In the paired case-control study of 84 older adults (84 years old, IQR 69-81; 52% female), EEF1E1 was significantly increased in those with sarcopenia compared to those without (14.68 [95%CI, 2.02-27.34] pg/mL, p = 0.03) and was associated with skeletal muscle index (R² = 0.51, p < 0.001) and hand grip strength (R² = 0.54, p < 0.001). In the preclinical study, aged mice exhibited higher EEF1E1 mRNA and protein levels in skeletal muscle compared to young mice, accompanied by a lower muscle mass and strength, increased cellular senescence and protein degradation markers and reduced muscle differentiation efficiency (all p < 0.05). HIIT reduced EEF1E1 expression and mitigated age-related muscle decline and atrophy in aged mice more effectively than MICT. Notably, EEF1E1 downregulation via siRNA significantly counteracted D-galactose-induced myoblast senescence as evidenced by reduced markers of muscle protein degradation and improved muscle differentiation efficiency (all p < 0.05). Conversely, treatments that increased EEF1E1 levels accelerated the senescence process (p < 0.05). Further exploration indicated that the decrease in EEF1E1 was associated with increased SIRT1 level and enhanced autophagy.

Conclusions: This study highlights the potential of HIIT as a promising approach to prevent and treat sarcopenia while also highlighting EEF1E1 as a potential intervention target.

Keywords: EEF1E1; autophagy; high‐intensity interval training; sarcopenia; senescence.

FIGURE 1

Evaluating plasma protein level changes in sedentary adults following MICT and HIIT. MICT refers to moderate‐intensity continuous training, whereas HIIT denotes high‐intensity interval training. (a) Randomized crossover trial flowchart: Outline of the study design aimed at identifying differentially expressed plasma proteins in sedentary adults. (b) Protein expression heatmaps and Venn diagram: Heatmaps of proteins with differential expression after MICT and HIIT. A Venn diagram is included to show proteins uniquely or commonly affected by MICT and HIIT. (c) EEF1E1‐associated biological processes: Outline of the biological processes related to EEF1E1. (d) EEF1E1 plasma levels: Comparison of the relative plasma concentrations of EEF1E1 before and after MICT and HIIT. (e) EEF1E1 protein structure prediction: Illustration depicting the predicted EEF1E1 protein structure based on UniProt data. Results are shown as the median with the interquartile range (IQR) (n = 10). The significance of changes in EEF1E1 levels due to MICT or HIIT was assessed using a paired t‐test.

FIGURE 2

Linking EEF1E1 with sarcopenia in older adults. EEF1E1 represents the eukaryotic translation elongation factor 1 epsilon 1. This figure summarizes results from a sarcopenia case–control study in a population of community‐dwelling older adults (n = 84). (a) Study flowchart: Illustration of the design and progression of the sarcopenia case–control study. (b) Comparisons of EEF1E1 plasma levels, appendicular skeletal muscle mass, SMMI values, left calf circumference measurements, right calf circumference measurements, handgrip strength assessment, 6‐m walk test speeds and 30‐s chair stand test performance between the control and sarcopenia groups. Comparative analysis: An independent t‐test was used for comparing the difference in outcomes between the control and sarcopenia groups. (c) Correlation analysis: A Pearson correlation, adjusted for biological sex and age, was used to assess the relationship between EEF1E1 levels and appendicular skeletal muscle mass, skeletal muscle mass index, handgrip strength and 6‐m walk test speed. (d) Correlation analysis: A Pearson correlation, adjusted for biological sex, was used to assess the relationship between age and EEF1E1 levels, hand grip strength and 6‐m walk speed.

FIGURE 3

HIIT counteracts muscle mass and strength loss in aged mice. (a) Animal protocol: The diagram illustrates the protocol used. Aged mice (20 months old) were divided into three groups: sedentary (A‐Sed), moderate‐intensity continuous training (A‐MICT) and high‐intensity interval training (A‐HIIT). These groups underwent respective 4‐week training programs. Young mice (3 months old) served as the normal control group (Young). (b) Physical performance tests: The grip strength, hanging time and maximum running speed of the mice were evaluated (n = 8). (c) Body and muscle weight assessment: body weight in grams, muscle complex mass (gastrocnemius, plantaris and soleus) of a single leg in milligrams and the ratio of muscle complex mass to body weight were assessed (n = 8). (d) myofibre analysis: The cross‐sectional area (CSA) of gastrocnemius myofibres was measured using haematoxylin–Eosin (HE) and wheat germ agglutinin (WGA) staining (scale bar = 100 μm, n = 8). (e) Fibre type identification: Fast (red) and slow (green) muscle fibres were identified using stained with immunofluorescence staining (scale bar = 100 μm). The CSA of fast muscle fibres was analysed (n = 8). Data are presented as the mean ± SD. An independent two‐tailed t‐test was used for comparisons between the Young and A‐Sed groups. An ANOVA with Bonferroni multiple comparisons test was applied for the A‐Sed, A‐MICT and A‐HIIT groups.

FIGURE 4

HIIT mitigates EEF1E1 expression and atrophy in aged mice. (a) Eef1e1 mRNA expression in young and aged mice: Eef1e1 mRNA levels in various main organs were assessed (n = 4). (b) Plasma EEF1E1 protein levels: Plasma EEF1E1 protein expression was determined (n = 6). (c,d) Muscle EEF1E1 protein and Eef1e1 mRNA expression: The EEF1E1 protein and Eef1e1 mRNA levels in each group were measured (n = 6). (e) Protein expression of atrophy and differentiation markers: The expression levels of the atrophic factors Atrogin1 and MuRF1 and the differentiation markers MyoD, myogenin and MyHC II were determined (n = 6). Data are presented as the mean ± SD. An independent two‐tailed t‐test was used for comparisons between the Young and A‐Sed groups. An ANOVA with Bonferroni multiple comparison test was applied for the A‐Sed, A‐MICT and A‐HIIT groups.

FIGURE 5

Eef1e1 suppression ameliorates measures of senescence, reduces protein degradation and accelerates fusion in skeletal muscle cells. (a) SA‐β‐galactosidase staining in myoblasts: Myoblasts incubated with varying concentrations of D‐galactose (D‐gal) underwent SA‐β‐ galactosidase staining. Analysis was performed using a scale bar of 100 μm (n = 5). Protein expression analysis: The protein expression levels of EEF1E1, p21 and p53 were determined (n = 3). (b) Effects of Eef1e1‐siRNA transfection: Cells were transfected with Eef1e1‐siRNA (si‐Eef1e1) or control‐siRNA (si‐NC) in the presence of D‐gal or phosphate‐buffered saline (PBS). SA‐β‐Galactosidase staining assessment: Staining was performed and analysed (scale bar = 100 μm, n = 5). The protein and mRNA expression levels of EEF1E1 were measured (n = 3). Atrophy markers: The protein expression of Atrogin1, MuRF1 and MyHC II was determined (n = 3). (c) MyHCII immunofluorescence: Cellular immunofluorescent staining of MyHCII was conducted post‐differentiation into myotubes (scale bar = 100 μm, n = 3). Myotube index: The ratio of nuclei in myotubes to total nuclei was analysed as an index of myotube formation (n = 3). Myogenic factors: mRNA levels of myogenin and MyoD were measured (n = 3). (d) Cell cycle analysis: The cell cycle was assessed using flow cytometry (n = 3). Data are presented as the mean ± SD. ANOVA with Bonferroni multiple comparison test was applied to (a). For (b,c), ANOVA and independent two‐tailed t‐tests were used as deemed appropriate.

FIGURE 6

Eef1e1 overexpression and supplementation exacerbate measures of senescence, promote protein degradation and inhibit fusion in skeletal muscle cells. (a) EEF1E1 protein expression in myoblasts: Myoblasts were treated with phosphate‐buffered saline (PBS), recombinant EEF1E1 protein (rEEF1E1) or transfected with an Eef1e1 plasmid (oeEef1e1) and the EEF1E1 protein expression was determined (n = 3). (b) SA‐β‐Galactosidase staining was performed on treated myoblasts; scale bar = 100 μm (n = 5). (c) Protein expression: Determination of protein levels of Atrogin1, MuRF1 and MyHCII (n = 3). (d) MyHCII immunofluorescence was performed post‐differentiation into myotubes; scale bar = 100 μm. The ratio of nuclei in myotubes to total nuclei was analysed (n = 5). (e) mRNA expression: mRNA levels of MyoD and Myogenin (n = 3). (f) Cell cycle analysis was performed using flow cytometry (n = 3). Data are expressed as the mean ± SD. Statistical analysis was performed using ANOVA with Bonferroni multiple comparison test.

FIGURE 7

Correlation between sarcopenia in mice and D‐gal‐induced myoblast senescence with decreased SIRT1 levels and autophagy activity. (a) Protein–protein docking: Illustration of the interaction between EEF1E1 and SIRT1. (b) Experimental groups and training protocols: Aged mice were categorized into sedentary (A‐Sed), moderate‐intensity continuous training (A‐MICT) and high‐intensity interval training (A‐HIIT) groups, each undergoing a 4‐week training programme. Young mice served as the normal control group (Young). Electron microscopy of skeletal muscle samples: Transmission electron microscope images highlighting damaged mitochondria (yellow arrows) and autophagy vesicles (av); scale bars = 2 or 1 μm. Protein expressions of SIRT1, pAMPK, AMPK, LC3I, LC3II, p62, pULK and ULK in muscle tissues were analysed (n = 6). (c) Cellular immunofluorescence in muscle cells: Following transfection with Eef1e1‐siRNA (si‐Eef1e1) or control‐siRNA (si‐NC) and treatment with D‐gal or phosphate‐buffered saline (PBS), cells were stained for LAMP and LC3; scale bar = 100 μm (n = 6). (d) Protein expression analysis in transfected cells: Levels of SIRT1, pAMPK and AMPK were determined (n = 3). Then, following transfection with si‐Eef1e1 or si‐NC and treatment with phosphate‐buffered saline (PBS) or bafilomycin A1 (BafA1), LC3I, LC3II and p62 protein expression were determined (n = 3). (e) Cellular immunofluorescence post‐treatment: Following incubation with PBS, BafA1, rEEF1E1 protein or Eef1e1 plasmid transfection, LAMP and LC3 staining were conducted; scale bar = 100 μm (n = 6). (f) Protein expression post‐treatment: Quantification of SIRT1, pAMPK, AMPK, LC3I, LC3II and p62 levels (n = 3). Data are presented as the mean ± SD. For (b), an independent two‐tailed t‐test was used for comparisons between the Young and A‐Sed groups. For (c–f), ANOVA with Bonferroni multiple comparison test was used for the comparisons.