Gymnastik- och idrottshögskolan, GIH

Age-related loss of muscle mass and function is underpinned by changes at the myocellular level. However, our understanding of the aged muscle phenotype might be confounded by factors secondary to ageing per se, such as inactivity and adiposity. Here, using healthy, lean, recreationally active, older men, we investigated the impact of ageing on myocellular properties in skeletal muscle. Muscle biopsies were obtained from young men (22 ± 3 years, n = 10) and older men (69 ± 3 years, n = 11) matched for health status, activity level and body mass index. Immunofluorescence was used to assess myofibre composition, morphology (size and shape), capillarization, the content of satellite cells and myonuclei, the spatial relationship between satellite cells and capillaries, denervation and myofibre grouping. Compared with young muscle, aged muscle contained 53% more type I myofibres, in addition to smaller (-32%) and misshapen (3%) type II myofibres (P < 0.05). Aged muscle manifested fewer capillaries (-29%) and satellite cells (-38%) surrounding type II myofibres (P < 0.05); however, the spatial relationship between these two remained intact. The proportion of denervated myofibres was ∼2.6-fold higher in old than young muscle (P < 0.05). Aged muscle had more grouped type I myofibres (∼18-fold), primarily driven by increased size of existing groups rather than increased group frequency (P < 0.05). Aged muscle displayed selective deterioration of type II myofibres alongside increased denervation and myofibre grouping. These data are key to understanding the cellular basis of age-related muscle decline and reveal a pressing need to fine-tune strategies to preserve type II myofibres and innervation status in ageing populations.

Background

Sarcopenia is thought to be underlined by age-associated anabolic resistance and dysregulation of intracellular signalling pathways. However, it is unclear whether these phenomena are driven by ageing per se or other confounding factors.

Methods

Lean and healthy young (n = 10, 22 ± 3 years, BMI; 23.4 ± 0.8 kg/m2) and old men (n = 10, 70 ± 3 years, BMI; 22.7 ± 1.3 kg/m2) performed unilateral resistance exercise followed by intake of essential amino acids (EAA). Muscle biopsies were collected from the rested and the exercised leg before, immediately after and 60 and 180 min after EAA intake. Muscle samples were analysed for amino acid concentrations, muscle protein synthesis (MPS) and associated anabolic signalling.

Results

Following exercise, peak plasma levels of EAA and leucine were similar between groups, but the area under the curve was ~11% and ~28% lower in Young (p < 0.01). Absolute levels of muscle EAA and leucine peaked 60 min after exercise, with ~15 and ~21% higher concentrations in the exercising leg (p < 0.01) but with no difference between groups. MPS increased in both the resting (~0.035%·h−1 to 0.056%·h−1, p < 0.05) and exercising leg (~0.035%·h−1 to 0.083%·h−1, p < 0.05) with no difference between groups. Phosphorylation of S6K1Thr389 increased to a similar extent in the exercising leg in both groups but was 2.8-fold higher in the resting leg of Old at the 60 min timepoint (p < 0.001). Phosphorylation of 4E-BP1Ser65 increased following EAA intake and exercise, but differences between legs were statistically different only at 180 min (p < 0.001). However, phosphorylation of this site was on average 78% greater across all timepoints in Old (p < 0.01). Phosphorylation of eEF2Thr56 was reduced (~66% and 39%) in the exercising leg at both timepoints after EAA intake and exercise, with no group differences (p < 0.05). However, phosphorylation at this site was reduced by ~27% also in the resting leg at 60 min, an effect that was only seen in Old (p < 0.01). Total levels of Rheb (~45%), LAT1 (~31%) and Rag B (~31%) were higher in Old (p < 0.001).

Conclusion

Lean and healthy old men do not manifest AR as evidenced by potent increases in MPS and mTORC1 signalling following EAA intake and exercise. Maintained anabolic sensitivity with age appears to be a function of a compensatory increase in basal levels of proteins involved in anabolic signalling. Therefore, our results suggest that age per se does not appear to cause AR in human skeletal muscle.

Human skeletal muscle comprises slow-twitch (type I) and fast-twitch (type II) fibers. Fiber type-specific analyses often require manual isolation of fibers, necessitating effective tissue preservation. While freeze-drying remains the standard, alternative preservation methods such as RNAlater and RNAlater-ICE are increasingly used. Besides their utility in preserving RNA, it needs to be determined whether RNAlater and RNAlater-ICE can be utilized for broader downstream biochemical analyses in skeletal muscle tissue. In this study, we compared freeze-drying to RNAlater and three RNAlater-ICE-based protocols. We observed substantial and consistent alterations in protein content, amino acid levels, and enzyme activity depending on the preservation method. Notably, all RNAlater-ICE protocols abolished citrate synthase activity, and branched-chain amino acid levels were markedly reduced in both RNAlater and RNAlater-ICE-treated samples relative to freeze-dried tissue. Total protein concentration was comparable between freeze-dried and RNAlater-preserved muscle, whereas RNAlater-ICE protocols yielded lower values. After centrifugation, supernatant protein concentration was higher in RNAlater-treated samples, but consistently lowest following RNAlater-ICE treatment. Our results demonstrate the importance of choosing an appropriate preservation method for skeletal muscle prior to downstream biochemical analysis and that care should be taken when using RNAlater and RNAlater-ICE for protein or amino acid analysis.

The muscular and myocellular adaptations to low-load resistance exercise training (LL-RET) remain incompletely understood in the trained state. The primary aim of this study was to examine adaptations to an LL-RET regimen, comparing these to a high-load training regimen (HL-RET). Fourteen resistance-trained males and females (26.4 ± 4.4 years) participated in a 9-week RET program (twice per week). Using a within-subject design, each individual trained one leg with HL-RET (3-5 repetitions), and the other with LL-RET (20-25 repetitions), all sets performed to volitional failure. Maximal strength (1 RM) and muscle thickness were assessed. Muscle biopsies were analyzed for fiber type composition, fiber cross-sectional area (fCSA), and satellite cell- and myonuclear content using immunofluorescence. The training regimens led to comparable increases in 1 RM in multi-joint movements (21 ± 10%), but not in single-joint movements where HL-RET was superior (9 ± 13% vs -3 ± 10%). Regardless of training regimen, muscle thickness increased pre- to post-intervention by 7 ± 17% at the mid-thigh site and 8 ± 8% at the distal site. However, this was not accompanied by changes at the myocellular level, with no observed differences in fCSA and fiber type composition. Satellite cell content increased by 25 ± 57% in type I fibers, independent of training regimen, but no changes were noted in myonuclear content. LL-RET can replicate many aspects of HL-RET leading to similar increases in muscle hypertrophy and strength. Our study supports the notion that comparable adaptations to RET can be achieved using widely distinct loading regimens.

BACKGROUND: Exercising with low muscle glycogen content can improve training adaptation, but the mechanisms underlying the muscular adaptation are still largely unknown. In this study, we measured substrate utilization and cell signaling in different muscle fiber types during exercise and investigated a possible link between these variables.

METHODS: Five subjects performed a single leg cycling exercise in the evening (day 1) with the purpose of reducing glycogen stores. The following morning (day 2), they performed two-legged cycling at ∼70% of VO_2peak for 1 h. Muscle biopsies were taken from both legs pre- and post-exercise for enzymatic analyses of glycogen, metabolite concentrations using LC-MS/MS-based quantification, and protein signaling using Western blot in pools of type I or type II fibers.

RESULTS: Glycogen content was 60-65% lower for both fiber types (P < 0.01) in the leg that exercised on day 1 (low leg) compared to the other leg with normal level of glycogen (normal leg) before the cycling exercise on day 2. Glycogen utilization during exercise was significantly less in both fiber types in the low compared to the normal leg (P < 0.05). In the low leg, there was a 14- and 6-fold increase in long-chain fatty acids conjugated to carnitine in type I and type II fibers, respectively, post-exercise. This increase was 3-4 times larger than in the normal leg (P < 0.05). Post-exercise, mTOR^Ser2448 phosphorylation was increased in both fiber types in the normal leg (P < 0.05) but remained unchanged in both fiber types in the low leg together with an increase in eEF2^Thr56 phosphorylation in type I fibers (P < 0.01). Exercise induced a reduction in the autophagy marker LC3B-II in both fiber types and legs, but the post-exercise level was higher in both fiber types in the low leg (P < 0.05). Accordingly, the LC3B-II/I ratio decreased only in the normal leg (75% for type I and 87% for type II, P < 0.01).

CONCLUSIONS: Starting an endurance exercise session with low glycogen availability leads to profound changes in substrate utilization in both type I and type II fibers. This may reduce the mTORC1 signaling response, primarily in type I muscle fibers, and attenuate the normally observed reduction in autophagy.

Cerebral palsy (CP), the most prevalent childhood-onset motor disability, frequently entails progressive musculoskeletal complications. This comprehensive review synthesizes existing knowledge of microscopic and molecular alterations in CP skeletal muscle. Considerable methodological variability, heterogeneous patient cohorts, and inconsistent control groups significantly complicate comparative interpretations across studies. Nonetheless, some structural abnormalities consistently emerge, including increased variability in muscle fibre size, altered fibre type distribution, long sarcomeres at standardized joint positions, increased collagen content, disrupted neuromuscular junction integrity, reduced capillary density, and mitochondrial and satellite cell impairments. Investigations of satellite cell function in vitro further underscore potential mechanistic alterations, although findings remain inconsistent. Remarkably, few studies have systematically explored the cellular and molecular consequences of standard clinical interventions, revealing a notable research gap. In conclusion, the overall literature reveals considerable divergence in reported outcomes, reflecting the profound complexity of CP muscle biology. We believe that resolving this complexity will require more coordinated and collaborative research approaches.

Muscle disuse has rapid and debilitating effects on muscle mass and overall health, making it an important issue from both scientific and clinical perspectives. However, the myocellular adaptations to muscle disuse are not yet fully understood, particularly those related to the myonuclear permanence hypothesis. Therefore, in this study, we assessed fiber size, number of myonuclei, satellite cells, and capillaries in human gastrocnemius muscle after a period of immobilization following an Achilles tendon rupture. Six physically active patients (5M/1F, 43 {plus minus} 15 years) were recruited to participate after sustaining an acute unilateral Achilles tendon rupture. Muscle biopsies were obtained from the lateral part of the gastrocnemius before and after six weeks of immobilization using a plaster cast and orthosis. Muscle fiber characteristics were analyzed in tissue cross-sections and isolated single fibers using immunofluorescence and high-resolution microscopy. Immobilization did not change muscle fiber type composition nor cross-sectional area of type I or type II fibers, but muscle fiber volume tended to decline by 13% (p=0.077). After immobilization, the volume per myonucleus was significantly reduced by 20% (p=0.008). Myonuclei were not lost in response to immobilization but tended to increase in single fibers and type II fibers. No significant changes were observed for satellite cells or capillaries. Myonuclei were not lost in the gastrocnemius muscle after a prolonged period of immobilization, which may provide support to the myonuclear permanence hypothesis in human muscle. Capillaries remained stable throughout the immobilization period, whereas the response was variable for satellite cells, particularly in type II fibers.

OBJECTIVE: Here we use skeletal muscle fiber composition to investigate whether defects in amino acid metabolism are involved in the early development of IR in healthy young individuals before onset of clinical manifestations.

DESIGN: Two groups consisting of healthy young men and women, insulin-sensitive and insulin resistant, were studied using a cross-sectional design.

METHODS: Biopsies were obtained from the vastus lateralis muscle and an intravenous glucose tolerance test was performed. Plasma and muscle tissue were analyzed by metabolomics.

RESULTS: Subjects in group 1 (n=20; age 28±5 yrs; body mass index 22.3±2.7 kg/m2) had an expression of type I muscle fibers and whole-body insulin sensitivity, respectively, of 58.8±5.7% and 1.8±0.7 units. Subjects in group 2 (n=16; age 25±6 yrs; body mass index 22.6±3.0 kg/m2) had an expression of type I muscle fibers and whole-body insulin sensitivity, respectively, of 29.8±6.6% and 0.8±0.3 units (P<0.001 vs. group 1 for both). Anserine and β-alanine contents in muscle were significantly higher and taurine lower in group 2 vs. 1, consistent with the differences in muscle fiber composition between groups. Taurine correlated well with insulin sensitivity and expression of type I muscle fibers (r=0.63; P<0.001 for both). In contrast, there were no significant differences in plasma or tissue contents of glutamine, arginine, or branch-chain amino acids between groups.

CONCLUSIONS: These data demonstrate that the early development of IR is not a consequence of defects in amino acid metabolism. Rather, defects in amino acid metabolism in diseased states are more likely a consequence of IR.