Gymnastik- och idrottshögskolan, GIH

Regular exercise using assistive movement devices, such as running frames, has emerged as a promising strategy to improve cardiorespiratory fitness in individuals with cerebral palsy (CP). However, the molecular pathways underlying these adaptations remain poorly understood. Here, we examined a novel class of signalling molecules, mitochondrial-derived microproteins (MDPs), and assessed whether individuals with CP exhibit altered circulating levels compared with typically developing (TD) individuals at rest and following an acute bout of endurance exercise. Three groups were included: TD adults (31 ± 6 years), TD adolescents (16 ± 1 years) and adults with CP (25 ± 6 years). Individuals with CP were classified as Gross Motor Function Classification System (GMFCS) levels II-IV and had at least 3 months of frame running experience. Habitual physical activity, ultrasound-derived muscle thickness, and peak oxygen uptake were assessed. The exercise session consisted of 45 min of frame running for individuals with CP and conventional running for TD participants. Blood samples were obtained before and 1 h after exercise, and plasma MDP concentrations were measured using in-house enzyme-linked immunosorbent assay. Adults with CP had reduced muscle mass and maximal oxygen uptake compared to TD individuals. Despite this, they exhibited basal circulating levels of MDPs, including humanin, MOTS-c and SHMOOSE, comparable to TD adults and adolescents, with no associations with CP subtype or motor impairment severity. Following exercise, circulating MDPs showed no or only modest changes across groups, with no differences between CP and TD individuals. Overall, these findings suggest preserved mitochondrial-derived signalling via MDPs in individuals with CP.

Cerebral palsy (CP) is the most common cause of childhood-onset physical disability. It results from injury to the developing brain and is characterized by motor impairments, muscle weakness, and fatigue. CP is commonly associated with marked deficits in muscle mass and function, and many individuals experience early declines in physical performance and functional ability as they age. These features resemble changes observed in age-related muscle loss, that is, sarcopenia, raising the possibility of shared underlying mechanisms. This paper hypothesizes that skeletal muscles of individuals with CP undergo accelerated aging, driven by cellular and molecular pathways similar to those implicated in sarcopenia. To support this hypothesis, we highlight emerging evidence of phenotypic overlap between CP and aging muscle, including neuromuscular changes, impaired satellite cell function, altered niche components, chronic inflammation, and metabolic deficits such as reduced capillarization and mitochondrial dysfunction. To test this hypothesis, we propose cross-sectional and longitudinal studies targeting both baseline aging markers and the rate of aging-related changes. These studies should focus on established hallmarks of aging, such as mitochondrial dysfunction, DNA methylation, and markers of cellular senescence. If confirmed, this hypothesis could reshape our understanding of muscle pathology in CP. It may also open up the possibility of repurposing therapeutic strategies demonstrated to be effective in geriatric care for children and young adults with CP.

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.

mRNA increased transiently, followed by increased ribosomal DNA transcription, leading to elevated total RNA levels. Skeletal muscle-specific ribosomal protein paralog RPL3L was replaced by the ubiquitously expressed RPL3 during the initial phases of recovery, but this shift was reversed by day 28. A substantial transcriptomic response was observed in human muscle injury, with heavy emphasis on MYC-induced anabolism and inflammation. This supports a model in which MYC-driven changes in ribosomal content and composition form a core anabolic module in skeletal muscle repair, potentially representing a targetable axis to enhance recovery after muscle injury.

Introduction/Aims Traditional methods of sampling skeletal muscle tissue are invasive. This study aimed to evaluate a sub-millimeter core-biopsy (microbiopsy) as a potentially more tolerable method, with further regard to tissue yield and analyzability of RNA expression.Methods Children (9-13 years, n = 11) and adults (18-50 years, n = 16) were recruited. Microbiopsy and venipuncture were performed, with prior application of local anesthesia cream. Additionally, adults underwent a Bergstr & ouml;m muscle biopsy, with infiltrative local anesthesia. Pain was rated using the visual analog scale (VAS), reported as medians (95% CI). Microbiopsy samples were freeze-dried and weighed. To evaluate RNA sequencing performance at low tissue sample weights, a six-step incremental tissue ladder (10-500 mu g) was analyzed.Results Children rated venipunctures and microbiopsies low, at VAS = 0.1 (0.0-0.6) and 1.6 (0.9-3.9), respectively. Microbiopsy pain ratings were slightly higher than venipuncture, p < 0.001. Pain ratings in adults were 0.0 (0.0-0.5), 1.8 (1.3-2.4), 2.9 (2.4-3.8), and 2.7 (2.2-3.8) for venipuncture, microbiopsy, Bergstr & ouml;m biopsy, and infiltrative local anesthesia, respectively. Microbiopsy was rated less painful than Bergstr & ouml;m biopsy and local anesthesia (p < 0.05). Children did not rate microbiopsy more painful than adults (p = 0.82). Microbiopsies yielded on average 303 (SD 121.8) mu g. RNA sequencing detected similar transcriptomic signatures across the tissue ladder.Discussion The generally low pain ratings for the microbiopsy procedure support its use as a tolerable method of acquiring skeletal muscle samples in both children and adults. It represents a less painful alternative to Bergstr & ouml;m biopsies while still rendering adequate material for RNA sequencing.

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.

Sprint interval training (SIT) is a time-efficient type of endurance training that involves large type 2 muscle fibre recruitment. Effective antioxidant supplementation may mitigate positive training adaptations by limiting the oxidant challenge. Our aim was to test whether SIT affects type 2 more than type 1 muscle fibres, and whether the muscular training response is mitigated by antioxidant treatment. Young men performed three weekly SIT sessions (4-6 x 30 s all-out cycling) for 3 weeks while treated with antioxidants (vitamin C, 1 g day(-1); vitamin E, 235 mg day(-1)) or placebo. Vastus lateralis biopsies were taken to measure (i) activation of genes for reactive oxygen/nitrogen species (ROS) sensors and inflammatory mediators with quantitative RT-PCR and (ii) fibre type-specific proteome adaptations using MS-based proteomics. Vitamin treatment decreased the upregulation of genes for ROS sensors and inflammatory regulators during the first SIT session. The 3 weeks of SIT caused generally larger proteome adaptations in type 2 than in type 1 fibres, and this included larger increases in abundance of proteins involved in mitochondrial energy production. Vitamin treatment blunted the SIT-induced proteome adaptations, whereas it did not affect the training-induced improvement in maximal cycling performance. In conclusion, (i) the large type 2 fibre recruitment and resulting proteome adaptations are instrumental to the effectiveness of SIT and (ii) antioxidant supplementation counteracts positive muscular adaptations to SIT, which would blunt any improvement in submaximal endurance performance, whereas it does not affect the improvement in maximal cycling performance, where O-2 delivery to muscle would be limiting.

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.

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.