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Aim: To provide a detailed gene and protein expression analysis related to mitochondrial biogenesis and assess mitochondrial content in skeletal muscle of children with cerebral palsy (CP)., Method: Biceps brachii muscle samples were collected from 19 children with CP (mean [SD] age 15y 4mo [2y 6mo], range 9-18y, 16 males, three females) and 10 typically developing comparison children (mean [SD] age 15y [4y], range 7-21y, eight males, two females). Gene expression (quantitative reverse transcription polymerase chain reaction [PCR]), mitochondrial DNA (mtDNA) to genomic DNA ratio (quantitative PCR), and protein abundance (western blotting) were analyzed. Microarray data sets (CP/aging/bed rest) were analyzed with a focused query investigating metabolism- and mitochondria-related gene networks., Results: The mtDNA to genomic DNA ratio was lower in the children with CP compared to the typically developing group (-23%, p=0.002). Out of five investigated complexes in the mitochondrial respiratory chain, we observed lower protein levels of all complexes (I, III, IV, V, -20% to -37%; p<0.05) except complex II. Total peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) messenger RNA (p<0.004), isoforms PGC1α1 (p=0.05), and PGC1α4 (p<0.001) were reduced in CP. Transcriptional similarities were observed between CP, aging, and 90 days' bed rest., Interpretation: Mitochondrial biogenesis, mtDNA, and oxidative phosphorylation protein content are reduced in CP muscle compared with typically developing muscle. Transcriptional pathways shared between aging and long-term unloading suggests metabolic dysregulation in CP, which may guide therapeutic strategies for combatting CP muscle pathology. What this paper adds Cerebral palsy (CP) muscle contains fewer energy-generating organelles than typically developing muscle. Gene expression in CP muscle is similar to aging and long-term bed rest., (© 2021 The Authors. Developmental Medicine & Child Neurology published by John Wiley & Sons Ltd on behalf of Mac Keith Press.)

Cerebral palsy (CP) is a non-progressive motor disorder that affects posture and gait due to contracture development. The purpose of this study is to analyze a possible relation between muscle stiffness and gene expression levels in muscle tissue of children with CP. Next-generation sequencing (NGS) of gene transcripts was carried out in muscle biopsies from gastrocnemius muscle (n = 13 children with CP and n = 13 typical developed (TD) children). Passive stiffness of the ankle plantarflexors was measured. Structural changes of the basement membranes and the sarcomere length were measured. Twelve pre-defined gene target sub-categories of muscle function, structure and metabolism showed significant differences between muscle tissue of CP and TD children. Passive stiffness was significantly correlated to gene expression levels of HSPG2 (p = 0.02; R 2  = 0.67), PRELP (p = 0.002; R 2  = 0.84), RYR3 (p = 0.04; R 2  = 0.66), C COL5A3 (p = 0.0007; R 2  = 0.88), ASPH (p = 0.002; R 2  = 0.82) and COL4A6 (p = 0.03; R 2  = 0.97). Morphological differences in the basement membrane were observed between children with CP and TD children. The sarcomere length was significantly increased in children with CP when compared with TD (p = 0.04). These findings show that gene targets in the categories: calcium handling, basement membrane and collagens, were significantly correlated to passive muscle stiffness. A Reactome pathway analysis showed that pathways involved in DNA repair, ECM proteoglycans and ion homeostasis were amongst the most upregulated pathways in CP, while pathways involved in collagen fibril crosslinking, collagen fibril assembly and collagen turnover were amongst the most downregulated pathways when compared with TD children. These results underline that contracture formation and motor impairment in CP is an interplay between multiple factors.

Muscle contracture development is a major complication for individuals with cerebral palsy (CP) and has lifelong implications. In order to recognize contracture development early and to follow up on preventive interventions aimed at muscle health development, non-invasive, and easy to use methods are needed. The aim of the present study was to assess whether multi-frequency Bioimpedance (mfBIA) can be used to detect differences between skeletal muscle of individuals with CP and healthy controls. The mfBIA technique was applied to the medial gastrocnemius muscle of n = 24 adults with CP and n = 20 healthy controls of both genders. The phase angle (PA) and the centre frequency (fc) were significantly lower in individuals with CP when compared to controls; PA: - 25% for women and - 31.8% for men (P < 0.0001); fc: - 5.6% for women and - 5.2% for men (P < 0.009). The reactance (Xc) and the extracellular resistance (Re) of skeletal muscle from individuals with CP were significantly higher when compared to controls; Xc: + 9.9% for women and + 28.9% for men (P < 0.0001); Re: + 39.7% for women and + 91.2% for men (P < 0.0001). The present study shows that several mfBIA parameters differ significantly between individuals with CP and healthy controls. Furthermore, these changes correlated significantly with the severity of CP, as assessed using the GMFCS scale. The present data indicate that mfBIA shows promise in terms of being a useful diagnostic tool, capable of characterizing muscle health and its development in individuals with cerebral palsy.

Plastic adaptations are known to take place in muscles, tendons, joints, and the nervous system in response to changes in muscle activity. However, few studies have addressed how these plastic adaptations are related. Thus this study focuses on changes in the mechanical properties of the ankle plantarflexor muscle-tendon unit, stretch reflex activity, and spinal neuronal pathways in relation to cast immobilization. The left rat hindlimb from toes to hip was immobilized with a plaster cast for 1, 2, 4, or 8 wk followed by acute electrophysiological recordings to investigate muscle stiffness and stretch reflex torque. Moreover, additional acute experiments were performed after 4 wk of immobilization to investigate changes in the central gain of the stretch reflex. Monosynaptic reflexes (MSR) were recorded from the L4 and L5 ventral roots following stimulation of the corresponding dorsal roots. Rats developed reduced range of movement in the ankle joint 2 wk after immobilization. This was accompanied by significant increases in the stiffness of the muscle-tendon complex as well as an arthrosis at the ankle joint at 4 and 8 wk following immobilization. Stretch reflexes were significantly reduced at 4-8 wk following immobilization. This was associated with increased central gain of the stretch reflex. These data show that numerous interrelated plastic changes occur in muscles, connective tissue, and the central nervous system in response to changes in muscle use. The findings provide an understanding of coordinated adaptations in multiple tissues and have important implications for prevention and treatment of the negative consequences of immobilization following injuries of the nervous and musculoskeletal systems. NEW & NOTEWORTHY Immobilization leads to multiple simultaneous adaptive changes in muscle, connective tissue, and central nervous system.

Individuals with cerebral palsy (CP) participate in reduced levels of physical activity and spend an increased amount of time in a sedentary state compared with healthy control subjects. Whether this in part can be explained by impaired muscle function is still unclear. The aim of the present study was to elucidate differences in muscle fibre recruitment during treadmill exercise between CP subjects and healthy age-, sex- and BMI-matched controls. This is a case-control study. Acoustic myography (AMG), a method recording fibre use and efficiency from contracting muscles, was applied during a period of treadmill exercise. The recorded AMG parameters revealed that the CP subjects had a significantly lower initial S-score (spatial summation) than the controls (P < 0.01). However, the T-score (temporal summation) and the E-score (efficiency) showed no significant differences between individuals with CP and the healthy control subjects. The present findings indicate that CP subjects use a higher degree of spatial summation (more fibres recruited) to keep up the same speed during treadmill exercise when compared to healthy matched control subjects. Our results suggest that individuals with CP have a tendency to recruit far more muscle fibres during bouts of exercise than healthy individuals. This may partly explain why CP subjects experience premature fatigue.

Muscle contractures are a common complication to cerebral palsy (CP). The purpose of this study was to evaluate whether individuals with CP carry specific gene variants of important structural genes that might explain the severity of muscle contractures. Next-generation-sequencing (NGS) of 96 candidate genes associated with muscle structure and metabolism were analyzed in 43 individuals with CP (Gross Motor Function classification system [GMFCS] I, n=10; GMFCS II, n=14; GMFCS III, n=19) and four control participants. In silico analysis of the identified variants was performed. The variants were classified into four categories ranging from likely benign (VUS0) to highly likely functional effect (VUS3). All individuals with CP were classified and grouped according to their GMFCS level: Statistical comparisons were made between GMFCS groups. Kruskal-Wallis tests showed significantly more VUS2 variants in the genes COL4 (GMFCS I-III; 1, 1, 5, respectively [p < .04]), COL5 (GMFCS I-III; 1, 1, 5 [p < .04]), COL6 (GMFCS I-III; 0, 4, 7 [p < .003]), and COL9 (GMFCS I-III; 1, 1, 5 [p < .04]), in individuals with CP within GMFCS Level III when compared to the other GMFCS levels. Furthermore, significantly more VUS3 variants in COL6 (GMFCS I-III; 0, 5, 2 [p < .01]) and COL7 (GMFCS I-III; 0, 3, 0 [p < .04]) were identified in the GMFCS II level when compared to the other GMFCS levels. The present results highlight several candidate gene variants in different collagen types with likely functional effects in individuals with CP., (© 2018 Wiley Periodicals, Inc.)

Botulinum-toxin A (BoNT/A) is used for a wide range of conditions. Intramuscular administration of BoNT/A inhibits the release of acetylcholine at the neuromuscular junction from presynaptic motor neurons causing muscle-paralysis. The aim of the present study was to investigate the effect of high dose intramuscular BoNT/A injections (6 UI = 60 pg) on muscle tissue. The gait pattern of the rats was significantly affected 3 weeks after BoNT/A injection. The ankle joint rotated externally, the rats became flat footed, and the stride length decreased after BoNT/A injection. Additionally, there was clear evidence of microstructural changes on the tissue level by as evidenced by 3D imaging of the muscles by Synchrotron Radiation X-ray Tomographic Microscopy (SRXTM). Both the fibrillar and the non-fibrillar tissues were affected. The volume fraction of fibrillary tissue was reduced significantly and the non-fibrillar tissue increased. This was accompanied by a loss of the linear structure of the muscle tissue. Furthermore, gene expression analysis showed a significant upregulation of COL1A1, MMP-2, TGF-b1, IL-6, MHCIIA and MHCIIx in the BoNT/A injected leg, while MHVIIB was significantly downregulated., In Conclusion: The present study reveals that high dose intramuscular BoNT/A injections cause microstructural damage of the muscle tissue, which contributes to impaired gait.

Botulinum toxin (Btx) is used in children with cerebral palsy and in other neurological patients to diminish spasticity and reduce the risk of development of contractures. We investigated changes in the central gain of the stretch reflex circuitry in response to Btx injection in the triceps surae muscle in rats. Experiments were performed in 21 rats. Eight rats were a control group, and 13 rats were injected with 6 IU of Btx in the left triceps surae muscle. Two weeks after Btx injection, larger monosynaptic reflexes (MSR) were recorded from the left (injected) than the right (noninjected) L4 + L5 ventral roots following stimulation of the corresponding dorsal roots. A similar increase on the left side was observed in response to stimulation of descending motor tracts, suggesting that increased excitability of spinal motor neurons may at least partly explain the increased reflexes. However, significant changes were also observed in postactivation depression of the MSR, suggesting that plastic changes in transmission from Ia afferent to the motor neurons also may be involved. The data demonstrate that muscle paralysis induced by Btx injection is accompanied by plastic adaptations in the central stretch reflex circuitry, which counteract the antispastic effect of Btx. NEW & NOTEWORTHY Injection of botulinum toxin into ankle muscles causes increased gain of stretch reflex. This is caused by adaptive changes in regulation of transmitter release from Ia afferents and increased excitability of spinal motor neurons., (Copyright © 2017 the American Physiological Society.)

Skeletal muscle tissue is mechanosensitive, as it is able to sense mechanical impacts and to translate these into biochemical signals making the tissue adapt. Among its mechanosensitive nature, skeletal muscle tissue is the largest metabolic organ of the human body. Disturbances in skeletal muscle mechanosensing and metabolism cause and contribute to many diseases, i.e. muscular dystrophies/myopathies, cardiovascular diseases, COPD or diabetes mellitus type 2. A less commonly focused muscle-related disorder is clinically known as muscle contractures that derive from cerebral palsy (CP) conditions in young and adults. Muscle contractures are characterized by gradually increasing passive muscle stiffness resulting in complete fixation of joints. Different mechanisms have been identified in CP-related contractures, i.e. altered calcium handling, altered metabolism or altered titin regulation. The muscle-related extracellular matrix (ECM), specifically collagens, plays a role in CP-related contractures. Herein, we focus on mechanically sensitive complexes, known as costameres (Cstms), and discuss their potential role in CP-related contractures. We extend our discussion to the ECM due to the limited knowledge of its role in CP-related contractures. The aims of this review are (1) to summarize CP-related contracture mechanisms, (2) to raise novel hypotheses on the genesis of contractures with a focus on Cstms, and (3) to stimulate novel approaches to study CP-related contractures.

Botulinum toxin is used with the intention of diminishing spasticity and reducing the risk of development of contractures. Here, we investigated changes in muscle stiffness caused by reflex activity or elastic muscle properties following botulinum toxin injection in the triceps surae muscle in rats. Forty-four rats received injection of botulinum toxin in the left triceps surae muscle. Control measurements were performed on the noninjected contralateral side in all rats. Acute experiments were performed, 1, 2, 4, and 8 wk following injection. The triceps surae muscle was dissected free, and the Achilles tendon was cut and attached to a muscle puller. The resistance of the muscle to stretches of different amplitudes and velocities was systematically investigated. Reflex-mediated torque was normalized to the maximal muscle force evoked by supramaximal stimulation of the tibial nerve. Botulinum toxin injection caused severe atrophy of the triceps surae muscle at all time points. The force generated by stretch reflex activity was also strongly diminished but not to the same extent as the maximal muscle force at 2 and 4 wk, signifying a relative reflex hyperexcitability. Passive muscle stiffness was unaltered at 1 wk but increased at 2, 4, and 8 wk (P < 0.01). These data demonstrate that botulinum toxin causes a relative increase in reflex stiffness, which is likely caused by compensatory neuroplastic changes. The stiffness of elastic elements in the muscles also increased. The data are not consistent with the ideas that botulinum toxin is an efficient antispastic medication or that it may prevent development of contractures., (Copyright © 2016 the American Physiological Society.)

Background: Limb immobilization causes a rapid loss of muscle mass and strength that requires appropriate rehabilitation to ensure restoration of normal function. Whereas the knowledge of muscle mass signaling with immobilization has increased in recent years, the molecular regulation in the rehabilitation of immobilization-induced muscle atrophy is only sparsely studied. To investigate the phosphorylation and expression of candidate key molecular muscle mass regulators after immobilization and subsequent rehabilitation we performed two separate studies., Methods: We immobilized the lower limb for 2 weeks followed by the in-house hospital standard physiotherapy rehabilitation (Study 1). Secondly, we conducted an intervention study using the same 2 weeks immobilization protocol during which protein/carbohydrate supplementation was given. This was followed by 6 weeks of rehabilitation in the form of resistance training and continued protein/carbohydrate supplementation (Study 2). We obtained muscle biopsies from the medial gastrocnemius prior to immobilization (PRE), post-immobilization (IMMO) and post-rehabilitation (REHAB) and measured protein expression and phosphorylation of Akt, mTOR, S6k, 4E-BP1, GSK3β, ubiquitin and MURF1 and mRNA expression of Atrogin-1, MURF1, FOXO1, 3 and 4 as well as appropriate housekeeping genes., Results: In both studies, no changes in protein expression or phosphorylation for any measured protein were observed. In Study 1, FOXO3 and FOXO4 mRNA expression decreased after IMMO and REHAB compared to PRE, whereas other mRNAs remained unchanged. Interestingly, we found significant changes in expression of the putative housekeeping genes GAPDH, HADHA and S26 with immobilization in both studies., Conclusions: In neither study, the changes in muscle mass associated with immobilization and rehabilitation were accompanied by expected changes in expression of atrophy-related genes or phosphorylation along the Akt axis. Unexpectedly, we observed significant changes in several of the so-called housekeeping genes GAPDH, HADHA and S26 with immobilization in both studies, thereby questioning the usefulness of these genes for normalization of RNA data purposes in muscle immobilization studies.

Skeletal muscles fatigue after exercise, and reductions in maximal force appear. A difference in training status between the legs was introduced by unilateral immobilization of the calf muscles for 2 weeks in young men, who were randomly assigned to two groups, either a RUN group (n = 8) that was exposed to prolonged exercise (1-h running: individual pace) or a REST group (n = 12) that did no exercise after immobilization. Cross-sectional area (CSA) of the triceps-surae muscles was calculated by magnetic resonance imaging (MRI), and maximal voluntary contraction (MVC) force of the plantar flexors was measured before and after immobilization and after the running protocol. The CSA of triceps-surae muscles decreased significantly with a 7% reduction in both groups. A significant drop in the MVC of the triceps-surae muscle (10%; P < 0.05) was observed in response to immobilization. When subjected to running exercise immediately after immobilization, the muscle strength of the triceps-surae muscles dropped even further, but just in the immobilized leg (41%; P < 0.05). The present study highlights the importance of determining the muscle endurance when evaluating the effect of immobilization on muscle parameters.