Cerebral palsy (CP) is the most common cause of motor impairment in children.¹ Although the initial brain injury is non-progressive, skeletal muscle function typically deteriorates over time.² Skeletal muscles of individuals with CP are small, weak, and compositionally different compared with typically developing muscle. Specifically, increased collagen content³ and intramuscular adipose tissue⁴ are believed to contribute to poor muscle function in this population. Poor muscle quality is a well-described risk factor for insulin resistance and development of type 2 diabetes^{5, 6} and is typically associated with a reduced mitochondrial content of the muscle.⁷ Whether the described pathological findings in CP muscle influences metabolic capacity of the tissue is not known.

A recent study by Zogby et al. investigated skeletal muscle mitochondrial characteristics in CP muscle using an immunohistochemical approach. According to fiber-type-specific succinate dehydrogenase activity,⁸ their results showed a similar succinate dehydrogenase activity per unit area in both type I and type II fibers in individuals with CP and typically developing individuals, suggesting that muscle oxidative potential is preserved in children with CP. Conversely, other studies reporting on fiber types in the muscles of children with CP describe a clear slow to fast-fiber-type shift,^{9, 10} indicative of a less oxidative phenotype of the muscle, as fast type IIx fibers contain fewer mitochondria than slow type I fibers. Moreover, two skeletal muscle transcriptomics studies in children with CP^{11, 12} indicate reduced messenger RNA levels of key genes involved in skeletal muscle mitochondrial biogenesis, such as AMP-activated protein kinase (AMPK) and transcription factor A (Tfam), contrasting with the data of Zogby et al. An accurate picture of the metabolic characteristics of CP muscle is of great importance for developing the appropriate therapeutics to counteract muscle dysfunction.

Given the discrepancies in the literature, we wanted to use genomic, transcriptomic, targeted messenger RNA, and protein-based approaches to investigate whether: (1) mitochondrial DNA (mtDNA) abundance and protein levels of oxidative phosphorylation (OXPHOS) enzymes, as markers of mitochondrial content, are reduced in CP muscle compared with typically developing muscle; (2) gene and protein expression of factors involved in mitochondrial biogenesis are reduced in CP muscle compared with typically developing muscle; and (3) gene networks linked to mitochondrial biology and glucose metabolism are downregulated in CP muscle compared with typically developing muscle. In addition, CP gene networks were compared with severe inactivity and aging data sets to identify common themes with respect to metabolic dysregulation and provide translatable insight into appropriate therapeutic targets.

METHOD

RESULTS

To investigate oxidative capacity in CP muscle, we quantified levels of five OXPHOS proteins: complex I (NDUFB8), complex II (SDHB), complex III (UQCRC2), complex IV (COXII), and complex V (ATP5A). Four out of five complexes (I, III, IV, V) showed a significant reduction of −20% to −37% (p<0.05) in content in CP muscle, whereas complex II tended to be lower in CP but was not statistically different to typically developing muscle (−33%, p=0.07; Fig. 1). We observed a lower mtDNA to genomic DNA ratio (−23%, p=0.002) in CP biceps brachii muscle compared with typically developing biceps brachii muscle (Fig. 2a). Skeletal muscle protein levels of Tfam were not significantly different in CP muscle compared with typically developing muscle (p=0.15, Fig 2b,c).

Consistent with evidence of reduced mitochondrial content, PGC1α gene expression was suppressed (−24%, 1.60 [0.45] vs 0.84 [0.55], p=0.004) in CP muscle compared with typically developing muscle (Fig. 3a). Splice variants displayed a non-homogenous expression in CP muscle; PGC1α1 and PGC1α4 were reduced (−36%, 1.15 [0.28] vs 0.74 [0.54], p=0.05; −64%, 1.94 [0.84] vs 0.70 [0.53], p<0.001 respectively) compared with typically developing muscle, whereas PGC1α2 (−69%, 0.42 [0.65] vs 0.13 [0.29], p=0.18) and PGC1α3 (+22, 0.53 [0.58] vs 0.65 [0.82], p=0.70) were not (Fig. 3b–e).

A secondary analysis of a publicly available microarray data set comparing CP and typically developing hamstrings muscles¹² produced 2290 significantly downregulated genes in CP muscle. Gene ontology and pathway analyses revealed glycolytic and mitochondrial transcriptional networks to be suppressed in CP skeletal muscle (Fig. 4a,b). The gene set enriching the mitochondrial biogenesis node is shown in Figure 4c and the gene set enriching the glycolytic transcriptional network is shown in Figure 4d. We also compared the CP skeletal muscle transcriptome with that of older adults¹⁶ and individuals subjected to prolonged bed rest¹⁷ to determine whether a similar metabolic signature existed between conditions. CP shared more genes with the aging data set (332 genes) compared with the bed rest data set (109 genes). We found that 28 downregulated genes were shared between the three conditions (Fig. S1, online supporting information) and that mitochondrial and aerobic respiration transcriptional networks were suppressed under all three conditions (Fig. S2a,b, online supporting information), including gene ontologies (Reactome) such as mitochondrial translation initiation, elongation, and termination (MRPS15, MRPL2, MRPL35) and tricarboxylic acid cycle and electron transport chain (CYCS, OGDH, SLC16A1, ATP5MPL, NNT, DLAT). For a complete list of genes shared between CP and/or aging/bed rest see Table S4 (online supporting information).

DISCUSSION

This study addresses biological processes involved in mitochondrial biogenesis, mtDNA content, and OXPHOS protein abundance in CP skeletal muscle. We show that key upstream regulators of metabolism and mitochondrial biogenesis are reduced in CP skeletal muscle compared with typically developing controls, and that this is associated with downregulation of large gene networks affecting glucose metabolism and mitochondrial biology. Skeletal muscle in individuals with CP also contains lower amounts of mtDNA, potentially indicating fewer mitochondria in CP skeletal muscle compared with typically developing muscle. Moreover, we also observed lower levels of OXPHOS proteins in CP skeletal muscle that, together with reduced overall mitochondrial content, suggests a reduced oxidative capacity in CP muscle compared with typically developing muscle.

PGC1α is a key regulator of mitochondrial biogenesis in skeletal muscle.²⁰ Transgenic mice that overexpress PGC1α in skeletal muscle display an oxidative phenotype with higher exercise tolerance than control mice and greater mitochondrial content.^{21, 22} On the contrary, several studies that have used transgenic mice lacking PGC1α have reported intact exercise adaptations including mitochondrial biogenesis,^23-25 indicating a high level of biological complexity that is still not fully elucidated. Acute exercise studies in humans have also identified PGC1α as an early response gene to aerobic exercise, preceding mitochondria-related gene expression.²⁶ PGC1α indirectly influences mtDNA transcription via increased expression of mitochondrial Tfam, which is coactivated by nuclear respiratory factor-1.²⁰ In our study, we observed reduced abundance of both total levels of PGC1α and isoforms PGC1α1 and PGC1α4. PGC1α1 is believed to be the splice isoform most involved in mitochondrial biogenesis,²⁷ whereas PGC1α4 has been implicated in skeletal muscle size regulation.¹³ Contrasting data from two previous skeletal muscle transcriptomics studies in children with CP,^{11, 12} we did not observe a significant reduction in Tfam protein in skeletal muscle potentially owing to low sample number for this specific analysis. In conjunction with these data, we observed lower levels of mtDNA/genomic DNA, indicating fewer mitochondria in CP skeletal muscle than in typically developing muscle. Our skeletal muscle data are supported by recent findings that children with CP also have reduced mitochondrial content in peripheral blood cells.²⁸ Blood-based bioenergetic profiling correlates to physical function in older individuals²⁹ and has been suggested as a proxy for skeletal muscle oxidative capacity.³⁰ Taken together, our data suggest a reduced activity of mitochondrial biogenesis in individuals with CP compared with typically developing children.

Mitochondrial content in skeletal muscle has been linked to aerobic capacity^{31, 32} and shown to increase in response to aerobic training.³³ Individuals with CP have lower VO₂ max than typically developing peers.^{34, 35} Aerobic capacity depends on central (cardiac output, ventilation) and peripheral (perfusion, oxygen extraction, mitochondrial content) factors. For individuals with CP, it is mostly unknown how these parameters are influenced by increased exercise intensity and thereby an increased demand for energy (i.e. ATP). Interestingly, lactate production is higher than expected at moderate exercise intensities in individuals with CP, indicating that an insufficient ATP production is evident already at relatively light exercise intensities.³⁶ The knowledge about metabolic capacity of CP skeletal muscle is scarce. Transcriptomics studies have indicated reduced expression of gene ontologies related to oxidative metabolism.^{11, 12} We observed reduced levels of four out of five electron transport chain complexes (I, III, IV, V) with only succinate dehydrogenase (II) not reaching statistically significant levels. Interestingly, complex II does not include any mtDNA encoded subunits, tentatively suggesting that reduced mtDNA content primarily affects the other complexes more as they are dependent on mitochondrially translated proteins. Our data support the previous observation of a reduced aerobic capacity in skeletal muscle and matches previously published fiber-type data from CP biceps muscle that shows a shift to faster myosin isoforms (IIx).¹⁰ Thus, our data suggest a role for decreased skeletal muscle mitochondrial content and potentially also capacity in the reduced exercise tolerance typically observed in individuals with CP.

Early metabolic dysregulation of skeletal muscle most likely plays a role in the increased risk of metabolic and cardiovascular disease that has been observed in the adult population with CP.^{37, 38} To gain further insight into metabolism-related gene expression in CP skeletal muscle, we performed a secondary analysis of publicly available hamstrings muscle microarray data. A gene ontology analysis (biological processes, reactome) on all significantly downregulated genes showed that glucose metabolism- and mitochondria-related gene networks were enriched in CP muscle. Surprisingly, CP muscle shared more downregulated genes with aged skeletal muscle than with long-term disuse. This is intriguing and challenges the belief that CP skeletal muscle pathophysiology is due to disuse per se and suggests common mechanisms with aging, most likely involving altered skeletal muscle neural input. We found that 28 downregulated genes were shared between the three conditions. Mitochondrial and aerobic respiration transcriptional networks were suppressed under all three conditions and out of the 28 common genes, 14 had mitochondrial localization as indicated by Mitocarta 3.0.³⁹ This focused analysis on gene expression supports the biochemical data presented in this paper and further suggests that individuals with CP, already at a young age, display evidence of metabolic dysregulation in skeletal muscle, as previously described with aging and long-term bed rest.^{17, 40, 41} Collectively, our findings stress the need for the development of intervention strategies, including but not limited to endurance exercise training performed at a sufficient frequency and intensity for children with the ability to voluntarily activate the muscle.⁴²

The results of the current investigation should be viewed in light of its limitations. Skeletal muscle samples from CP were collected from children and adolescents with a fixed contracture of the elbow flexors in most cases. Thus, our findings related to mitochondrial abundance and capacity in skeletal muscle from individuals with CP could be either due to the pathophysiology of the contracture per se or a more generalized feature of skeletal muscle in CP. We also recognize the fact that our study focuses on just one muscle which is a limitation for generalizability. Skeletal muscle samples from the typically developing comparison group were obtained post-mortem and represent a cumulative sample of children and adolescents after an accidental death. Thus, there was no age matching between CP and typically developing children. It should also be noted that we report measures of mtDNA/genomic DNA and OXPHOS protein abundance in CP versus typically developing muscle. For a more complete picture, ideally mitochondrial function should also have been measured. Moreover, the publicly available microarray data sets are not sampled from the biceps muscle, and this should be considered when comparing our biochemical data on biceps muscle samples with the gene network analysis on CP muscle (hamstrings muscle), aging, and bed rest (vastus lateralis muscle).

In conclusion, we observed evidence of reduced mtDNA and OXPHOS protein content in CP skeletal muscle, indicating reduced mitochondrial abundance. Moreover, transcriptional similarities to aging and long-term unloading suggests signs of metabolic alterations in CP skeletal muscle. Altogether, these aberrations in children with CP potentially influence future metabolic health of individuals with CP and tentatively contributes to the risk for metabolic diseases such as type 2 diabetes.