Alterations in Satellite Cell Differentiation from Young Patients with Cerebral Palsy

Research Article

Phys Med Rehabil Int. 2023; 10(2): 1217.

Alterations in Satellite Cell Differentiation from Young Patients with Cerebral Palsy

Marlies Corvelyn¹; Domiziana Costamagna1,2; Justine Meirlevede1; Robin Duelen1; Jorieke Deschrevel3; Ghislaine Gayan-Ramirez3; Hannah De Houwer4; Anja Van Campenhout4,5; Kaat Desloovere2; Maurilio Sampaolesi1*

1Stem Cell and Developmental Biology, Dept. of Development and Regeneration, KU Leuven, Belgium

2Research Group for Neurorehabilitation, Dept. of Rehabilitation Sciences, KU Leuven, Belgium

3Laboratory of Respiratory Diseases and Thoracic Surgery, Dept. of Chronic Diseases and Metabolism, KU Leuven, Belgium

4Department of Orthopaedic Surgery, University Hospitals Leuven, Belgium

5Department of Development and Regeneration, KU Leuven, Belgium

*Corresponding author: Maurilio Sampaolesi Stem Cell and Developmental Biology, Department of Development and Regeneration, KU Leuven, Belgium. Tel: +3216373132 Email: maurilio.sampaolesi@kuleuven.be

Received: July 03, 2023 Accepted: July 31, 2023 Published: August 07, 2023

Abstract

Cerebral Palsy (CP) is one of the most common lifelong conditions leading to childhood physical disability, affecting approximately 1.6 in 1000 live births. Literature reported muscle alterations of CP patients in comparison to Typically Developing (TD) children such as fibrotic tissue accumulation and reduced Satellite Cell (SC) numbers with altered fusion capacity. To better understand the observed CP muscle phenotype, we quantified and investigated SC-progenitors of the Medial Gastrocnemius from young patients with CP (n=17, aged 3-9 years, GMFCS levels I-III) and age-matched TD children (n=12). We found an increased myotube diameter and a higher amount of nucleus clusters in CP-derived myotubes compared to TD-derived myotubes. Additionally, these nucleus accumulations were larger and less linear in SCs from children with CP compared to TD. Further, no altered expression levels of multiple genes associated with SC differentiation, fusion and nuclear positioning involved in other muscle disorders have been observed. In conclusion, we unprecedently quantified altered differentiation features of SCs from CP muscles with unaltered gene hallmarks of myopathies. Further research would be needed to elucidate pathophysiological mechanisms related to the altered SC properties observed in CP muscles and its impact on in vivo muscle functioning.

Keywords: Satellite cells; Cerebral palsy; Young patients; Myogenesis; Myotube

Abbreviations: BL: Bilateral; BoNT: Botulinum Neurotoxin A; CP: Cerebral Palsy; DMD: Duchenne Muscular Dystrophy; FACS: Fluorescent Activated Cell Sorting; FI: Fusion Index; F: Female; GMFCS: Gross Motor Function Classification System; IF: Immunofluorescent; IQR: Interquartile Range; M: Male; MG: Medial Gastrocnemius; MyHC: Myosin Heavy Chain; RMSE: Rooth Mean Square Error; SC: Satellite Cell; SD: Standard Deviation; ST: Semitendinosus; TD: Typically Developing; UL: Unilateral

Introduction

Cerebral Palsy (CP) is one of the most common lifelong conditions leading to childhood physical disability, with an incidence of around 1.6 in 1000 live births [1]. CP originates from a neural lesion in the immature brain, leading to progressive musculoskeletal symptoms. Clinically, CP manifests itself on both neural and muscular level (spasticity, muscle weakness and decreased muscular control), resulting in decreased functional ability such as disturbed gait [2-4]. These patients can be classified following the Gross Motor Function Classification System (GMFCS), from levels I to V, based on their functional abilities [5,6]. The lower the level the more functional the child is. Treatment mainly consists of management of the symptoms at the muscle level, including physiotherapy, orthoses, Botulinum Neurotoxin A (BoNT) injections and orthopaedic surgery [4,7].

Previous investigations on CP microscopic muscle properties reported altered muscle composition with i.e. an increased collagen content, accumulation of adipose tissue [8-10] and a significant reduction in the number of Satellite Cells (SCs) in contractured muscles of patients with CP [11-13]. SCs are quiescent adult stem cells located between the sarcolemma and the basal lamina of the muscle and are primarily involved in adult myogenesis [14,15]. Unfortunately, research on the functionality of SCs in CP muscle pathology is scarce [16,17] and requires further examinations. Domenighetti et al. described lower Fusion Index (FI) values in myotubes from CP-derived SCs (patients aged 3-18 years) compared to TD adolescent cells (aged 14-18 years), based on hamstring biopsies [16,18]. Furthermore, they described these myotubes as thinner and more spindle-shaped compared to those of TD adolescents. Our group previously reported higher FI values based on Myosin Heavy Chain (MyHC) expression of SCs from younger patients with CP (aged 3-9 years) derived from microbiopsies of the Medial Gastrocnemius (MG) compared to aged-matched TD children [17]. These SC-derived myotubes from children with CP seemed to be larger and contained more accumulations of myonuclei within the myotubes compared to those of TD children. Interestingly, studies regarding other myopathologies, such as facioscapulohumeral and Duchenne Muscular Dystrophies (DMD), have described similar in vitro SC alterations in myotube morphology, diameter and/or branching [19-22]. Moreover, also nuclear positioning within the myotube seemed to be pivotal, as impairments in these processes were associated with centronuclear myopathies or Emery-Dreifuss muscular dystrophy [23,24]. These studies while linking in vitro SC features to muscle weakness emphasize the relevance of the SC alterations in myotube morphology. Whether the in vitro SC-derived myotube alterations can be linked to muscle alterations in CP children is not yet known. This is for a part due to the fact that the measurements up to now were purely descriptive. We, however, believe that the quantification of the myotubes and myonucleus features in vitro would help providing a more complete picture of the SC behaviour alterations, ultimately leading to better understanding of the observed muscle alterations. In this regard, Kahn and colleagues also hypothesised altered SC behaviour based on ex vivo muscle sections via differences in myonuclear domain in contractured muscles from patients with CP [25].

The current study aimed to employ a software tool developed in our laboratory [26] to quantify the SC-derived myotube phenotype from two different muscles in young patients with CP in comparison to age-matched TD children. We also examined potential associations with clinical parameters and performed explorative assessments of gene expression to determine potentially involved actors in altered SC functioning.

Materials and Methods

Muscle Microbiopsy Collection

This study protocol was approved by the Ethical Committee of the University Hospitals of Leuven, Belgium (S61110 and S62645). Written informed consent was provided by the parents or next of kin. Children with CP were recruited from the CP Reference Centre, whereas TD children were recruited from the Traumatology Unit for upper limb surgeries, or from the Ear, Nose and Throat Unit for other procedures at the University Hospitals Leuven (Belgium). A group of patients with CP (n=17, age 3-9 years; GMFCS I-III) and age-matched TD children (n=12) were included in this study (Table 1). In- and exclusion criteria for both the CP and the TD groups were as described before [17]. All biopsies were collected during interventions requiring general anaesthesia related to orthopaedic interventions (including BoNT-injections). Microbiopsies from the muscle mid-belly of the MG were obtained from all enrolled children, while for a subgroup of subjects (CP: n=6, TD: n=3) also a second biopsy of the Semitendinosus (ST) was collected. The biopsy collections were performed percutaneously under ultrasound guidance, with a microbiopsy needle (16-gauge, Bard) as described previously [17].

Citation: Corvelyn M, Costamagna D, Meirlevede J, Duelen R, Deschrevel J, et al. Alterations in Satellite Cell Differentiation from Young Patients with Cerebral Palsy. Phys Med Rehabil Int. 2023; 10(2): 1217.