Project Description:
CP is the leading cause of physical disability in childhood. While CP results from a neurologic insult, negative consequences in skeletal muscle, including hypoplasia, stiffness, fibrosis, contractures and weakness, are the primary contributors to decreased function. Muscle contractures that limit joint mobility and patient function are the primary focus of many clinical and surgical interventions for CP, however the specific tissue elements responsible for contractures are unknown. Current treatments available to address the functional consequences of weak, stiff muscles in CP broadly include aggressive physical therapy, neurotoxin injections, and surgical intervention, including tendon lengthening and osseous deformity correction, neither of which
address the inherent weakness and both of which can increase inflammation, worsen fibrosis 1,2, and/or lead to
a decrease in available contractile muscle 3. Determination of the mechanisms and signaling pathway(s) resulting
in fibrosis and muscle weakness is necessary to advance the development of preventative and restorative biologic treatments aimed at improving patient care, function and quality of life. Muscle dysfunction in CP muscle is observed as early as 12 months of age 4,5. Previous studies have focused on factors affecting muscle growth, demonstrating diminished satellite cells in CP muscle. However, further investigation of satellite cell-depleted murine models indicates that satellite cells are not solely responsible for resultant muscle pathology8. Previous research has shown an increase in fibrosis, or excess extracellular
matrix (ECM) deposition9-11, in affected muscles. We hypothesize that the clinical manifestation of muscular fibrosis in spastic CP contractures and weakness are directly related to the pathology initiated and promoted by the ECM, as has been implicated in other muscle diseases12,13. ECM production, organization, and breakdown is tightly regulated through bidirectional communication between cells and their surrounding ECM in an effort to maintain mechanical homeostasis 14,15. Disruption of mechanical homeostasis alters the degradation, synthesis, and organization of the ECM, often leading to increased ECM deposition 16. Abundant ECM deposition has been associated with elevated ECM stiffness 17-19, and ECM mechanical properties are emerging as a critical factor directing cell behavior. Although no previous study has directly assessed ECM mechanical properties in CP, fibrosis has been grossly correlated to in vivo musculotendinous stiffness. The current proposal builds upon the principal observation of increased ECM deposition in CP muscle by interrogating the ECM in isolation and determining the association between ECM mechanical properties, passive muscle stiffness, and CP clinical severity. This study is part of a larger research program in our group aimed at investigating the molecular basis of fibrosis in CP. As such, we have assembled a diverse team to investigate muscle fibrosis in CP as a determinant of disease severity and as a target for biological, preventative
intervention. Our expertise in CP surgery, neuromuscular disease, biomechanics, molecular biology, muscle pathology and physiology will advance our proposed investigation of CP fibrosis and its resultant clinical manifestations. Findings from this research will identify ECM alterations and targets in CP muscle that will serve as the foundation for future studies aimed at identifying the key molecular drivers of muscle fibrosis in CP and the development of interventions at improving muscle contractures and patient function. In this proposal, we aim to (1) define the role of in vitro ECM mechanical properties in passive muscle stiffness, (2) evaluate ECM organization and crosslinking across disease severity and age, and (3) develop a
meaningful clinical outcome measure of fibrosis. Each subject with CP will be classified by age and gross motor functional classification system (GMFCS), as a measure of overall musculoskeletal disease burden, and passive muscle stiffness will be evaluated on a biodex isokinetic dynamometer prior to surgery. Muscle biopsies measuring 1cm3 will be obtained during planned lower extremity surgeries in patients with spastic CP and in typically developing control subjects. Samples will be decellularized22 to allow for quantification of ECM composition and organization and evaluation ECM mechanical properties using atomic force microscopy.
Samples will additionally be sent for analysis of ECM crosslinking, which will be correlated with both in vivo and in vitro stiffness, controlling for age and GMFCS level. Defining the relative importance of fibrosis, quantifying its contribution to clinical function and severity, and developing a non-invasive means to measure ECM pathology are key to our ultimate goals: identification of clinically-relevant molecular targets for intervention and development of novel biological treatments for fibrosis and testing of their efficacy so that we may protect muscles at risk in newly-diagnosed infants and young children and restore diseased muscle in older children and adolescents to strikingly alter the natural course of disease.
