Loss of myogenic potential and fusion capacity of muscle stem cells isolated from contractured muscle in children with cerebral palsy.

Cerebral palsy (CP) is the most common cause of pediatric neurodevelopmental and physical disability in the United States. It is defined as a group of motor disorders caused by a nonprogressive perinatal insult to the brain. Although the brain lesion is nonprogressive, there is a progressive, lifelong impact on skeletal muscles, which are shorter, spastic, and may develop debilitating contractures. Satellite cells are resident muscle stem cells that are indispensable for postnatal growth and regeneration of skeletal muscles. Here we measured the myogenic potential of satellite cells isolated from contractured muscles in children with CP. When compared with typically developing (TD) children, satellite cell-derived myoblasts from CP differentiated more slowly (slope: 0.013 (SD 0.013) CP vs. 0.091 (SD 0.024) TD over 24 h, P < 0.001) and fused less (fusion index: 21.3 (SD 8.6) CP vs. 81.3 (SD 7.7) TD after 48 h, P < 0.001) after exposure to low-serum conditions that stimulated myotube formation. This impairment was associated with downregulation of several markers important for myoblast fusion and myotube formation, including DNA methylation-dependent inhibition of promyogenic integrin-ß 1D (ITGB1D) protein expression levels (-50% at 42 h), and ~25% loss of integrin-mediated focal adhesion kinase phosphorylation. The cytidine analog 5-Azacytidine (5-AZA), a demethylating agent, restored ITGB1D levels and promoted myogenesis in CP cultures. Our data demonstrate that muscle contractures in CP are associated with loss of satellite cell myogenic potential that is dependent on DNA methylation patterns affecting expression of genetic programs associated with muscle stem cell differentiation and muscle fiber formation.

High resolution muscle measurements provide insights into equinus contractures in patients with cerebral palsy.

Muscle contractures that occur after upper motor neuron lesion are often surgically released or lengthened. However, surgical manipulation of muscle length changes a muscle's sarcomere length (Ls ), which can affect force production. To predict effects of surgery, both macro- (fascicle length (Lf )) and micro- (Ls ) level structural measurements are needed. Therefore, the purpose of this study was to quantify both Ls and Lf in patients with cerebral palsy (CP) as well as typically developing (TD) children. Soleus ultrasound images were obtained from children with CP and TD children. Lf was determined and, with the joint in the same position, CP biopsies were obtained and formalin fixed, and Ls was measured by laser diffraction. Since soleus Ls values were not measurable in TD children, TD Ls values were obtained using three independent methods. While average Lf did not differ between groups (CP=3.6±1.2 cm, TD=3.5±0.9 cm; p>0.6), Ls was dramatically longer in children with CP (4.07±0.45 µm vs. TD=2.17±0.24 µm; p<0.0001). While Lf values were similar between children with CP and TD children, this was due to highly stretched sarcomeres within the soleus muscle. Surgical manipulation of muscle-tendon unit length will thus alter muscle sarcomere length and change force generating capacity of the muscle.

Pathophysiology of muscle contractures in cerebral palsy.

Patients with cerebral palsy present with a variety of adaptations to muscle structure and function. These pathophysiologic symptoms include functional deficits such as decreased force production and range of motion, in addition to changes in muscle structure such as decreased muscle belly size, increased sarcomere length, and altered extracellular matrix structure and composition. On a cellular level, patients with cerebral palsy have fewer muscle stem cells, termed satellite cells, and altered gene expression. Understanding the nature of these changes may present opportunities for the development of new muscle treatment therapies.

Stiff muscle fibers in calf muscles of patients with cerebral palsy lead to high passive muscle stiffness.

Cerebral palsy (CP), caused by an injury to the developing brain, can lead to alterations in muscle function. Subsequently, increased muscle stiffness and decreased joint range of motion are often seen in patients with CP. We examined mechanical and biochemical properties of the gastrocnemius and soleus muscles, which are involved in equinus muscle contracture. Passive mechanical testing of single muscle fibers from gastrocnemius and soleus muscle of patients with CP undergoing surgery for equinus deformity showed a significant increase in fiber stiffness (p<0.01). Bundles of fibers that included their surrounding connective tissues showed no stiffness difference (p=0.28).). When in vivo sarcomere lengths were measured and fiber and bundle stiffness compared at these lengths, both fibers and bundles of patients with CP were predicted to be much stiffer in vivo compared to typically developing (TD) individuals. Interestingly, differences in fiber and bundle stiffness were not explained by typical biochemical measures such as titin molecular weight (a giant protein thought to impact fiber stiffness) or collagen content (a proxy for extracellular matrix amount). We suggest that the passive mechanical properties of fibers and bundles are thus poorly understood.

Comparison of pelvic muscle architecture between humans and commonly used laboratory species.

INTRODUCTION AND HYPOTHESIS: Pelvic floor muscles (PFM) are deleteriously affected by vaginal birth, which contributes to the development of pelvic floor disorders. To mechanistically link these events, experiments using animal models are required, as access to human PFM tissue is challenging. In choosing an animal model, a comparative study of PFM design is necessary, since gross anatomy alone is insufficient to guide the selection. METHODS: Human PFM architecture was measured using micromechanical dissection and then compared with mouse (n = 10), rat (n = 10), and rabbit (n = 10) using the Architectural Difference Index (ADI) (parameterizing a combined measure of sarcomere length-to-optimal-sarcomere ratio, fiber-to-muscle-length ratio, and fraction of total PFM mass and physiological cross-sectional area (PCSA) contributed by each muscle). Coccygeus (C), iliocaudalis (IC), and pubocaudalis (PC) were harvested and subjected to architectural measurements. Parameters within species were compared using repeated measures analysis of variance (ANOVA) with post hoc Tukey's tests. The scaling relationships of PFM across species were quantified using least-squares regression of log-10-transformed variables. RESULTS: Based on the ADI, rat was found to be the most similar to humans (ADI = 2.5), followed by mouse (ADI = 3.3). When animals' body mass was regressed against muscle mass, muscle length, fiber length, and PCSA scaling coefficients showed a negative allometric relationship or smaller increase than predicted by geometric scaling. CONCLUSION: In terms of muscle design among commonly used laboratory animals, rat best approximates the human PFM, followed by mouse. Negative allometric scaling of PFM architectural parameters is likely due to the multifaceted function of these muscles.

Comparison of rotator cuff muscle architecture between humans and other selected vertebrate species.

In this study, we compare rotator cuff muscle architecture of typically used animal models with that of humans and quantify the scaling relationships of these muscles across mammals. The four muscles that correspond to the human rotator cuff - supraspinatus, infraspinatus, subscapularis and teres minor - of 10 commonly studied animals were excised and subjected to a series of comparative measurements. When body mass among animals was regressed against physiological cross-sectional area, muscle mass and normalized fiber length, the confidence intervals suggested geometric scaling but did not exclude other scaling relationships. Based on the architectural difference index (ADI), a combined measure of fiber length-to-moment arm ratio, fiber length-to-muscle length ratio and the fraction of the total rotator cuff physiological cross-sectional area contributed by each muscle, chimpanzees were found to be the most similar to humans (ADI=2.15), followed closely by capuchins (ADI=2.16). Interestingly, of the eight non-primates studied, smaller mammals such as mice, rats and dogs were more similar to humans in architectural parameters compared with larger mammals such as sheep, pigs or cows. The force production versus velocity trade-off (indicated by fiber length-to-moment arm ratio) and the excursion ability (indicated by fiber length-to-muscle length ratio) of humans were also most similar to those of primates, followed by the small mammals. Overall, primates provide the best architectural representation of human muscle architecture. However, based on the muscle architectural parameters of non-primates, smaller rather than larger mammals may be better models for studying muscles related to the human rotator cuff.

Anatomical, architectural, and biochemical diversity of the murine forelimb muscles.

We characterized the architecture, fiber type, titin isoform distribution, and collagen content of 27 portions of 22 muscles in the murine forelimb. The mouse forelimb was different from the human arm in that it had the extensor digitorum lateralis muscle and no brachioradialis muscle. Architecturally, the mouse forelimb differed from humans with regard to load bearing, having a much larger contribution from extensors than flexors. In mice, the extensor : flexor PCSA ratio is 2.7, whereas in humans it is only 1.4. When the architectural difference index was calculated, similarities became especially apparent between flexors and extensors of the distal forelimb, as well as pronators. Discriminant analysis revealed that biochemical measures of collagen, titin, and myosin heavy chain were all strong between-species discriminators. In terms of composition, when compared with similar muscles in humans, mice had, on average, faster muscles with higher collagen content and larger titin isoforms. This report establishes the anatomical and biochemical properties of mouse forelimb muscles. Given the prevalence of this species in biological studies, these data will be invaluable for studying the biological basis of mouse muscle structure and function.

Flagella and pili-mediated near-surface single-cell motility mechanisms in P. aeruginosa.

Bacterial biofilms are structured multicellular communities that are responsible for a broad range of infections. Knowing how free-swimming bacteria adapt their motility mechanisms near a surface is crucial for understanding the transition from the planktonic to the biofilm phenotype. By translating microscopy movies into searchable databases of bacterial behavior and developing image-based search engines, we were able to identify fundamental appendage-specific mechanisms for the surface motility of Pseudomonas aeruginosa. Type IV pili mediate two surface motility mechanisms: horizontally oriented crawling, by which the bacterium moves lengthwise with high directional persistence, and vertically oriented walking, by which the bacterium moves with low directional persistence and high instantaneous velocity, allowing it to rapidly explore microenvironments. The flagellum mediates two additional motility mechanisms: near-surface swimming and surface-anchored spinning, which often precedes detachment from a surface. Flagella and pili interact cooperatively in a launch sequence whereby bacteria change orientation from horizontal to vertical and then detach. Vertical orientation facilitates detachment from surfaces and thereby influences biofilm morphology.

Bacteria use type IV pili to walk upright and detach from surfaces.

Bacterial biofilms are structured multicellular communities involved in a broad range of infections. Knowing how free-swimming bacteria adapt their motility mechanisms near surfaces is crucial for understanding the transition between planktonic and biofilm phenotypes. By translating microscopy movies into searchable databases of bacterial behavior, we identified fundamental type IV pili-driven mechanisms for Pseudomonas aeruginosa surface motility involved in distinct foraging strategies. Bacteria stood upright and "walked" with trajectories optimized for two-dimensional surface exploration. Vertical orientation facilitated surface detachment and could influence biofilm morphology.