Hypermotility Rescues Paralysis-Inhibited Embryonic Tendon Development in a Stage-Dependent Manner

Introduction: Tendons transmit forces from muscle to bone, enabling skeletal movements. During in utero development, reduced fetal movements are associated with tendon-related musculoskeletal birth deformities, including clubfoot which affects up to 1 in 100 live births. These deformities involve formation of abnormal limb tendons that possess inferior mechanical properties, thereby compromising normal life functions including walking and standing. Unfortunately, abnormal tendons are incapable of regenerating, and there are no tendon therapeutics available. To develop treatment strategies that promote normal tendon formation, studies to understand the regulation of embryonic tendon development are needed. The chick embryo is a useful model to study musculoskeletal development because it shares molecular, biochemical, and structural similarities with mammals. It is also more amenable to experimental manipulation since it is contained in an eggshell, rather than in utero. Our studies in the chick have shown that kicking frequency regulates calcaneal tendon mechanical properties during development. Specifically, during later developmental days (D)17-D19, paralysis inhibited embryonic tendon mechanical property development, whereas higher-than-normal kicking frequency (hypermotility) enhanced mechanical properties. Based on our previous discoveries that substantial embryonic tendon mechanical property elaboration begins earlier (D14), this work aimed to investigate how kicking frequency affects younger embryo tendons and whether hypermotility rescues paralysis-inhibited mechanical development. We also examined genes that may regulate these movement frequency effects. We hypothesize that paralysis inhibits mechanical development of the chick calcaneal tendon as early as D13, that hypermotility rescues mechanical development after paralysis, and that these are due to movement frequency-dependent regulation of molecular pathways.

Materials and

Methods: White Leghorn chick embryos were treated in ovo with either pancuronium bromide (PB) to induce flaccid paralysis, 4-aminopyridine (4-AP) to induce hypermotility, or saline for natural motility. Calcaneal tendons were harvested for tensile testing to evaluate mechanical properties or analyzed using bulk RNAseq for differentially expressed genes (DEGs). Conditions included: i) 48h paralysis at different developmental stages (D13-D15, D15-D17, D17-D19), and ii) paralysis for different durations from D13-D15 (48h) or D13-D17 (96h), followed by 48h rescue with saline or 4-AP to induce natural or hypermotile movement, respectively. Controls for the rescue experiments included both saline and sustained paralysis groups, representative of normal and paralyzed tendons. We used One-way ANOVA with Tukey’s multiple comparison tests to analyze pairwise statistical differences within 3 or more groups. DEGs were determined using DESeq2. Gene ontology pathway analysis was performed using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) to determine biological processes associated with up- or down-regulated DEGs in a given treatment group. A minimum of N=3 (biological replicates) was analyzed for each treatment group.

Results, Conclusions, and Discussions: Paralysis during D13-D15, D15-D17, and D17-D19 each resulted in significantly lower tendon elastic modulus and ultimate stress, but not peak strain, compared to control. Paralysis effects were stage-dependent, with the greatest differences in mechanical properties after D13-D15 paralysis (Fig. 1A). Following 48h paralysis, both natural motility and hypermotility increased modulus and ultimate stress compared to continuous paralysis. However, only hypermotility restored mechanical properties to saline control levels (Fig. 1B). In contrast, following 96h paralysis, neither rescue condition improved mechanical properties compared to continuous paralysis (Fig. 1C). Peak strain was not affected in any groups (not shown). Interestingly, RNAseq showed that hypermotility rescue involved fewer up- and down-regulated DEGs than natural motility rescue, relative to sustained paralysis (Fig. 1D). Pathway analysis of DEGs unique to each rescue group revealed different biological processes involved in hypermotility versus natural motility rescue (not shown).

This study revealed that paralysis leads to inferior calcaneal tendon mechanical properties in a stage-dependent manner, with the greatest effects during D13-D15. Tendons paralyzed during D13-D15 had 75% lower modulus and peak stress than normal, whereas tendons paralyzed during D15-D17 and D17-D19 possessed ~30-40% lower modulus and peak stress than normal. To test the possibility of restoring the mechanical properties of paralyzed tendons by increasing embryo movement frequency, we induced natural motility or hypermotility rescue following a shorter (48h) or longer (96h) period of paralysis. Hypermotility and natural motility rescued mechanical properties fully and partially, respectively, but only after the shorter paralysis period (48h). In our previous work, paralysis reduced lysyl oxidase (LOX) activity levels, thus it is possible LOX-mediated collagen crosslinking is involved in mechanoregulation of tendon development. However, other mechanisms may also be involved given the numerous differences in DEGs between natural motility and hypermotility rescue as compared to sustained paralysis. Differences in DEG profiles also suggest natural motility and hypermotility rescues after paralysis involve divergent molecular mechanisms.

These exciting results show that embryo movement frequency directly impacts tendon mechanical properties, apparently through differential regulation of molecular pathways, during development and establish a model for mechanistic studies that may lead to clinical solutions for tendon-related birth defects.

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