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Active and Passive Mechanical Deficits Precede Spinal Curvature in a Zebrafish Model of Idiopathic Scoliosis

ABSTRACT: Idiopathic scoliosis is a common spinal disorder characterized by progressive three-dimensional curvature of unknown cause. Although biomechanical imbalance has long been proposed to contribute to scoliosis, the early physiological states that precede curvature onset remain poorly understood. Here, we investigated this problem using zebrafish uts2r3 mutants, which develop fully penetrant juvenile-onset spinal curvature following disruption of urotensin signaling. Transcriptomic analysis before curvature revealed altered expression of muscle-associated genes, suggesting that Uts2r3 influences axial muscle development or function. However, immunofluorescence, birefringence imaging, and quantitative analysis of myotome morphology showed that mutants lack overt muscle architectural defects or dystrophic pathology. By contrast, direct measurements of isolated larval trunks revealed pre-curvature biomechanical abnormalities: namely, uts2r3 mutants generated reduced active force following electrical stimulation while also exhibiting increased passive resistance to stretch. These findings identify urotensin signaling as a regulator of axial tissue biomechanics during growth and suggest that scoliosis-like curvature can arise from an early imbalance between active force generation and passive tissue stiffness.

ORGANISM(S): Danio rerio

PROVIDER: GSE334452 | GEO | 2026/06/05

REPOSITORIES: GEO

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Project description:Critically ill intensive care unit (ICU) patients commonly develop severe muscle wasting and impaired muscle function, leading to delayed recovery, with subsequent increased morbidity and financial costs, and decrease quality of life of survivors. Acute Quadriplegic Myopathy (AQM) is one of the most common neuromuscular disorders associated with ICU-acquired muscle weakness. Although there are no available treatments for the ICU-acquired muscle weakness, it has been demonstrated that early mobilization can improve its prognosis and functional outcomes. This study aims at improving our understanding of the effects of passive mechanical loading on skeletal muscle structure and function by using a unique experimental rat ICU model allowing analyses of the temporal sequence of changes in mechanically ventilated and pharmacologically paralyzed animals at durations varying from 6 h to 14 days. Results show that passive mechanical loading alleviated the muscle wasting and the loss of force-generation associated with the ICU intervention, resulting in a doubling of the functional capacity of the loaded vs. unloaded muscles after a 2-week ICU intervention. We demonstrated that the improved maintenance of muscle structure and function is likely a consequence of a reduced oxidative stress, and a reduced loss of the molecular motor protein myosin. A complex temporal gene expression pattern, delineated by microarray analysis, was observed with loading-induced changes in transcript levels of sarcomeric proteins, muscle developmental processes, stress response, ECM/cell adhesion proteins and metabolism. Thus, the results from this study show that passive mechanical loading alleviates the severe negative consequences on muscle structure and function associated with mechanical silencing in ICU patients, strongly supporting early and intense physical therapy in immobilized ICU patients. This study aims to unravel the effects of passive mechanical loading on skeletal muscle structure and function in an experimental rat ICU model at duration varying between 6h and 14 days. A total of 23 experimental female Sprague-Dawley rats were included in this study. The experimental rats were anaesthetized, treated with the neuromuscular blocking agent (NMBA) M-NM-1-cobrotoxin, mechanically ventilated and monitored for durations varying from 6h to 4 days (n=13), from 5 to 8 days (n=4), and from 9 to 14 days (n=6). The left leg of the animal was activated for 6 hours at the shortest duration and 12 hours per day at durations 12 hours and longer throughout the experiment, using a mechanical lever arm that produced a continuous passive maximal ankle joint flexions-extensions at a speed of 13.3 cycles per minute. Muscle biopsies were obtained from gastrocnemius muscle (proximal part) immediately after euthanasia, were quickly frozen in liquid propane cooled by liquid nitrogen, and stored at -80M-BM-0C. RNA was extracted.

Project description:Background Three-dimensional (3D) bioengineered models of human skeletal muscle are a promising approach for studying muscle development, function and disease in vitro. These models more closely resemble the complexity of native muscle tissue than two-dimensional (2D) monolayer culture and allow for functional measurements to be performed. However, a more complete understanding of how culture condition and duration impacts the myotube maturity and function is required to validate the transition from 2D to 3D culture of muscle cells. Methods Human skeletal muscle cells were cultured as either 2D monolayers or within 3D fibrin-based hydrogels as muscle constructs for up to 21 days. Quantitative proteomic analysis and functional assessments, including contractile force and cross-sectional area measurements, were conducted to evaluate the impact of culture conditions and duration on muscle cell differentiation. Results Proteomic analysis revealed myoblasts differentiated into myotubes by 8 days of differentiation in both 2D and 3D environments. However, the proteomic profiles of myotubes varied significantly between the two culture environments. At day 8 of differentiation, myosin heavy chain isotype abundance indicated a predominantly slow-twitch phenotype in 3D constructs, compared to a mixed fibre type phenotype in 2D monolayers. By day 21 of differentiation, 3D muscle constructs displayed improved mitochondrial maturity, extracellular matrix remodelling, and signs of transitioning towards a fast-twitch phenotype. This prolonged culture duration also resulted in increased passive tension but decreased peak contractile force in 3D muscle constructs. Conclusions This study demonstrates that 3D culture promotes maturity in human skeletal muscle cells, mimicking the biochemical cues and energy demands seen in native muscle tissue. The data highlights the importance of selecting appropriate culture conditions and durations when studying skeletal muscle cells in vitro and suggests 8 days of differentiation as optimal for achieving peak contractile force in 3D muscle constructs.

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Active and Passive Mechanical Deficits Precede Spinal Curvature in a Zebrafish Model of Idiopathic Scoliosis

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