Development of an platform for the analysis of contractile and calcium dynamics in single human myotubes.

myotube cultures are widely used as models for studying muscle pathophysiology, but their limited maturation and heterogeneity pose significant challenges for functional analyses. While they remain the gold standard for studying muscle function , myotube cultures do not fully recapitulate the complexity and native features of muscle fibers, which may compromise their ability to predict outcomes. To promote maturation and decrease heterogeneity, we have incorporated engineered structures into myotube cultures, based on a PDMS thin layer with micrometer-sized grooves (μGrooves) placed over a glass substrate. Different sizes and shapes of μGrooves were tested for their ability to promote alignment and fusion of myoblasts and enhance their differentiation into myotubes. A 24 hour electrical field stimulation protocol (4 V, 6 ms, 0.1 Hz) was used to further promote myotube maturation, after which several myotube features were assessed, including myotube alignment, width, fusion index, contractile function, and calcium handling. Our results indicate superior calcium and contractile performance in μGrooved myotubes, particularly with the 100 μm-width 700 μm-long geometry (7 : 1). This platform generated homogeneous and isolated myotubes that reproduced native muscle features, such as excitation-contraction coupling and force-frequency responses. Overall, our 2D muscle platform enables robust high-content assays of calcium dynamics and contractile readouts with increased sensitivity and reproducibility compared to traditional myotube cultures, making it particularly suitable for screening therapeutic candidates for different muscle pathologies.