Genotype‐Dependent Morpho‐Mechanical Profiling of Patient‐Derived Human Myotubes on Nanogrooved Substrates

ABSTRACT Muscle disorders such as myofibrillar myopathies and Duchenne muscular dystrophy involve mutations in key cytoskeletal proteins and lead to progressive muscle degeneration. Yet, the mechanical characterization of affected muscle cells has relied mainly on immature or non‐human models. Here, we introduce a human in vitro platform based on patient‐derived immortalized myoblasts differentiated into myotubes on nanogrooved substrates, which promote alignment and organotypic maturation. Using immunostaining and atomic force microscopy (AFM), we show that desmin‐ and dystrophin‐mutated myotubes exhibit distinct morphological and mechanical phenotypes compared to wild‐type myotubes. We developed an AFM stiffness pipeline to quantify cell body stiffness across myotubes of variable thickness. Desmin‐ and dystrophin‐mutated myotubes are stiffer than controls, with desmin mutants also displaying cytoskeletal disorganization. A dynamic fatigue assay (cyclic AFM indentations over time) further revealed impaired stiffening and faster mechanical fatigue in desmin mutants, while dystrophin mutants preserved resilience. This set of results establishes a reproducible and human‐relevant system to probe muscle mechanics in disease, offering a unique intermediate model between conventional immortalized lines and complex iPSC‐derived tissues, and enabling future quantitative screening and translational applications.