Advanced modelling of RYR1-related myopathies using human iPS cells and 3D engineered skeletal muscles

<title>Abstract</title> <p> Background Ryanodine receptor type 1-related myopathies (RYR1-RM) represent a clinically and genetically heterogeneous group of congenital skeletal muscle disorders caused by pathogenic variants in the ryanodine receptor type 1 ( <italic>RYR1</italic> ) gene, which encodes a calcium channel critical for skeletal muscle excitation-contraction coupling. Dysregulated calcium handling is a central feature of these conditions. However, little is known on the precise mechanisms by which specific mutations disrupt muscle development, maturation, and contractile function in human. Furthermore, the lack of physiologically relevant human models has hindered the development of effective therapeutic strategies. In this study, we investigated whether dominant pathogenic <italic>RYR1</italic> variants, leading to gain of function, affect myogenic capacity and excitation-contraction coupling during human skeletal muscle development and maturation in patient-derived iPSC platforms. Methods We generated patient-specific induced pluripotent stem cells (iPSCs) carrying pathogenic gain of function <italic>RYR1</italic> variants in position R2452W and A4894P, followed by transgene-free, lineage-directed differentiation into skeletal myogenic derivatives. These <italic>RYR1</italic> pathogenic variants underwent morphological and functional characterisation in two-dimensional (2D) cultures and 3D engineered muscle tissues. Results The <italic>RYR1</italic> gain-of-function variants R2452W and A4894P did not significantly impact reprogramming or exit from pluripotency. Differentiation of <italic>RYR1</italic> -mutant iPSCs recapitulated histological, disease-associated phenotypes of RYR1-RM, including altered myofibre size and alignment. Functional assessment of electromechanical coupling in 2D and 3D cultures revealed altered calcium dynamics and reduced force of contraction in RYR1-RM models vs. healthy controls, demonstrating that the disrupted excitation–contraction coupling associated with dysregulated RYR1 channel activity can be faithfully recapitulated <italic>in vitro</italic> in a humanised setting. Conclusion Collectively, these findings establish a disease modelling platform providing the foundation for genotype–phenotype correlation studies, precision medicine and preclinical therapeutic evaluation for RYR1-RM. </p>