3D human skeletal muscle organoids reveal distinct effects of high-dose dihydronicotinamide riboside on muscle development

Abstract The evaluation of NAD+-boosting compounds in human skeletal muscle is hindered by limitations of traditional 2D cultures and animal models. Human-relevant, three-dimensional (3D) engineered skeletal muscle organoids offer a promising platform to assess the biological effects of metabolic modulation. Here we engineered 3D human skeletal muscle organoids to investigate the impact of dihydronicotinamide riboside (NRH), a potent NAD+ precursor. Sustained exposure to high NRH concentrations (500 µM) enhanced early differentiation markers, including increased myotube fusion and fast-twitch fiber area, but concurrently induced structural defects such as disrupted sarcomeric organization, enlarged acetylcholine receptor clusters, and impaired acetylcholine-stimulated calcium signaling. These results reveal that excessive and sustained NAD+ elevation can uncouple rapid differentiation from proper maturation in muscle tissue. Our results highlight the importance of dose and duration optimization for NAD+-boosting compounds and establish 3D engineered muscle organoids as a valuable non-animal platform for mechanistic toxicology and preclinical safety assessment. Competing Interest Statement The authors have declared no competing interest.