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

Ramanathan, A.; Venkateshvaran, A.; Pundlik, S. S.; Suresh, Y.; Hegde, A.; Rajalakshmi, S.; Barik, A.; Lal, H.; Zachariah, N.; Pranesh, G.; Subramanian, M.

doi:10.64898/2025.11.28.690663

bioRxiv · 10.64898/2025.11.28.690663

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

Ramanathan, A.·Venkateshvaran, A.·Pundlik, S. S.·Suresh, Y.·Hegde, A.·Rajalakshmi, S.·Barik, A.·Lal, H.·Zachariah, N.·Pranesh, G.·Subramanian, M.

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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 {micro}M) 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.

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Ramanathan, A., Venkateshvaran, A., Pundlik, S. S., Suresh, Y., Hegde, A., Rajalakshmi, S., Barik, A., Lal, H., Zachariah, N., Pranesh, G., Subramanian, M.. 2025-12-03. 3D human skeletal muscle organoids reveal distinct effects of high-dose dihydronicotinamide riboside on muscle development. https://doi.org/10.64898/2025.11.28.690663

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