Dose-Dependent Effects of Dihydronicotinamide Riboside on Human Engineered Skeletal Muscle Development

Nicotinamide adenine dinucleotide (NAD+) precursors are explored for metabolic and longevity interventions; however, their impact on human skeletal muscle development remains unclear due to limitations of conventional 2D cultures and animal models. Here, we employed three-dimensional engineered skeletal muscle tissues derived from primary human myoblasts to investigate dose-dependent effects of dihydronicotinamide riboside (NRH), a potent NAD+ precursor. Moderate NRH exposure (25 μM) enhanced myogenic differentiation and fusion without affecting viability, aligning with physiologically relevant plasma levels. In contrast, a sustained high NRH concentration (500 μM) increased the myotube number and fast-twitch fiber area but disrupted sarcomeric organization, acetylcholine receptor clustering, and calcium signaling, with transcriptomic profiling revealing downregulation of myogenesis and metabolic pathways. These findings demonstrate that NRH exerts biphasic effects on human skeletal muscle differentiation and maturation, underscoring the utility of 3D engineered muscle tissues as a human-relevant platform to evaluate therapeutic and toxic doses of NAD+-boosting compounds.