Early life lipid overload in Native American Myopathy is phenocopied by stac3 knockout in zebrafish.

Understanding the early stages of human congenital myopathies is critical for proposing strategies for improving musculoskeletal muscle performance, such as restoring the functional integrity of the cytoskeleton. SH3 and cysteine-rich domain 3 (STAC3) are proteins involved in nutrient regulation and are an essential component of the excitation-contraction (EC) coupling machinery for Ca²⁺ releasing. A mutation in STAC3 causes debilitating Native American Myopathy (NAM) in humans, while loss of this gene in mice and zebrafish (ZF) results in premature death. Clinically, NAM patients demonstrated increased lipids in skeletal muscle, but it is unclear if neutral lipids are associated with altered muscle function in NAM. Using a CRISPR/Cas9 induced stac3^-/- knockout (KO) zebrafish model, we determined that loss of stac3 leads to delayed larval hatching which corresponds with muscle weakness and decreased whole-body Ca²⁺ level during early skeletal development. Specifically, we observed defects in the cytoskeleton in F-actin and slow muscle fibers at 5 and 7 days post-fertilizations (dpf). Myogenesis regulators such as myoD and myf5, mstnb were significantly altered in stac3^-/- larvae. These muscle alterations were associated with elevated neutral lipid levels starting at 5 dpf and persisting beyond 7 dpf. Larva lacking stac3 had reduced viability with no larva knockouts surviving past 11 dpf. This data suggests that our stac3^-/- zebrafish serve as an alternative model to study the diminished muscle function seen in NAM patients. The data gathered from this new model over time supports a mechanistic view of lipotoxicity as a critical part of the pathology of NAM and the associated loss of function in muscle.