Decoding Giant Muscle Protein Dynamics

About This Grant

Nebulin is a giant actin-binding protein critical for sarcomere structure and function. Mutations in the NEB gene cause congenital myopathies, often recessive and associated with reduced nebulin levels and thin filament instability. However, recent findings highlight a new class of dominant NEB variants involving large in-frame deletions, such as NEBΔ14–89, which remove a substantial fraction of the super-repeats (SRs) but still result in high levels of mutant protein expression. Unlike recessive mutations, NEBΔ14–89 nebulin incorporates efficiently into thin filaments and displaces the wild-type isoform, suggesting a novel disease mechanism rooted in altered protein dynamics rather than haploinsufficiency. Despite the deletion of 17 of 29 SRs, the mutant protein aligns correctly with the thin filament and accumulates to high levels in sarcomeres. These findings raise important questions about how altered translation efficiency and replacement kinetics of truncated nebulin contribute to myopathy. Aim 1 investigates whether elevated NEBΔ14–89 levels arise from increased transcript abundance and/or translation efficiency. Patient biopsy-derived RNA and protein levels will be quantified, and ribosome profiling (Ribo-seq) will assess translational output. STORM microscopy and contractile assays will examine structural and functional impact. Aim 2 addresses nebulin replacement kinetics using novel Dendra2-tagged mouse models and in vivo imaging. We investigate nebulin turnover dynamics in adult skeletal muscle using a mouse model in which the N-terminus of WT nebulin is tagged with Dendra2. We will apply in vivo fluorescence recovery after photoconversion using surgically implanted imaging windows in mice, allowing us to monitor nebulin replacement up to months. Pilot study show that this work is feasible and support slow replacement kinetics (1-2%/day). We will study nebulin's cytosolic diffusion kinetics and the cytosolic vs. incorporated fraction using single molecule localization microscopy. Sub-aim 2a will define replacement kinetics of wild-type nebulin in adult muscle. Sub-aim 2b will assess how NebΔ14–89, NebΔ14– 77, and NebΔ53–64 variants affect nebulin's cytosolic pool and thin filament incorporation dynamics and will establish thin filament length and contractile function. We hypothesize that deletion size correlates with slower turnover, greater cytosolic accumulation, and more severe dysfunction. Aim 3 tests the therapeutic potential of allele-specific knockdown using shRNA against NEBΔ14–89. Rescue of the myopathy in mice by reducing mutant transcript levels would provide a compelling strategy for clinical intervention. Supported by robust pilot data, access to patient biopsies, validated mouse models, and advanced imaging technologies that, for the first time, enable real-time visualization of muscle protein dynamics in living animals, this project promises to uncover new mechanisms of dominant nebulin myopathy and guide future therapies.