Zebrafish as a model to understand the impact of inactivity and neuromuscular electrical stimulation on neuromuscular plasticity in Duchenne Muscular Dystrophy

Skeletal muscle plasticity is imperative for functional adaptation to changing demands. Although a great deal is known about the structural and functional plasticity of healthy skeletal muscle, far less is known about plasticity in diseased muscle. Here, we created an experimental design to better understand how dystrophin-deficient zebrafish muscle responds to inactivity versus activity. We tested two inactivity paradigms, intermittent versus extended inactivity, and found that extended inactivity, defined as 72 hours in low-dose tricaine, worsens muscle structure and decreases survival. Strikingly, extended inactivity followed by a single session of neuromuscular electrical stimulation (NMES) further worsens muscle structure and obliterates its ability to regenerate. Next, we tested four unique NMES paradigms that varied in their frequency and voltage, and found that each NMES paradigm differentially affected muscle structure, function, and survival. However, one NMES paradigm, which we refer to as endurance NMES, improved all three components of muscle health. Accompanying these improvements, we observed increased sarcomere lengths as well as larger surface areas, volumes, and filament indices of muscle nuclei. Additionally, time-lapse imaging reveals less degeneration and improved regeneration of the fast-twitch muscle fibers. RNAseq analysis highlights two potential mechanisms, one in which gene expression levels are returned to wild-type levels as well as remodeling of the extracellular matrix. Collectively, these data provide a new methodology with which to study neuromuscular plasticity in healthy versus diseased muscle. In addition, our data clearly indicate that, at least in the zebrafish model, NMES is beneficial whereas inactivity is deleterious for dystrophic muscle. Overall design: Gene expression was assayed using RNA sequencing in dmd mutant and wild-type sibling control whole zebrafish larvae at 7 days post fertilization with and without neuromuscular electrical stimultation (NMES). Each biological replicate consisted of two zebrafish larvae. For wild-type siblings, there were 4 biological replicates for the control group and 3 biological replicates for the NMES group. For the dmd mutants, there were 8 biological replicates in the dmd mutant control group, and 10 biological replicates in the dmd NMES group.