Dystroglycanopathies represent forms of muscular dystrophy, which present as severe mental retardation and brain and eye malformations. The rare genetic disorder results in muscle development abnormalities and can be lethal. Scientists from the University of Maine published a study in Skeletal Muscle where they found supplementing with a molecule called nicotinamide adenine dinucleotide (NAD+) improved neuromuscular development in zebrafish with dystroglycanopathy. 

Muscle constitutes an essential component of the body used for strength and locomotion with muscle fibers overlapping and pulling together as rope-like structures that contract when flexed. Two parts of the muscle flexion system, the neuromuscular junction and the myotendinous junction, link action planning in the brain to actual movements through muscle flexing. The neuromuscular junction initiates muscle contractions as a chemical connection between a neuron and a muscle fiber, while the myotendinous junction transmits force from muscle to the skeletal system as the connection between a tendon and muscle.

By injecting zebrafish with morpholinos, a molecule that causes neuromuscular development dysfunction through decreasing the activity of the FKRP gene, the researchers created fish with muscle degeneration. In these diseased zebrafish, supplementation with NAD+ or B3 vitamins prior to muscle development reduced muscle degeneration and improved motility. 

Along with NAD+’s role as an essential molecule for metabolism and cellular health, previous studies have also established NAD+ as an essential molecule for muscle development. B3 vitamins boost NAD+ levels, the essential molecule that helps regulate metabolic functions, by providing the body with the molecules needed to synthesize NAD+. The increased NAD+ levels from NAD+ or B3 vitamins supplementation reversed the deleterious effects of the morpholinos and restored muscle function during development.

(Bailey et al., 2019 | Skeletal Muscle) NAD+ supplementation given prior to muscle development improves muscle structure and function. The left column represents normal zebrafish muscle structure, the middle column reduced fkrp gene activity, and the right column reduced fkrp gene activity with NAD+ supplementation given prior to muscle development.

Cells require NAD+ to perform a cellular function essential for neuromuscular junction maturation called “glycosylation of dystroglycan.” Treatments of NAD+ improved neuromuscular junction formation and maturation during development in the neuromuscular dysfunctioning zebrafish with reduced FKRP gene activity. These results suggest that at certain times during development, supplementation with NAD+ or B3 vitamins can improve neuromuscular junction and overall muscle structure and function in zebrafish with dystroglycanopathies.

(Bailey et al., 2019 | Skeletal Muscle) NAD+ and B3 vitamin supplementation improve neuromuscular junction formation in zebrafish. The yellow-colored stain in the lower left of each of the four images shows neuromuscular junctions. The top left image shows a normal zebrafish, the top right image shows the reduced neuromuscular junction formation with morpholino injections that reduce fkrp activity. The bottom left image shows improvements in neuromuscular junction formation with NAD+ supplementation in zebrafish with reduced fkrp gene activity. The bottom right image shows improved neuromuscular junction formation with B3 vitamin supplementation in diseased zebrafish with reduced fkrp gene activity.

NAD+ and B3 vitamins’ ability to improve neuromuscular dysfunction comes with a catch — the treatment needs to be administered in the early stages of muscle development. Although NAD+ and B3 vitamin supplementation after initial muscle development improve myotendinous junctions, they do not help with muscle structure and function.  These results were in contrast to the outcome of giving supplementation prior to muscle development, suggesting early intervention is most beneficial in neuromuscular dysfunctions.

(Bailey et al., 2019 | Skeletal Muscle) NAD+ and B3 vitamin supplementation do not confer improvements in muscle structure or function when given after initiation of muscle development. The leftmost picture is normal muscle, the left middle picture shows muscle with less fkrp gene activity, the right middle picture shows muscle with less fkrp gene activity and NAD+ supplementation, and the far right picture shows muscle structure with less fkrp gene activity and B3 vitamin supplementation.

NAD+ supplementation prior to muscle development improved muscle formation along with neuromuscular and myotendinous junction formation. In contrast, supplementing with NAD+ or B3 vitamins to boost NAD+ levels after muscle development did not improve muscle structure or function in the zebrafish. These results support the notion that a time window exists where increasing NAD+ levels can improve muscle structure and function in dystroglycanopathies.

“Taken together, our data indicate that Fkrp plays an early and crucial role in muscle, [myotendinous junction], and [neuromuscular junction] development,” noted the researchers in their study.