A sandwich ELISA kit reveals marked elevation of titin N‐terminal fragment levels in the urine of mdx mice

The mdx mouse is a model of Duchenne muscular dystrophy (DMD), a fatal progressive muscle wasting disease caused by dystrophin deficiency, and is used most widely in preclinical studies. Mice with dystrophin deficiency, however, show milder muscle strength phenotypes than humans. In human, the introduction of a sandwich enzyme‐linked immunosorbent assay (ELISA) kit revealed a more than 700‐fold increase in titin N‐terminal fragment levels in the urine of pediatric patients with DMD. Notably, the urinary titin level declines with aging, reflecting progression of muscle wasting. In mouse, development of a highly sensitive ELISA kit has been awaited. Here, a sandwich ELISA kit to measure titin N‐terminal fragment levels in mouse urine was developed. The developed kit showed good linearity, recovery, and repeatability in measuring recombinant or natural mouse titin N‐terminal fragment levels. The titin N‐terminal fragment concentration in the urine of mdx mice was more than 500‐fold higher than that of normal mice. Urinary titin was further analyzed by extending the collection of urine samples to both young (3–11 weeks old) and aged (56–58 weeks old) mdx mice. The concentration in the young group was significantly higher than that in the aged group. It was concluded that muscle protein breakdown is active and persistent in mdx mice even though the muscle phenotype is mild. Our results provide an opportunity to develop DMD treatments that aim to alleviate muscle protein breakdown by monitoring urinary titin levels.